register interest

Professor Nicholas J White FRS

Research Area: Global Health
Technology Exchange: Mass spectrometry
Scientific Themes: Tropical Medicine & Global Health and Immunology & Infectious Disease
Keywords: malaria, drug discovery and therapeutics
Web Links:
Proposed mechanism of periodic Plasmodium vivax relapse activation

Proposed mechanism of periodic Plasmodium vivax relapse activation

Professor White’s diverse interests include the epidemiology, pathophysiology and management of uncomplicated and severe malaria, meliodosis, enteric fever, tetanus, dengue haemorrhagic fever, Japanese encephalitis and tuberculosis. His particular interests at present include the pathophysiology and treatment of severe malaria,  the prevention of antimalarial drug resistance using artemisinin-based combinations. and the biology of relapse in vivax malaria.

Name Department Institution Country
Professor Nicholas PJ Day FMedSci FRCP Tropical Medicine University of Oxford United Kingdom
Professor François H Nosten Tropical Medicine University of Oxford United Kingdom
Professor Adrianus Dondorp Tropical Medicine University of Oxford United Kingdom
Professor Jeremy Farrar Tropical Medicine University of Oxford United Kingdom
Professor Paul Newton Tropical Medicine University of Oxford United Kingdom
Professor Dominic Kwiatkowski Wellcome Trust Centre for Human Genetics University of Oxford United Kingdom
Dr Olivo Miotto Tropical Medicine University of Oxford United Kingdom
Professor Philippe J Guerin Tropical Medicine University of Oxford United Kingdom
Dr Gareth Turner Tropical Medicine University of Oxford United Kingdom
Professor Paul Turner Tropical Medicine University of Oxford United Kingdom
Cheah PY, White NJ. 2016. Antimalarial mass drug administration: ethical considerations. Int Health, 8 (4), pp. 235-238. | Show Abstract | Read more

Falciparum malaria is a major cause of death and illness in tropical countries, particularly in childhood. In endemic countries, a significant proportion of the community is infected with malaria asymptomatically. One promising way to eliminate malaria is to give the entire population malaria treatment. This is called mass drug administration (MDA) and it raises a number of ethical issues, as possible long-term benefits are uncertain. The effectiveness of MDA is critically dependent on level of participation, so the promised benefits to the community can be annulled by non-participation of a small number of individuals. These potential benefits range a wide spectrum, from the permanent elimination of malaria (success) to a transient reduction in the prevalence of infection and the incidence of illness (failure). The drawbacks of MDA are: inconvenience, potential toxicity, loss of confidence in the elimination campaign, possible drug resistance (though highly unlikely), and the potential for a rebound of malaria illness (if immunity is lost and malaria is reintroduced later). Other ethical issues are related to balancing individual and public health interests, and potentially limiting individual autonomy by making MDA compulsory.

Pearson RD, Amato R, Auburn S, Miotto O, Almagro-Garcia J, Amaratunga C, Suon S, Mao S et al. 2016. Genomic analysis of local variation and recent evolution in Plasmodium vivax. Nat Genet, 48 (8), pp. 959-964. | Show Abstract | Read more

The widespread distribution and relapsing nature of Plasmodium vivax infection present major challenges for the elimination of malaria. To characterize the genetic diversity of this parasite in individual infections and across the population, we performed deep genome sequencing of >200 clinical samples collected across the Asia-Pacific region and analyzed data on >300,000 SNPs and nine regions of the genome with large copy number variations. Individual infections showed complex patterns of genetic structure, with variation not only in the number of dominant clones but also in their level of relatedness and inbreeding. At the population level, we observed strong signals of recent evolutionary selection both in known drug resistance genes and at new loci, and these varied markedly between geographical locations. These findings demonstrate a dynamic landscape of local evolutionary adaptation in the parasite population and provide a foundation for genomic surveillance to guide effective strategies for control and elimination of P. vivax.

Phyo AP, Ashley EA, Anderson TJ, Bozdech Z, Carrara VI, Sriprawat K, Nair S, White MM et al. 2016. Declining Efficacy of Artemisinin Combination Therapy Against P. Falciparum Malaria on the Thai-Myanmar Border (2003-2013): The Role of Parasite Genetic Factors. Clin Infect Dis, pp. ciw388-ciw388. | Show Abstract | Read more

BACKGROUND:  Deployment of mefloquine-artesunate (MAS3) on the Thailand-Myanmar border has led to a sustained reduction in falciparum malaria, although antimalarial efficacy has declined substantially in recent years. The role of Plasmodium falciparum K13 mutations (a marker of artemisinin resistance) in reducing treatment efficacy remains controversial. METHODS:  Between 2003 and 2013, we studied the efficacy of MAS3 in 1005 patients with uncomplicated P. falciparum malaria in relation to molecular markers of resistance. RESULTS:  Polymerase chain reaction (PCR)-adjusted cure rates declined from 100% in 2003 to 81.1% in 2013 as the proportions of isolates with multiple Pfmdr1 copies doubled from 32.4% to 64.7% and those with K13 mutations increased from 6.7% to 83.4%. K13 mutations conferring moderate artemisinin resistance (notably E252Q) predominated initially but were later overtaken by propeller mutations associated with slower parasite clearance (notably C580Y). Those infected with both multiple Pfmdr1 copy number and a K13 propeller mutation were 14 times more likely to fail treatment. The PCR-adjusted cure rate was 57.8% (95% confidence interval [CI], 45.4, 68.3) compared with 97.8% (95% CI, 93.3, 99.3) in patients with K13 wild type and Pfmdr1 single copy. K13 propeller mutation alone was a strong risk factor for recrudescence (P = .009). The combined population attributable fraction of recrudescence associated with K13 mutation and Pfmdr1 amplification was 82%. CONCLUSIONS:  The increasing prevalence of K13 mutations was the decisive factor for the recent and rapid decline in efficacy of artemisinin-based combination (MAS3) on the Thailand-Myanmar border.

Abdulla S, Achan J, Yeka A, D'Alessandro U, Adam I, Alemayehu BH, Allan R, Temu EA et al. 2016. Gametocyte carriage in uncomplicated Plasmodium falciparum malaria following treatment with artemisinin combination therapy: a systematic review and meta-analysis of individual patient data BMC Medicine, 14 (1), | Show Abstract | Read more

© 2016 WWARN Gametocyte Study Group.Background: Gametocytes are responsible for transmission of malaria from human to mosquito. Artemisinin combination therapy (ACT) reduces post-treatment gametocyte carriage, dependent upon host, parasite and pharmacodynamic factors. The gametocytocidal properties of antimalarial drugs are important for malaria elimination efforts. An individual patient clinical data meta-analysis was undertaken to identify the determinants of gametocyte carriage and the comparative effects of four ACTs: artemether-lumefantrine (AL), artesunate/amodiaquine (AS-AQ), artesunate/mefloquine (AS-MQ), and dihydroartemisinin-piperaquine (DP). Methods: Factors associated with gametocytaemia prior to, and following, ACT treatment were identified in multivariable logistic or Cox regression analysis with random effects. All relevant studies were identified through a systematic review of PubMed. Risk of bias was evaluated based on study design, methodology, and missing data. Results: The systematic review identified 169 published and 9 unpublished studies, 126 of which were shared with the WorldWide Antimalarial Resistance Network (WWARN) and 121 trials including 48,840 patients were included in the analysis. Prevalence of gametocytaemia by microscopy at enrolment was 12.1 % (5887/48,589), and increased with decreasing age, decreasing asexual parasite density and decreasing haemoglobin concentration, and was higher in patients without fever at presentation. After ACT treatment, gametocytaemia appeared in 1.9 % (95 % CI, 1.7-2.1) of patients. The appearance of gametocytaemia was lowest after AS-MQ and AL and significantly higher after DP (adjusted hazard ratio (AHR), 2.03; 95 % CI, 1.24-3.12; P = 0.005 compared to AL) and AS-AQ fixed dose combination (FDC) (AHR, 4.01; 95 % CI, 2.40-6.72; P < 0.001 compared to AL). Among individuals who had gametocytaemia before treatment, gametocytaemia clearance was significantly faster with AS-MQ (AHR, 1.26; 95 % CI, 1.00-1.60; P = 0.054) and slower with DP (AHR, 0.74; 95 % CI, 0.63-0.88; P = 0.001) compared to AL. Both recrudescent (adjusted odds ratio (AOR), 9.05; 95 % CI, 3.74-21.90; P < 0.001) and new (AOR, 3.03; 95 % CI, 1.66-5.54; P < 0.001) infections with asexual-stage parasites were strongly associated with development of gametocytaemia after day 7. Conclusions: AS-MQ and AL are more effective than DP and AS-AQ FDC in preventing gametocytaemia shortly after treatment, suggesting that the non-artemisinin partner drug or the timing of artemisinin dosing are important determinants of post-treatment gametocyte dynamics.

Nakeesathit S, Saralamba N, Pukrittayakamee S, Dondorp A, Nosten F, White NJ, Imwong M. 2016. Limited Polymorphism of the Kelch Propeller Domain in Plasmodium malariae and P. ovale Isolates from Thailand. Antimicrob Agents Chemother, 60 (7), pp. 4055-4062. | Show Abstract | Read more

Artemisinin resistance in Plasmodium falciparum, the agent of severe malaria, is currently a major obstacle to malaria control in Southeast Asia. A gene named "kelch13" has been associated with artemisinin resistance in P. falciparum The orthologue of the kelch gene in P. vivax was identified and a small number of mutations were found in previous studies. The kelch orthologues in the other two human malaria parasites, P. malariae and P. ovale, have not yet been studied. Therefore, in this study, the orthologous kelch genes of P. malariae, P. ovale wallikeri, and P. ovale curtisi were isolated and analyzed for the first time. The homologies of the kelch genes of P. malariae and P. ovale were 84.8% and 82.7%, respectively, compared to the gene in P. falciparum kelch polymorphisms were studied in 13 P. malariae and 5 P. ovale isolates from Thailand. There were 2 nonsynonymous mutations found in these samples. One mutation was P533L, which was found in 1 of 13 P. malariae isolates, and the other was K137R, found in 1 isolate of P. ovale wallikeri (n = 4). This result needs to be considered in the context of widespread artemisinin used within the region; their functional consequences for artemisinin sensitivity in P. malariae and P. ovale will need to be elucidated.

Thuan PD, Ca NTN, Toi PV, Nhien NTT, Thanh NV, Anh ND, Phu NH, Thai CQ et al. 2016. A Randomized Comparison of Chloroquine versus Dihydroartemisinin-Piperaquine for the Treatment of Plasmodium vivax Infection in Vietnam AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 94 (4), pp. 879-885. | Read more

Tun KM, Jeeyapant A, Imwong M, Thein M, Aung SS, Hlaing TM, Yuentrakul P, Promnarate C et al. 2016. Parasite clearance rates in Upper Myanmar indicate a distinctive artemisinin resistance phenotype: a therapeutic efficacy study. Malar J, 15 (1), pp. 185. | Show Abstract | Read more

BACKGROUND: Artemisinin resistance in Plasmodium falciparum extends across Southeast Asia where it is associated with worsening partner drug resistance and a decline in the efficacy of frontline artemisinin-based combination therapy. Dihydroartemisinin-piperaquine (DP) is an essential component of preventive and curative treatment in the region, but its therapeutic efficacy has fallen in Cambodia. METHODS: A prospective clinical and parasitological evaluation of DP was conducted at two sites in Upper Myanmar between August 2013 and December 2014, enrolling 116 patients with acute uncomplicated falciparum malaria. Patients received DP orally for 3 days together with primaquine 0.25 mg/kg on admission. Parasite clearance half-lives based on 6 hourly blood smears, and day 42 therapeutic responses were assessed as well as parasite K13 genotypes. RESULTS: Median parasite clearance half-life was prolonged, and clearance half-life was greater than 5 h in 21% of patients. Delayed parasite clearance was significantly associated with mutations in the propeller region of the parasite k13 gene. The k13 F446I mutation was found in 25.4% of infections and was associated with a median clearance half-life of 4.7 h compared with 2.7 h for infections without k13 mutations (p < 0.001). There were no failures after 42 days of follow-up, although 18% of patients had persistent parasitaemia on day 3. CONCLUSION: The dominant k13 mutation observed in Upper Myanmar, F446I, appears to be associated with an intermediate rate of parasite clearance compared to other common mutations described elsewhere in the Greater Mekong Subregion. Discerning this phenotype requires relatively detailed clearance measurements, highlighting the importance of methodology in assessing artemisinin resistance.

Boni MF, White NJ, Baird JK. 2016. The Community As the Patient in Malaria-Endemic Areas: Preempting Drug Resistance with Multiple First-Line Therapies. PLoS Med, 13 (3), pp. e1001984. | Show Abstract | Read more

Maciej F. Boni and colleagues propose deploying multiple first-line combination therapies against malaria within a community to delay drug-resistance evolution.

Thuan PD, Ca NT, Van Toi P, Nhien NT, Thanh NV, Anh ND, Phu NH, Thai CQ et al. 2016. A Randomized Comparison of Chloroquine Versus Dihydroartemisinin-Piperaquine for the Treatment of Plasmodium vivax Infection in Vietnam. Am J Trop Med Hyg, 94 (4), pp. 879-885. | Show Abstract | Read more

A total of 128 Vietnamese patients with symptomatic Plasmodium vivax mono-infections were enrolled in a prospective, open-label, randomized trial to receive either chloroquine or dihydroartemisinin-piperaquine (DHA-PPQ). The proportions of patients with adequate clinical and parasitological responses were 47% in the chloroquine arm (31 of 65 patients) and 66% in the DHA-PPQ arm (42 of 63 patients) in the Kaplan-Meier intention-to-treat analysis (absolute difference 19%, 95% confidence interval = 0-37%), thus establishing non-inferiority of DHA-PPQ. Fever clearance time (median 24 versus 12 hours,P= 0.02), parasite clearance time (median 36 versus 18 hours,P< 0.001), and parasite clearance half-life (mean 3.98 versus 1.80 hours,P< 0.001) were all significantly shorter in the DHA-PPQ arm. All cases of recurrent parasitemia in the chloroquine arm occurred from day 33 onward, with corresponding whole blood chloroquine concentration lower than 100 ng/mL in all patients. Chloroquine thus remains efficacious for the treatment of P. vivax malaria in southern Vietnam, but DHA-PPQ provides more rapid symptomatic and parasitological recovery.

Moore KA, Simpson JA, Paw MK, Pimanpanarak M, Wiladphaingern J, Rijken MJ, Jittamala P, White NJ, Fowkes FJ, Nosten F, McGready R. 2016. Safety of artemisinins in first trimester of prospectively followed pregnancies: an observational study. Lancet Infect Dis, 16 (5), pp. 576-583. | Show Abstract | Read more

BACKGROUND: Artemisinins, the most effective antimalarials available, are not recommended for falciparum malaria during the first trimester of pregnancy because of safety concerns. Therefore, quinine is used despite its poor effectiveness. Assessing artemisinin safety requires weighing the risks of malaria and its treatment. We aimed to assess the effect of first-trimester malaria and artemisinin treatment on miscarriage and major congenital malformations. METHODS: In this observational study, we assessed data from antenatal clinics on the Thai-Myanmar border between Jan 1, 1994, and Dec 31, 2013. We included women who presented to antenatal clinics during their first trimester with a viable fetus. Women were screened for malaria, and data on malaria, antimalarial treatment, and birth outcomes were collected. The relationship between artemisinin treatments (artesunate, dihydroartemisinin, or artemether) and miscarriage or malformation was assessed using Cox regression with left-truncation and time-varying exposures. FINDINGS: Of 55 636 pregnancies registered between 1994 and 2013, 25 485 pregnancies were analysed for first-trimester malaria and miscarriage, in which 2558 (10%) had first-trimester malaria. The hazard of miscarriage increased 1·61-fold after an initial first-trimester falciparum episode (95% CI 1·32-1·97; p<0·0001), 3·24-fold following falciparum recurrence (2·24-4·68; p<0·0001), and 2·44-fold (1·01-5·88; p=0·0473) following recurrent symptomatic vivax malaria. No difference was noted in miscarriage in first-line falciparum treatments with artemisinin (n=183) versus quinine (n=842; HR 0·78 [95% CI 0·45-1·34]; p=0·3645) or in risk of major congenital malformations (two [2%] of 109 [95% CI 0·22-6·47] versus eight (1%) of 641 [0·54-2·44], respectively). INTERPRETATION: First-trimester falciparum and vivax malaria both increase the risk of miscarriage. We noted no evidence of an increased risk of miscarriage or of major congenital malformations associated with first-line treatment with an artemisinin derivative compared with quinine. In view of the low efficacy of quinine and wide availability of highly effective artemisinin-based combination therapies, it is time to reconsider first-trimester antimalarial treatment recommendations. FUNDING: The Wellcome Trust and The Bill & Melinda Gates Foundation.

Chairat K, Jittamala P, Hanpithakpong W, Day NP, White NJ, Pukrittayakamee S, Tarning J. 2016. Population pharmacokinetics of oseltamivir and oseltamivir carboxylate in obese and non-obese volunteers. Br J Clin Pharmacol, 81 (6), pp. 1103-1112. | Show Abstract | Read more

AIMS: The aims of the present study were to compare the pharmacokinetics of oseltamivir and its active antiviral metabolite oseltamivir carboxylate in obese and non-obese individuals and to determine the effect of obesity on the pharmacokinetic properties of oseltamivir and oseltamivir carboxylate. METHODS: The population pharmacokinetic properties of oseltamivir and oseltamivir carboxylate were evaluated in 12 obese [body mass index (BMI) ≥30 kg m(-2) ) and 12 non-obese (BMI <30 kg m(-2) ) Thai adult volunteers receiving a standard dose of 75 mg and a double dose of 150 mg in a randomized sequence. Concentration-time data were collected and analysed using nonlinear mixed-effects modelling. RESULTS: The pharmacokinetics of oseltamivir and oseltamivir carboxylate were described simultaneously by first-order absorption, with a one-compartment disposition model for oseltamivir, followed by a metabolism compartment and a one-compartment disposition model for oseltamivir carboxylate. Creatinine clearance was a significant predictor of oseltamivir carboxylate clearance {3.84% increase for each 10 ml min(-1) increase in creatinine clearance [95% confidence interval (CI) 0.178%, 8.02%]}. Obese individuals had an approximately 25% (95% CI 24%, 28%) higher oseltamivir clearance, 20% higher oseltamivir volume of distribution (95% CI 19%, 23%) and 10% higher oseltamivir carboxylate clearance (95% CI 9%, 11%) compared with non-obese individuals. However, these altered pharmacokinetic properties were small and did not change the overall exposure to oseltamivir carboxylate. CONCLUSIONS: The results confirmed that a dose adjustment for oseltamivir in obese individuals is not necessary on the basis of its pharmacokinetics.

Peto TJ, Tripura R, Lee SJ, Althaus T, Dunachie S, Nguon C, Dhorda M, Promnarate C et al. 2016. Association between Subclinical Malaria Infection and Inflammatory Host Response in a Pre-Elimination Setting. PLoS One, 11 (7), pp. e0158656. | Show Abstract | Read more

BACKGROUND: Subclinical infections in endemic areas of Southeast Asia sustain malaria transmission. These asymptomatic infections might sustain immunity against clinical malaria and have been considered benign for the host, but if they are associated with chronic low-grade inflammation this could be harmful. We conducted a case-control study to explore the association between subclinical malaria and C-reactive protein (CRP), an established biomarker of inflammation. METHODS: Blood samples from asymptomatic villagers in Pailin, Western Cambodia were tested for malaria by high-volume ultra-sensitive polymerase chain reaction (uPCR) to determine the Plasmodium species. Plasma CRP concentration was measured in 328 individuals with parasitaemia (cases) and compared with: i) the same individual's value at the first time point when they had no detectable parasites (n = 282); and ii) age- sex- and village-matched controls (n = 328) free of Plasmodium infection. Plasma CRP concentrations were compared against thresholds of 3mg/L and 10mg/L. Subgroup analysis was carried out for cases with P vivax and P falciparum mono-infections. RESULTS: Median plasma CRP level for all samples was 0.59mg/L (interquartile range: 0.24-1.64mg/L). CRP concentrations were higher in parasitaemic individuals compared with same-person-controls (p = 0.050); and matched-controls (p = 0.025). 4.9% of samples had CRP concentrations above 10mg/L and 14.6% were above 3mg/L. Cases were more likely to have plasma CRP concentrations above these thresholds than age/sex matched controls, odds ratio 3.5 (95%CI 1.5-9.8) and 1.8 (95%CI 1.1-2.9), respectively. Amongst cases, parasite density and CRP were positively correlated (p<0.001), an association that remained significant when controlling for age and fever. Individuals with P.vivax mono-infections had the highest plasma CRP concentrations with the greatest association with parasitaemia. DISCUSSION: In this setting persistent malaria infections in asymptomatic individuals were associated with moderately elevated plasma CRP concentrations; chiefly evident in cases with P.vivax mono-infections. As well as interrupting malaria transmission within the community, treatment of asymptomatic malaria infections, in particular radical cure of vivax malaria, may benefit the health of infected individuals.

Phommasone K, Adhikari B, Henriques G, Pongvongsa T, Phongmany P, von Seidlein L, White NJ, Day NP et al. 2016. Asymptomatic Plasmodium infections in 18 villages of southern Savannakhet Province, Lao PDR (Laos). Malar J, 15 (1), pp. 296. | Show Abstract | Read more

BACKGROUND: A large fraction of Plasmodium infections do not cause clinical signs and symptoms of disease and persist at densities in blood that are not detectable by microscopy or rapid diagnostic tests. These infections may be critical as a transmission reservoir in areas of low malaria endemicity. Understanding the epidemiology of these infections would be helpful for malaria elimination. METHODS: A cross-sectional survey was conducted in Thapangthong and Nong Districts of Savannakhet Province, Lao PDR, to determine the prevalence of parasitaemia. A total of 888 blood samples were collected from afebrile volunteers aged ≥15 years in 18 villages during March and July 2015. Plasmodium infections were diagnosed by rapid diagnostic tests (RDT) and high volume, ultra-sensitive quantitative polymerase chain reaction (uPCR). RESULTS: uPCR detected Plasmodium infections in 175 of 888 samples (20 %). The species distribution was Plasmodium falciparum 3.6 % (32/888), Plasmodium vivax 11.1 % (99/888), mixed infections with P. falciparum and P. vivax 1.6 % (14/888) and Plasmodium of undetermined species 3.4 % (30/888). RDT identified only 2 % (18/888) positive cases. Using uPCR as reference, the sensitivity and specificity of RDTs were 28 and 100 %, respectively, in detecting P. falciparum infections, and 3 and 99 % in detecting asymptomatic P. vivax infections. The K13 kelch propeller domain C580Y mutation, associated with reduced susceptibility to artemisinin derivatives, was found in 75 % (12/18) of P. falciparum isolates from Thapangthong and in 7 % (2/28) from Nong (p < 0.001). In a multivariate analysis, males were more likely to have P. vivax infections [adjusted odds ratio (aOR) 4.76 (95 % CI 2.84-8.00)] while older villagers were at lower risk for parasitaemia [aOR for increasing age 0.98 (95 % CI 0.96-0.99)]. CONCLUSION: There is a high prevalence of asymptomatic Plasmodium infections in southern Savannakhet. Artemisinin-resistant P. falciparum strains form an increasing proportion of the parasite population in Thapangthong District and are already present in the more remote Nong District. This worrying trend has wider implications for Laos and could reverse the gains achieved by the successful control of malaria in Laos and the Greater Mekong Sub-region (GMS). Rapid elimination of P. falciparum has to be a top priority in Laos as well as in the wider GMS.

Peto TJ, Kloprogge SE, Tripura R, Nguon C, Sanann N, Yok S, Heng C, Promnarate C et al. 2016. History of malaria treatment as a predictor of subsequent subclinical parasitaemia: a cross-sectional survey and malaria case records from three villages in Pailin, western Cambodia. Malar J, 15 (1), pp. 240. | Show Abstract | Read more

BACKGROUND: Treatment of the sub-clinical reservoir of malaria, which may maintain transmission, could be an important component of elimination strategies. The reliable detection of asymptomatic infections with low levels of parasitaemia requires high-volume quantitative polymerase chain reaction (uPCR), which is impractical to conduct on a large scale. It is unknown to what extent sub-clinical parasitaemias originate from recent or older clinical episodes. This study explored the association between clinical history of malaria and subsequent sub-clinical parasitaemia. METHODS: In June 2013 a cross-sectional survey was conducted in three villages in Pailin, western Cambodia. Demographic and epidemiological data and blood samples were collected. Blood was tested for malaria by high-volume qPCR. Positive samples were analysed by nested PCR to determine the Plasmodium species. To identify previous episodes of malaria, case records were collected from village malaria workers and local health facilities and linked to study participants. RESULTS: Among 1343 participants, 40/122 (32.8 %) with a history of clinical malaria were parasitaemic during the cross-sectional survey, compared to 172/1221 (14.1 %) without this history (p < 0.001). Among the 212 parasitaemic participants in the survey, 40 (18.9 %) had a history of clinical malaria, compared to 87 out of 1131 (7.7 %) parasite-negative participants; p < 0.001, adjusted OR 3.3 (95 % CI; 2.1-5.1). A history of Plasmodium vivax was associated with sub-clinical P. vivax parasitaemia in the survey (p < 0.001), but this association was not seen with Plasmodium falciparum (p = 0.253); only three participants had both P. falciparum parasites in the survey and a clinical history of P. falciparum. CONCLUSIONS: A clinical episode of vivax malaria was associated with subsequent sub-clinical parasitaemia. Treatment of P. vivax with artemisinin-based combination therapy without primaquine often resulted in recurrent episodes. Targeting individuals with a history of clinical malaria will be insufficient to eliminate the sub-clinical reservoir as they constitute a minority of parasitaemias.

Tripura R, Peto TJ, Chalk J, Lee SJ, Sirithiranont P, Nguon C, Dhorda M, von Seidlein L et al. 2016. Persistent Plasmodium falciparum and Plasmodium vivax infections in a western Cambodian population: implications for prevention, treatment and elimination strategies. Malar J, 15 (1), pp. 181. | Show Abstract | Read more

BACKGROUND: Subclinical Plasmodium parasitaemia is an important reservoir for the transmission and persistence of malaria, particularly in low transmission areas. METHODS: Using ultrasensitive quantitative PCR (uPCR) for the detection of parasitaemia, the entire population of three Cambodian villages in Pailin province were followed for 1 year at three-monthly intervals. A cohort of adult participants found initially to have asymptomatic malaria parasitaemia was followed monthly over the same period. RESULTS: The initial cross sectional survey in June 2013 (M0) of 1447 asymptomatic residents found that 32 (2.2%) had Plasmodium falciparum, 48 (3.3%) had P. vivax, 4 (0.3%) had mixed infections and in 142/1447 (9.8%) malaria was detected but there was insufficient DNA to identify the species (Plasmodium. species). Polymorphisms in the 'K13-propeller' associated with reduced susceptibility to artemisinin derivatives (C580Y) were found in 17/32 (51%) P. falciparum strains. Monthly follow-up without treatment of 24 adult participants with asymptomatic mono or mixed P. falciparum infections found that 3/24 (13%) remained parasitaemic for 2-4 months, whereas the remaining 21/24 (87%) participants had cleared their parasitaemia after 1 month. In contrast, 12/34 (35%) adult participants with P. vivax mono-infection at M0 had malaria parasites (P. vivax or P. sp.) during four or more of the following 11 monthly surveys. CONCLUSIONS: This longitudinal survey in a low transmission setting shows limited duration of P. falciparum carriage, but prolonged carriage of P. vivax infections. Radical treatment of P. vivax infections by 8-aminoquinoline regimens may be required to eliminate all malaria from Cambodia. Trial registration ClinicalTrials.gov NCT01872702.

Wattanakul T, Teerapong P, Plewes K, Newton PN, Chierakul W, Silamut K, Chotivanich K, Ruengweerayut R, White NJ, Dondorp AM, Tarning J. 2016. Pharmacokinetic properties of intramuscular versus oral syrup paracetamol in Plasmodium falciparum malaria. Malar J, 15 (1), pp. 244. | Show Abstract | Read more

BACKGROUND: Fever is an inherent symptom of malaria in both adults and children. Paracetamol (acetaminophen) is the recommended antipyretic as it is inexpensive, widely available and has a good safety profile, but patients may not be able to take the oral drug reliably. A comparison between the pharmacokinetics of oral syrup and intramuscular paracetamol given to patients with acute falciparum malaria and high body temperature was performed. METHODS: A randomized, open-label, two-treatment, crossover, pharmacokinetic study of paracetamol dosed orally and intramuscularly was conducted. Twenty-one adult patients with uncomplicated falciparum malaria were randomized to receive a single 600 mg dose of paracetamol either as syrup or intramuscular injection on day 0 followed by a single dose administered by the alternative route on day 1. Paracetamol plasma concentrations were quantified frequently and modelled simultaneously using nonlinear mixed-effects modelling. The final population pharmacokinetic model was used for dose optimization simulations. Relationships between paracetamol concentrations with temperature and parasite half-life were investigated using linear and non-linear regression analyses. RESULTS: The population pharmacokinetic properties of paracetamol were best described by a two-compartment disposition model, with zero-order and first-order absorption for intramuscular and oral syrup administration, respectively. The relative bioavailability of oral syrup was 84.4 % (95 % CI 68.2-95.1 %) compared to intramuscular administration. Dosing simulations showed that 1000 mg of intramuscular or oral syrup administered six-hourly reached therapeutic steady state concentrations for antipyresis, but more favourable concentration-time profiles were achieved with a loading dose of 1500 mg, followed by a 1000 mg maintenance dose. This ensured that maximum therapeutic concentrations were reached rapidly during the first 6 h. No significant relationships between paracetamol concentrations and temperature or parasite half-life were found. CONCLUSIONS: Paracetamol plasma concentrations after oral syrup and intramuscular administration in patients with acute falciparum malaria were described successfully by a two-compartment disposition model. Relative oral bioavailability compared to intramuscular dosing was estimated as 84.4 % (95 % CI 68.2-95.1 %). Dosing simulations showed that a loading dose followed by six-hourly dosing intervals reduced the time delay to reach therapeutic drug levels after both routes of administration. The safety and efficacy of loading dose paracetamol antipyretic regimens now needs to be established in larger studies.

MalariaGEN Plasmodium falciparum Community Project. 2016. Genomic epidemiology of artemisinin resistant malaria. Elife, 5 (MARCH2016), | Show Abstract | Read more

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions.

Awab GR, Imwong M, Pukrittayakamee S, Alim F, Hanpithakpong W, Tarning J, Dondorp AM, Day NP, White NJ, Woodrow CJ. 2016. Clinical trials of artesunate plus sulfadoxine-pyrimethamine for Plasmodium falciparum malaria in Afghanistan: maintained efficacy a decade after introduction. Malar J, 15 (1), pp. 121. | Show Abstract | Read more

BACKGROUND: Combination therapy with artesunate plus sulfadoxine-pyrimethamine (SP) was adopted as recommended treatment for Plasmodium falciparum infection in Afghanistan in 2003. METHODS: A series of prospective clinical studies examining the efficacy of artesunate plus sulfadoxine-pyrimethamine (AS + SP) against P. falciparum were undertaken in sentinel sites in Afghanistan from 2007 to 2014, accompanied by relevant molecular studies. The first study was a randomized trial of AS + SP versus dihydroartemisinin-piperaquine, while two subsequent studies were standard therapeutic efficacy studies of AS + SP. RESULTS: Three hundred and three patients were enrolled across four provinces in the north and east of the country. Curative efficacy was high in all the trials, with an adequate clinical and parasitological response (ACPR) of more than 95 % in all groups and trial stages. Genotyping for drug-resistance alleles at dhfr indicated fixation of the S108 N mutation and a prevalence of the C59R mutation of approximately 95 % across all sites. Other mutations in dhfr and dhps remained rare or absent entirely, although five isolates from the first trial carried the dhps triple mutant SGEGA haplotype. In the last study undertaken in 2012-2014 the K13 artemisinin resistance marker was examined; only two of 60 successfully sequenced samples carried a K13-propeller mutation. CONCLUSIONS: These data confirm maintained efficacy 10 years after introduction of artesunate plus SP as combination treatment of P. falciparum in Afghanistan. The molecular data indicate that despite a substantial fall in incidence, resistance has not developed to artemisinins, or intensified to the ACT partner drug components. Trial Registration http://www.clinicaltrials.gov/ct NCT00682578, NCT01115439 and NCT01707199.

Grigg MJ, Barber BE, Marfurt J, Imwong M, William T, Bird E, Piera KA, Aziz A et al. 2016. Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah, Malaysia. PLoS One, 11 (3), pp. e0149519. | Show Abstract | Read more

BACKGROUND: Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission. METHODS: The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket. RESULTS: Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates. CONCLUSION: Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.

Ponsuwanna P, Kochakarn T, Bunditvorapoom D, Kümpornsin K, Otto TD, Ridenour C, Chotivanich K, Wilairat P, White NJ, Miotto O, Chookajorn T. 2016. Comparative genome-wide analysis and evolutionary history of haemoglobin-processing and haem detoxification enzymes in malarial parasites. Malar J, 15 (1), pp. 51. | Show Abstract | Read more

BACKGROUND: Malaria parasites have evolved a series of intricate mechanisms to survive and propagate within host red blood cells. Intra-erythrocytic parasitism requires these organisms to digest haemoglobin and detoxify iron-bound haem. These tasks are executed by haemoglobin-specific proteases and haem biocrystallization factors that are components of a large multi-subunit complex. Since haemoglobin processing machineries are functionally and genetically linked to the modes of action and resistance mechanisms of several anti-malarial drugs, an understanding of their evolutionary history is important for drug development and drug resistance prevention. METHODS: Maximum likelihood trees of genetic repertoires encoding haemoglobin processing machineries within Plasmodium species, and with the representatives of Apicomplexan species with various host tropisms, were created. Genetic variants were mapped onto existing three-dimensional structures. Genome-wide single nucleotide polymorphism data were used to analyse the selective pressure and the effect of these mutations at the structural level. RESULTS: Recent expansions in the falcipain and plasmepsin repertoires are unique to human malaria parasites especially in the Plasmodium falciparum and P. reichenowi lineage. Expansion of haemoglobin-specific plasmepsins occurred after the separation event of Plasmodium species, but the other members of the plasmepsin family were evolutionarily conserved with one copy for each sub-group in every Apicomplexan species. Haemoglobin-specific falcipains are separated from invasion-related falcipain, and their expansions within one specific locus arose independently in both P. falciparum and P. vivax lineages. Gene conversion between P. falciparum falcipain 2A and 2B was observed in artemisinin-resistant strains. Comparison between the numbers of non-synonymous and synonymous mutations suggests a strong selective pressure at falcipain and plasmepsin genes. The locations of amino acid changes from non-synonymous mutations mapped onto protein structures revealed clusters of amino acid residues in close proximity or near the active sites of proteases. CONCLUSION: A high degree of polymorphism at the haemoglobin processing genes implicates an imposition of selective pressure. The identification in recent years of functional redundancy of haemoglobin-specific proteases makes them less appealing as potential drug targets, but their expansions, especially in the human malaria parasite lineages, unequivocally point toward their functional significance during the independent and repetitive adaptation events in malaria parasite evolutionary history.

Zhu L, Mok S, Imwong M, Jaidee A, Russell B, Nosten F, Day NP, White NJ, Preiser PR, Bozdech Z. 2016. New insights into the Plasmodium vivax transcriptome using RNA-Seq. Sci Rep, 6 pp. 20498. | Show Abstract | Read more

Historically seen as a benign disease, it is now becoming clear that Plasmodium vivax can cause significant morbidity. Effective control strategies targeting P. vivax malaria is hindered by our limited understanding of vivax biology. Here we established the P. vivax transcriptome of the Intraerythrocytic Developmental Cycle (IDC) of two clinical isolates in high resolution by Illumina HiSeq platform. The detailed map of transcriptome generates new insights into regulatory mechanisms of individual genes and reveals their intimate relationship with specific biological functions. A transcriptional hotspot of vir genes observed on chromosome 2 suggests a potential active site modulating immune evasion of the Plasmodium parasite across patients. Compared to other eukaryotes, P. vivax genes tend to have unusually long 5' untranslated regions and also present multiple transcription start sites. In contrast, alternative splicing is rare in P. vivax but its association with the late schizont stage suggests some of its significance for gene function. The newly identified transcripts, including up to 179 vir like genes and 3018 noncoding RNAs suggest an important role of these gene/transcript classes in strain specific transcriptional regulation.

Bicanic T, Bottomley C, Loyse A, Brouwer AE, Muzoora C, Taseera K, Jackson A, Phulusa J et al. 2015. Toxicity of Amphotericin B Deoxycholate-Based Induction Therapy in Patients with HIV-Associated Cryptococcal Meningitis. Antimicrob Agents Chemother, 59 (12), pp. 7224-7231. | Show Abstract | Read more

Amphotericin B deoxycholate (AmBd) is the recommended induction treatment for HIV-associated cryptococcal meningitis (CM). Its use is hampered by toxicities that include electrolyte abnormalities, nephrotoxicity, and anemia. Protocols to minimize toxicity are applied inconsistently. In a clinical trial cohort of AmBd-based CM induction treatment, a standardized protocol of preemptive hydration and electrolyte supplementation was applied. Changes in blood counts, electrolyte levels, and creatinine levels over 14 days were analyzed in relation to the AmBd dose, treatment duration (short course of 5 to 7 days or standard course of 14 days), addition of flucytosine (5FC), and outcome. In the 368 patients studied, the hemoglobin levels dropped by a mean of 1.5 g/dl (95% confidence interval [CI], 1.0 to 1.9 g/dl) following 7 days of AmBd and by a mean of 2.3 g/dl (95% CI, 1.1 to 3.6 g/dl) after 14 days. Serum creatinine levels increased by 37 μmol/liter (95% CI, 30 to 45 μmol/liter) by day 7 and by 49 μmol/liter (95% CI, 35 to 64μmol/liter) by day 14 of AmBd treatment. Overall, 33% of patients developed grade III/IV anemia, 5.6% developed grade III hypokalemia, 9.5% had creatinine levels that exceeded 220 μmol, and 6% discontinued AmBd prematurely. The addition of 5FC was associated with a slight increase in anemia but not neutropenia. Laboratory abnormalities stabilized or reversed during the second week in patients on short-course induction. Grade III/IV anemia (adjusted odds ratio [aOR], 2.2; 95% CI, 1.1 to 4.3; P = 0.028) and nephrotoxicity (aOR, 4.5; 95% CI, 1.8 to 11; P = 0.001) were risk factors for 10-week mortality. In summary, routine intravenous saline hydration and preemptive electrolyte replacement during AmBd-based induction regimens for HIV-associated CM minimized the incidence of hypokalemia and nephrotoxicity. Anemia remained a concerning adverse effect. The addition of flucytosine was not associated with increased neutropenia. Shorter AmBd courses were less toxic, with rapid reversibility.

Reamtong O, Srimuang K, Saralamba N, Sangvanich P, Day NP, White NJ, Imwong M. 2015. Protein profiling of mefloquine resistant Plasmodium falciparum using mass spectrometry-based proteomics. Int J Mass Spectrom, 391 pp. 82-92. | Show Abstract | Read more

Malaria is a mosquito borne infectious disease caused by protozoa of genus Plasmodium. There are five species of Plasmodium that are found to infect humans. Plasmodium falciparum can cause severe malaria leading to higher morbidity and mortality of malaria than the other four species. Antimalarial resistance is the major obstacle to control malaria. Mefloquine was used in combination with Artesunate for uncomplicated P. falciparum in South East Asia and it has developed and established mefloquine resistance in this region. Here, gel-enhanced liquid chromatography/tandem mass spectrometry (GeLC-MS/MS)-based proteomics and label-free quantification were used to explore the protein profiles of mefloquine-sensitive and -induced resistant P. falciparum. A Thai P. falciparum isolate (S066) was used as a model in this research. Our data revealed for the first time that 69 proteins exhibited at least 2-fold differences in their expression levels between the two parasite lines. Of these, 36 were up-regulated and 33 were down-regulated in the mefloquine-resistant line compared with the mefloquine-sensitive line. These findings are consistent with those of past studies, where the multidrug resistance protein Pgh1 showed an up-regulation pattern consistent with that expected from its average 3-copy pfmdr1 gene number. Pgh1 and eight other up-regulated proteins (i.e., histo-aspartyl protease protein, exportin 1, eukaryotic translation initiation factor 3 subunit 8, peptidyl-prolyl cis-trans isomerase, serine rich protein homologue, exported protein 1, ATP synthase beta chain and phospholipid scramblase 1) were further validated for their expression levels using reverse transcriptase quantitative real-time PCR. The data support the up-regulation status in the mefloquine-resistant parasite line of all the candidate genes referred to above. Therefore, GeLC-MS/MS-based proteomics combined with label-free quantification is a reliable approach for exploring mefloquine resistance biomarkers in P. falciparum. Identification of these proteins leads to better understanding of mefloquine resistant mechanisms in malaria parasites.

Lubell Y, Blacksell SD, Dunachie S, Tanganuchitcharnchai A, Althaus T, Watthanaworawit W, Paris DH, Mayxay M et al. 2015. Performance of C-reactive protein and procalcitonin to distinguish viral from bacterial and malarial causes of fever in Southeast Asia. BMC Infect Dis, 15 (1), pp. 511. | Show Abstract | Read more

BACKGROUND: Poor targeting of antimicrobial drugs contributes to the millions of deaths each year from malaria, pneumonia, and other tropical infectious diseases. While malaria rapid diagnostic tests have improved use of antimalarial drugs, there are no similar tests to guide the use of antibiotics in undifferentiated fevers. In this study we estimate the diagnostic accuracy of two well established biomarkers of bacterial infection, procalcitonin and C-reactive protein (CRP) in discriminating between common viral and bacterial infections in malaria endemic settings of Southeast Asia. METHODS: Serum procalcitonin and CRP levels were measured in stored serum samples from febrile patients enrolled in three prospective studies conducted in Cambodia, Laos and, Thailand. Of the 1372 patients with a microbiologically confirmed diagnosis, 1105 had a single viral, bacterial or malarial infection. Procalcitonin and CRP levels were compared amongst these aetiological groups and their sensitivity and specificity in distinguishing bacterial infections and bacteraemias from viral infections were estimated using standard thresholds. RESULTS: Serum concentrations of both biomarkers were significantly higher in bacterial infections and malaria than in viral infections. The AUROC for CRP in discriminating between bacterial and viral infections was 0.83 (0.81-0.86) compared with 0.74 (0.71-0.77) for procalcitonin (p < 0.0001). This relative advantage was evident in all sites and when stratifying patients by age and admission status. For CRP at a threshold of 10 mg/L, the sensitivity of detecting bacterial infections was 95% with a specificity of 49%. At a threshold of 20 mg/L sensitivity was 86% with a specificity of 67%. For procalcitonin at a low threshold of 0.1 ng/mL the sensitivity was 90% with a specificity of 39%. At a higher threshold of 0.5 ng/ul sensitivity was 60% with a specificity of 76%. CONCLUSION: In samples from febrile patients with mono-infections from rural settings in Southeast Asia, CRP was a highly sensitive and moderately specific biomarker for discriminating between viral and bacterial infections. Use of a CRP rapid test in peripheral health settings could potentially be a simple and affordable measure to better identify patients in need of antibacterial treatment and part of a global strategy to combat the emergence of antibiotic resistance.

Herdman MT, Sriboonvorakul N, Leopold SJ, Douthwaite S, Mohanty S, Hassan MM, Maude RJ, Kingston HW et al. 2015. Erratum to: the role of previously unmeasured organic acids in the pathogenesis of severe malaria. Crit Care, 19 (1), pp. 382. | Read more

Ishioka H, Ghose A, Charunwatthana P, Maude R, Plewes K, Kingston H, Intharabut B, Woodrow C et al. 2016. Sequestration and Red Cell Deformability as Determinants of Hyperlactatemia in Falciparum Malaria. J Infect Dis, 213 (5), pp. 788-793. | Show Abstract | Read more

BACKGROUND: Hyperlactatemia is a strong predictor of mortality in severe falciparum malaria. Sequestered parasitized erythrocytes and reduced uninfected red blood cell deformability (RCD) compromise microcirculatory flow, leading to anaerobic glycolysis. METHODS: In a cohort of patients with falciparum malaria hospitalized in Chittagong, Bangladesh, bulk RCD was measured using a laser diffraction technique, and parasite biomass was estimated from plasma concentrations of Plasmodium falciparum histidine-rich protein 2 (PfHRP2). A multiple linear regression model was constructed to examine their associations with plasma lactate concentrations. RESULTS: A total of 286 patients with falciparum malaria were studied, of whom 224 had severe malaria, and 70 died. Hyperlactatemia (lactate level, ≥ 4 mmol/L) was present in 111 cases. RCD at shear stresses of 1.7 Pa and 30 Pa was reduced significantly in patients who died, compared with survivors, individuals with uncomplicated malaria, or healthy individuals (P < .05, for all comparisons). Multiple linear regression analysis showed that the plasma PfHRP2 level, parasitemia level, total bilirubin level, and RCD at a shear stress of 1.7 Pa were each independently correlated with plasma lactate concentrations (n = 278; R(2) = 0.35). CONCLUSIONS: Sequestration of parasitized red blood cells and reduced RCD both contribute to decreased microcirculatory flow in severe disease.

Kloprogge F, McGready R, Phyo AP, Rijken MJ, Hanpithakpon W, Than HH, Hlaing N, Zin NT et al. 2015. Opposite malaria and pregnancy effect on oral bioavailability of artesunate - a population pharmacokinetic evaluation. Br J Clin Pharmacol, 80 (4), pp. 642-653. | Show Abstract | Read more

AIM: The aim was to compare the pharmacokinetic properties of artesunate and dihydroartemisinin in the same women: i) pregnant with acute uncomplicated malaria on day 1 and 2, ii) pregnant with convalescent malaria on day 7 and iii) in a healthy state 3 months post-partum on day 1, 2 and 7. METHODS: Non-linear mixed-effects modelling was used to compare plasma concentration-time profiles of artesunate and dihydroartemisinin over 7 days of treatment following oral and intravenous artesunate administration to pregnant women with uncomplicated Plasmodium falciparum malaria during their second or third trimesters of pregnancy. The same women were restudied 3 months after delivery when fully recovered. Non-compartmental results of the same study have been published previously. RESULTS: Twenty pregnant patients on the Thailand-Myanmar border were studied and 15 volunteered to be restudied 3 months post-partum. Malaria and pregnancy had no effect on the pharmacokinetic properties of artesunate or dihydroartemisinin after intravenous artesunate administration. However, malaria and pregnancy had opposite effects on the absorption of orally administered artesunate. Malaria increased the absolute oral bioavailability of artesunate by 87%, presumably by inhibiting first pass effect, whereas pregnancy decreased oral bioavailability by 23%. CONCLUSIONS: The population pharmacokinetic analysis demonstrated opposite effects of malaria and pregnancy on the bioavailability of orally administered artesunate. Lower drug exposures during the second and third trimesters of pregnancy may contribute to lower cure rates and thus the development of drug resistance. Dose optimization studies are required for artesunate containing artemisinin-based combination therapies (ACTs) in later pregnancy.

Imwong M, Nguyen TN, Tripura R, Peto TJ, Lee SJ, Lwin KM, Suangkanarat P, Jeeyapant A et al. 2015. The epidemiology of subclinical malaria infections in South-East Asia: findings from cross-sectional surveys in Thailand-Myanmar border areas, Cambodia, and Vietnam. Malar J, 14 (1), pp. 381. | Show Abstract | Read more

BACKGROUND: The importance of the submicroscopic reservoir of Plasmodium infections for malaria elimination depends on its size, which is generally considered small in low transmission settings. The precise estimation of this reservoir requires more sensitive parasite detection methods. The prevalence of asymptomatic, sub-microscopic malaria was assessed by a sensitive, high blood volume quantitative real-time polymerase chain reaction method in three countries of the Greater Mekong Sub-region. METHODS: Cross-sectional surveys were conducted in three villages in western Cambodia, four villages along the Thailand-Myanmar border and four villages in southwest Vietnam. Malaria parasitaemia was assessed by Plasmodium falciparum/pan malaria rapid diagnostic tests (RDTs), microscopy and a high volume ultra-sensitive real-time polymerase chain reaction (HVUSqPCR: limit of detection 22 parasites/mL). All villagers older than 6 months were invited to participate. RESULTS: A census before the surveys identified 7355 residents in the study villages. Parasite prevalence was 224/5008 (4 %) by RDT, 229/5111 (5 %) by microscopy, and 988/4975 (20 %) when assessed by HVUSqPCR. Of these 164 (3 %) were infected with P. falciparum, 357 (7 %) with Plasmodium vivax, 56 (1 %) with a mixed infection, and 411 (8 %) had parasite densities that were too low for species identification. A history of fever, male sex, and age of 15 years or older were independently associated with parasitaemia in a multivariate regression model stratified by site. CONCLUSION: Light microscopy and RDTs identified only a quarter of all parasitaemic participants. The asymptomatic Plasmodium reservoir is considerable, even in low transmission settings. Novel strategies are needed to eliminate this previously under recognized reservoir of malaria transmission.

WWARN Parasite Clearance Study Group, Abdulla S, Ashley EA, Bassat Q, Bethell D, Björkman A, Borrmann S, D'Alessandro U et al. 2015. Baseline data of parasite clearance in patients with falciparum malaria treated with an artemisinin derivative: an individual patient data meta-analysis. Malar J, 14 (1), pp. 359. | Show Abstract | Read more

BACKGROUND: Artemisinin resistance in Plasmodium falciparum manifests as slow parasite clearance but this measure is also influenced by host immunity, initial parasite biomass and partner drug efficacy. This study collated data from clinical trials of artemisinin derivatives in falciparum malaria with frequent parasite counts to provide reference parasite clearance estimates stratified by location, treatment and time, to examine host factors affecting parasite clearance, and to assess the relationships between parasite clearance and risk of recrudescence during follow-up. METHODS: Data from 24 studies, conducted from 1996 to 2013, with frequent parasite counts were pooled. Parasite clearance half-life (PC1/2) was estimated using the WWARN Parasite Clearance Estimator. Random effects regression models accounting for study and site heterogeneity were used to explore factors affecting PC1/2 and risk of recrudescence within areas with reported delayed parasite clearance (western Cambodia, western Thailand after 2000, southern Vietnam, southern Myanmar) and in all other areas where parasite populations are artemisinin sensitive. RESULTS: PC1/2 was estimated in 6975 patients, 3288 of whom also had treatment outcomes evaluate d during 28-63 days follow-up, with 93 (2.8 %) PCR-confirmed recrudescences. In areas with artemisinin-sensitive parasites, the median PC1/2 following three-day artesunate treatment (4 mg/kg/day) ranged from 1.8 to 3.0 h and the proportion of patients with PC1/2 >5 h from 0 to 10 %. Artesunate doses of 4 mg/kg/day decreased PC1/2 by 8.1 % (95 % CI 3.2-12.6) compared to 2 mg/kg/day, except in populations with delayed parasite clearance. PC1/2 was longer in children and in patients with fever or anaemia at enrolment. Long PC1/2 (HR = 2.91, 95 % CI 1.95-4.34 for twofold increase, p < 0.001) and high initial parasitaemia (HR = 2.23, 95 % CI 1.44-3.45 for tenfold increase, p < 0.001) were associated independently with an increased risk of recrudescence. In western Cambodia, the region with the highest prevalence of artemisinin resistance, there was no evidence for increasing PC1/2 since 2007. CONCLUSIONS: Several factors affect PC1/2. As substantial heterogeneity in parasite clearance exists between locations, early detection of artemisinin resistance requires reference PC1/2 data. Studies with frequent parasite count measurements to characterize PC1/2 should be encouraged. In western Cambodia, where PC1/2 values are longest, there is no evidence for recent emergence of higher levels of artemisinin resistance.

Ashley EA, Aweeka F, Barnes KI, Bassat Q, Borrmann S, Dahal P, Davis TME, Deloron P et al. 2015. Artemether-lumefantrine treatment of uncomplicated Plasmodium falciparum malaria: a systematic review and meta-analysis of day 7 lumefantrine concentrations and therapeutic response using individual patient data BMC Medicine, 13 (1), | Show Abstract | Read more

© 2015 WorldWide Antimalarial Resistance Network (WWARN) Lumefantrine PK/PD Study Group.Background: Achieving adequate antimalarial drug exposure is essential for curing malaria. Day 7 blood or plasma lumefantrine concentrations provide a simple measure of drug exposure that correlates well with artemether-lumefantrine efficacy. However, the 'therapeutic' day 7 lumefantrine concentration threshold needs to be defined better, particularly for important patient and parasite sub-populations. Methods: The WorldWide Antimalarial Resistance Network (WWARN) conducted a large pooled analysis of individual pharmacokinetic-pharmacodynamic data from patients treated with artemether-lumefantrine for uncomplicated Plasmodium falciparum malaria, to define therapeutic day 7 lumefantrine concentrations and identify patient factors that substantially alter these concentrations. A systematic review of PubMed, Embase, Google Scholar, ClinicalTrials.gov and conference proceedings identified all relevant studies. Risk of bias in individual studies was evaluated based on study design, methodology and missing data. Results: Of 31 studies identified through a systematic review, 26 studies were shared with WWARN and 21 studies with 2,787 patients were included. Recrudescence was associated with low day 7 lumefantrine concentrations (HR 1.59 (95 % CI 1.36 to 1.85) per halving of day 7 concentrations) and high baseline parasitemia (HR 1.87 (95 % CI 1.22 to 2.87) per 10-fold increase). Adjusted for mg/kg dose, day 7 concentrations were lowest in very young children (<3 years), among whom underweight-for-age children had 23 % (95 % CI -1 to 41 %) lower concentrations than adequately nourished children of the same age and 53 % (95 % CI 37 to 65 %) lower concentrations than adults. Day 7 lumefantrine concentrations were 44 % (95 % CI 38 to 49 %) lower following unsupervised treatment. The highest risk of recrudescence was observed in areas of emerging artemisinin resistance and very low transmission intensity. For all other populations studied, day 7 concentrations ≥200 ng/ml were associated with >98 % cure rates (if parasitemia <135,000/μL). Conclusions: Current artemether-lumefantrine dosing recommendations achieve day 7 lumefantrine concentrations ≥200 ng/ml and high cure rates in most uncomplicated malaria patients. Three groups are at increased risk of treatment failure: very young children (particularly those underweight-for-age); patients with high parasitemias; and patients in very low transmission intensity areas with emerging parasite resistance. In these groups, adherence and treatment response should be monitored closely. Higher, more frequent, or prolonged dosage regimens should now be evaluated in very young children, particularly if malnourished, and in patients with hyperparasitemia.

Herdman MT, Sriboonvorakul N, Leopold SJ, Douthwaite S, Mohanty S, Hassan MM, Maude RJ, Kingston HW et al. 2015. The role of previously unmeasured organic acids in the pathogenesis of severe malaria. Crit Care, 19 (1), pp. 317. | Show Abstract | Read more

INTRODUCTION: Severe falciparum malaria is commonly complicated by metabolic acidosis. Together with lactic acid (LA), other previously unmeasured acids have been implicated in the pathogenesis of falciparum malaria. METHODS: In this prospective study, we characterised organic acids in adults with severe falciparum malaria in India and Bangladesh. Liquid chromatography-mass spectrometry was used to measure organic acids in plasma and urine. Patients were followed until recovery or death. RESULTS: Patients with severe malaria (n=138), uncomplicated malaria (n=102), sepsis (n=32) and febrile encephalopathy (n=35) were included. Strong ion gap (mean ± SD) was elevated in severe malaria (8.2 mEq/L ± 4.5) and severe sepsis (8.6 mEq/L ± 7.7) compared with uncomplicated malaria (6.0 mEq/L ± 5.1) and encephalopathy (6.6 mEq/L ± 4.7). Compared with uncomplicated malaria, severe malaria was characterised by elevated plasma LA, hydroxyphenyllactic acid (HPLA), α-hydroxybutyric acid and β-hydroxybutyric acid (all P<0.05). In urine, concentrations of methylmalonic, ethylmalonic and α-ketoglutaric acids were also elevated. Multivariate logistic regression showed that plasma HPLA was a strong independent predictor of death (odds ratio [OR] 3.5, 95 % confidence interval [CI] 1.6-7.5, P=0.001), comparable to LA (OR 3.5, 95 % CI 1.5-7.8, P=0.003) (combined area under the receiver operating characteristic curve 0.81). CONCLUSIONS: Newly identified acids, in addition to LA, are elevated in patients with severe malaria and are highly predictive of fatal outcome. Further characterisation of their sources and metabolic pathways is now needed.

Nguyen TD, Olliaro P, Dondorp A, Baird JK, Lam HM, Farrar J, Thwaites GE, White NJ, Boni MF. 2015. Optimal population-level deployment of artemisinin combination therapies TROPICAL MEDICINE & INTERNATIONAL HEALTH, 20 pp. 182-182.

White NJ, Chutasmit K, Jittamala P, Nosten F, Phyo AP, Pukrittayakamee S, Thanh DT, Uthaisin C et al. 2015. Efficacy, safety and pharmacokinetics of KAF156 in adult patients with acute, uncomplicated P. falciparum or vivax malaria: a proof-of-concept, open label study TROPICAL MEDICINE & INTERNATIONAL HEALTH, 20 pp. 18-18.

Kloprogge F, McGready R, Hanpithakpong W, Blessborn D, Day NP, White NJ, Nosten F, Tarning J. 2015. Lumefantrine and Desbutyl-Lumefantrine Population Pharmacokinetic-Pharmacodynamic Relationships in Pregnant Women with Uncomplicated Plasmodium falciparum Malaria on the Thailand-Myanmar Border. Antimicrob Agents Chemother, 59 (10), pp. 6375-6384. | Show Abstract | Read more

Artemether-lumefantrine is the most widely used antimalarial artemisinin-based combination treatment. Recent studies have suggested that day 7 plasma concentrations of the potent metabolite desbutyl-lumefantrine correlate better with treatment outcomes than those of lumefantrine. Low cure rates have been reported in pregnant women with uncomplicated falciparum malaria treated with artemether-lumefantrine in northwest Thailand. A simultaneous pharmacokinetic drug-metabolite model was developed based on dense venous and sparse capillary lumefantrine and desbutyl-lumefantrine plasma samples from 116 pregnant patients on the Thailand-Myanmar border. The best model was used to evaluate therapeutic outcomes with a time-to-event approach. Lumefantrine and desbutyl-lumefantrine concentrations, implemented in an Emax model, both predicted treatment outcomes, but lumefantrine provided better predictive power. A combined model including both lumefantrine and desbutyl-lumefantrine did not improve the model further. Simulations suggested that cure rates in pregnant women with falciparum malaria could be increased by prolonging the treatment course. (These trials were registered at controlled-trials.com [ISRCTN 86353884].).

Wuthiekanun V, Amornchai P, Langla S, White NJ, Day NP, Limmathurotsakul D, Peacock SJ. 2015. Antimicrobial Disk Susceptibility Testing of Leptospira spp. Using Leptospira Vanaporn Wuthiekanun (LVW) Agar. Am J Trop Med Hyg, 93 (2), pp. 241-243. | Show Abstract | Read more

Leptospira Vanaporn Wuthiekanun (LVW) agar was used to develop a disk diffusion assay for Leptospira spp. Ten pathogenic Leptospira isolates were tested, all of which were susceptible to 17 antimicrobial agents (amoxicillin/clavulanic acid, amoxicillin, azithromycin, cefoxitin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, doripenem, doxycycline, gentamicin, linezolid, nitrofurantoin, penicillin, piperacillin/tazobactam, and tetracycline). All 10 isolates had no zone of growth inhibition for four antimicrobials (fosfomycin, nalidixic acid, rifampicin, and trimethoprim/sulfamethoxazole). Of the ten Leptospira, seven had a growth inhibition zone of ≤ 21 mm for aztreonam, the zone diameter susceptibility break point for Enterobacteriaceae. This assay could find utility as a simple screening method during the epidemiological surveillance of antimicrobial resistance in Leptospira spp.

Price RN, von Seidlein L, Valecha N, Nosten F, Baird JK, White NJ. 2015. Global extent of chloroquine-resistant Plasmodium vivax - Authors' reply. Lancet Infect Dis, 15 (6), pp. 630-631. | Read more

Hanson J, Lee SJ, Hossain MA, Anstey NM, Charunwatthana P, Maude RJ, Kingston HW, Mishra SK et al. 2015. Microvascular obstruction and endothelial activation are independently associated with the clinical manifestations of severe falciparum malaria in adults: an observational study. BMC Med, 13 (1), pp. 122. | Show Abstract | Read more

BACKGROUND: Microvascular obstruction and endothelial dysfunction have both been linked to tissue hypoperfusion in falciparum malaria, but their relative contributions to the disease's pathogenesis and outcome are unknown. METHODS: Microvascular blood flow was quantified in adults with severe falciparum malaria on their admission to hospital; plasma biomarkers of endothelial function were measured simultaneously. The relationship between these indices and the patients' clinical findings and in-hospital course was examined. RESULTS: Microvascular obstruction was observed in 119/142 (84 %) patients; a median (interquartile range (IQR)) of 14.9 % (6.6-34.9 %) of capillaries were obstructed in patients that died versus 8.3 % (1.7-26.6 %) in survivors (P = 0.039). The proportion of obstructed capillaries correlated with the estimated parasite biomass (rs = 0.25, P = 0.004) and with plasma lactate (rs = 0.38, P <0.0001), the strongest predictor of death in the series. Plasma angiopoietin-2 (Ang-2) concentrations were markedly elevated suggesting widespread endothelial activation; the median (IQR) Ang-2 concentration was 21.9 ng/mL (13.4-29.4 ng/mL) in patients that died versus 14.9 ng/mL (9.8-29.3 ng/mL) in survivors (P = 0.035). Ang-2 concentrations correlated with estimated parasite biomass (rs = 0.35, P <0.001) and plasma lactate (rs = 0.37, P <0.0001). Microvascular obstruction and Ang-2 concentrations were not significantly correlated with each other (rs = 0.17, P = 0.06), but were independently associated with plasma lactate (P <0.001 and P = 0.002, respectively). CONCLUSIONS: Microvascular obstruction and systemic endothelial activation are independently associated with plasma lactate, the strongest predictor of death in adults with falciparum malaria. This supports the hypothesis that the two processes make an independent contribution to the pathogenesis and clinical manifestations of the disease.

Deen J, Dondorp AM, White NJ. 2015. Treatment of Ebola. N Engl J Med, 372 (17), pp. 1673-1674. | Read more

White LJ, Flegg JA, Phyo AP, Wiladpai-ngern JH, Bethell D, Plowe C, Anderson T, Nkhoma S et al. 2015. Defining the in vivo phenotype of artemisinin-resistant falciparum malaria: a modelling approach. PLoS Med, 12 (4), pp. e1001823. | Show Abstract | Read more

BACKGROUND: Artemisinin-resistant falciparum malaria has emerged in Southeast Asia, posing a major threat to malaria control. It is characterised by delayed asexual-stage parasite clearance, which is the reference comparator for the molecular marker 'Kelch 13' and in vitro sensitivity tests. However, current cut-off values denoting slow clearance based on the proportion of individuals remaining parasitaemic on the third day of treatment ('day-3'), or on peripheral blood parasite half-life, are not well supported. We here explore the parasite clearance distributions in an area of artemisinin resistance with the aim refining the in vivo phenotypic definitions. METHODS AND FINDINGS: Data from 1,518 patients on the Thai-Myanmar and Thai-Cambodian borders with parasite half-life assessments after artesunate treatment were analysed. Half-lives followed a bimodal distribution. A statistical approach was developed to infer the characteristics of the component distributions and their relative contribution to the composite mixture. A model representing two parasite subpopulations with geometric mean (IQR) parasite half-lives of 3.0 (2.4-3.9) hours and 6.50 (5.7-7.4) hours was consistent with the data. For individual patients, the parasite half-life provided a predicted likelihood of an artemisinin-resistant infection which depends on the population prevalence of resistance in that area. Consequently, a half-life where the probability is 0.5 varied between 3.5 and 5.5 hours. Using this model, the current 'day-3' cut-off value of 10% predicts the potential presence of artemisinin-resistant infections in most but not all scenarios. These findings are relevant to the low-transmission setting of Southeast Asia. Generalisation to a high transmission setting as in regions of Sub-Saharan Africa will need additional evaluation. CONCLUSIONS: Characterisation of overlapping distributions of parasite half-lives provides quantitative insight into the relationship between parasite clearance and artemisinin resistance, as well as the predictive value of the 10% cut-off in 'day-3' parasitaemia. The findings are important for the interpretation of in vitro sensitivity tests and molecular markers for artemisinin resistance and for contextualising the 'day 3' threshold to account for initial parasitaemia and sample size.

Cooper BS, Boni MF, Pan-ngum W, Day NP, Horby PW, Olliaro P, Lang T, White NJ, White LJ, Whitehead J. 2015. Evaluating clinical trial designs for investigational treatments of Ebola virus disease. PLoS Med, 12 (4), pp. e1001815. | Show Abstract | Read more

BACKGROUND: Experimental treatments for Ebola virus disease (EVD) might reduce EVD mortality. There is uncertainty about the ability of different clinical trial designs to identify effective treatments, and about the feasibility of implementing individually randomised controlled trials during an Ebola epidemic. METHODS AND FINDINGS: A treatment evaluation programme for use in EVD was devised using a multi-stage approach (MSA) with two or three stages, including both non-randomised and randomised elements. The probabilities of rightly or wrongly recommending the experimental treatment, the required sample size, and the consequences for epidemic outcomes over 100 d under two epidemic scenarios were compared for the MSA, a sequential randomised controlled trial (SRCT) with up to 20 interim analyses, and, as a reference case, a conventional randomised controlled trial (RCT) without interim analyses. Assuming 50% 14-d survival in the population treated with the current standard of supportive care, all designs had similar probabilities of identifying effective treatments correctly, while the MSA was less likely to recommend treatments that were ineffective. The MSA led to a smaller number of cases receiving ineffective treatments and faster roll-out of highly effective treatments. For less effective treatments, the MSA had a high probability of including an RCT component, leading to a somewhat longer time to roll-out or rejection. Assuming 100 new EVD cases per day, the MSA led to between 6% and 15% greater reductions in epidemic mortality over the first 100 d for highly effective treatments compared to the SRCT. Both the MSA and SRCT led to substantially fewer deaths than a conventional RCT if the tested interventions were either highly effective or harmful. In the proposed MSA, the major threat to the validity of the results of the non-randomised components is that referral patterns, standard of care, or the virus itself may change during the study period in ways that affect mortality. Adverse events are also harder to quantify without a concurrent control group. CONCLUSIONS: The MSA discards ineffective treatments quickly, while reliably providing evidence concerning effective treatments. The MSA is appropriate for the clinical evaluation of EVD treatments.

WorldWide Antimalarial Resistance Network (WWARN) AS-AQ Study Group, Adjuik MA, Allan R, Anvikar AR, Ashley EA, Ba MS, Barennes H, Barnes KI et al. 2015. The effect of dosing strategies on the therapeutic efficacy of artesunate-amodiaquine for uncomplicated malaria: a meta-analysis of individual patient data. BMC Med, 13 (1), pp. 66. | Show Abstract | Read more

BACKGROUND: Artesunate-amodiaquine (AS-AQ) is one of the most widely used artemisinin-based combination therapies (ACTs) to treat uncomplicated Plasmodium falciparum malaria in Africa. We investigated the impact of different dosing strategies on the efficacy of this combination for the treatment of falciparum malaria. METHODS: Individual patient data from AS-AQ clinical trials were pooled using the WorldWide Antimalarial Resistance Network (WWARN) standardised methodology. Risk factors for treatment failure were identified using a Cox regression model with shared frailty across study sites. RESULTS: Forty-three studies representing 9,106 treatments from 1999-2012 were included in the analysis; 4,138 (45.4%) treatments were with a fixed dose combination with an AQ target dose of 30 mg/kg (FDC), 1,293 (14.2%) with a non-fixed dose combination with an AQ target dose of 25 mg/kg (loose NFDC-25), 2,418 (26.6%) with a non-fixed dose combination with an AQ target dose of 30 mg/kg (loose NFDC-30), and the remaining 1,257 (13.8%) with a co-blistered non-fixed dose combination with an AQ target dose of 30 mg/kg (co-blistered NFDC). The median dose of AQ administered was 32.1 mg/kg [IQR: 25.9-38.2], the highest dose being administered to patients treated with co-blistered NFDC (median = 35.3 mg/kg [IQR: 30.6-43.7]) and the lowest to those treated with loose NFDC-25 (median = 25.0 mg/kg [IQR: 22.7-25.0]). Patients treated with FDC received a median dose of 32.4 mg/kg [IQR: 27-39.0]. After adjusting for reinfections, the corrected antimalarial efficacy on day 28 after treatment was similar for co-blistered NFDC (97.9% [95% confidence interval (CI): 97.0-98.8%]) and FDC (98.1% [95% CI: 97.6%-98.5%]; P = 0.799), but significantly lower for the loose NFDC-25 (93.4% [95% CI: 91.9%-94.9%]), and loose NFDC-30 (95.0% [95% CI: 94.1%-95.9%]) (P < 0.001 for all comparisons). After controlling for age, AQ dose, baseline parasitemia and region; treatment with loose NFDC-25 was associated with a 3.5-fold greater risk of recrudescence by day 28 (adjusted hazard ratio, AHR = 3.51 [95% CI: 2.02-6.12], P < 0.001) compared to FDC, and treatment with loose NFDC-30 was associated with a higher risk of recrudescence at only three sites. CONCLUSIONS: There was substantial variation in the total dose of amodiaquine administered in different AS-AQ combination regimens. Fixed dose AS-AQ combinations ensure optimal dosing and provide higher antimalarial treatment efficacy than the loose individual tablets in all age categories.

Tun KM, Imwong M, Lwin KM, Win AA, Hlaing TM, Hlaing T, Lin K, Kyaw MP et al. 2015. Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker. Lancet Infect Dis, 15 (4), pp. 415-421. | Show Abstract | Read more

BACKGROUND: Emergence of artemisinin resistance in southeast Asia poses a serious threat to the global control of Plasmodium falciparum malaria. Discovery of the K13 marker has transformed approaches to the monitoring of artemisinin resistance, allowing introduction of molecular surveillance in remote areas through analysis of DNA. We aimed to assess the spread of artemisinin-resistant P falciparum in Myanmar by determining the relative prevalence of P falciparum parasites carrying K13-propeller mutations. METHODS: We did this cross-sectional survey at malaria treatment centres at 55 sites in ten administrative regions in Myanmar, and in relevant border regions in Thailand and Bangladesh, between January, 2013, and September, 2014. K13 sequences from P falciparum infections were obtained mainly by passive case detection. We entered data into two geostatistical models to produce predictive maps of the estimated prevalence of mutations of the K13 propeller region across Myanmar. FINDINGS: Overall, 371 (39%) of 940 samples carried a K13-propeller mutation. We recorded 26 different mutations, including nine mutations not described previously in southeast Asia. In seven (70%) of the ten administrative regions of Myanmar, the combined K13-mutation prevalence was more than 20%. Geospatial mapping showed that the overall prevalence of K13 mutations exceeded 10% in much of the east and north of the country. In Homalin, Sagaing Region, 25 km from the Indian border, 21 (47%) of 45 parasite samples carried K13-propeller mutations. INTERPRETATION: Artemisinin resistance extends across much of Myanmar. We recorded P falciparum parasites carrying K13-propeller mutations at high prevalence next to the northwestern border with India. Appropriate therapeutic regimens should be tested urgently and implemented comprehensively if spread of artemisinin resistance to other regions is to be avoided. FUNDING: Wellcome Trust-Mahidol University-Oxford Tropical Medicine Research Programme and the Bill & Melinda Gates Foundation.

Mishra N, Prajapati SK, Kaitholia K, Bharti RS, Srivastava B, Phookan S, Anvikar AR, Dev V et al. 2015. Surveillance of artemisinin resistance in Plasmodium falciparum in India using the kelch13 molecular marker. Antimicrob Agents Chemother, 59 (5), pp. 2548-2553. | Show Abstract | Read more

Malaria treatment in Southeast Asia is threatened with the emergence of artemisinin-resistant Plasmodium falciparum. Genome association studies have strongly linked a locus on P. falciparum chromosome 13 to artemisinin resistance, and recently, mutations in the kelch13 propeller region (Pfk-13) were strongly linked to resistance. To date, this information has not been shown in Indian samples. Pfk-13 mutations were assessed in samples from efficacy studies of artemisinin combination treatments in India. Samples were PCR amplified and sequenced from codon 427 to 727. Out of 384 samples, nonsynonymous mutations in the propeller region were found in four patients from the northeastern states, but their presence did not correlate with ACT treatment failures. This is the first report of Pfk-13 point mutations from India. Further phenotyping and genotyping studies are required to assess the status of artemisinin resistance in this region.

Miotto O, Amato R, Ashley EA, MacInnis B, Almagro-Garcia J, Amaratunga C, Lim P, Mead D et al. 2015. Genetic architecture of artemisinin-resistant Plasmodium falciparum. Nat Genet, 47 (3), pp. 226-234. | Show Abstract | Read more

We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population.

White NJ, Hien TT, Nosten FH. 2015. A Brief History of Qinghaosu Trends in Parasitology, 31 (12), pp. 607-610. | Show Abstract | Read more

© 2015 Elsevier Ltd.The 2015 Nobel Prize for Medicine or Physiology was awarded to William C. Campbell and Satoshi Omura for their discovery of avermectins, and to Tu You You for her contribution to the discovery of artemisinin. The discovery and development of qinghaosu (artemisinin) as an antimalarial drug is a remarkable and convoluted tale.

Phyo AP, Jittamala P, Nosten FH, Pukrittayakamee S, Imwong M, White NJ, Duparc S, Macintyre F, Baker M, Möhrle JJ. 2016. Antimalarial activity of artefenomel (OZ439), a novel synthetic antimalarial endoperoxide, in patients with Plasmodium falciparum and Plasmodium vivax malaria: an open-label phase 2 trial. Lancet Infect Dis, 16 (1), pp. 61-69. | Show Abstract | Read more

BACKGROUND: Artefenomel (OZ439) is a novel synthetic trioxolane with improved pharmacokinetic properties compared with other antimalarial drugs with the artemisinin pharmacophore. Artefenomel has been generally well tolerated in volunteers at doses up to 1600 mg and is being developed as a partner drug in an antimalarial combination treatment. We investigated the efficacy, tolerability, and pharmacokinetics of artefenomel at different doses in patients with Plasmodium falciparum or Plasmodium vivax malaria. METHODS: This phase 2a exploratory, open-label trial was done at the Hospital for Tropical Diseases, Bangkok, and the Shoklo Malaria Research Unit in Thailand. Adult patients with acute, uncomplicated P falciparum or P vivax malaria received artefenomel in a single oral dose (200 mg, 400 mg, 800 mg, or 1200 mg). The first cohort received 800 mg. Testing of a new dose of artefenomel in a patient cohort was decided on after safety and efficacy assessment of the preceding cohort. The primary endpoint was the natural log parasite reduction per 24 h. Definitive oral treatment was given at 36 h. This trial is registered with ClinicalTrials.gov, number NCT01213966. FINDINGS: Between Oct 24, 2010, and May 25, 2012, 82 patients were enrolled (20 in each of the 200 mg, 400 mg, and 800 mg cohorts, and 21 in the 1200 mg cohort). One patient withdrew consent (before the administration of artefenomel) but there were no further dropouts. The parasite reduction rates per 24 h ranged from 0·90 to 1·88 for P falciparum, and 2·09 to 2·53 for P vivax. All doses were equally effective in both P falciparum and P vivax malaria, with median parasite clearance half-lives of 4·1 h (range 1·3-6·7) to 5·6 h (2·0-8·5) for P falciparum and 2·3 h (1·2-3·9) to 3·2 h (0·9-15·0) for P vivax. Maximum plasma concentrations, dose-proportional to 800 mg, occurred at 4 h (median). The estimated elimination half-life was 46-62 h. No serious drug-related adverse effects were reported; other adverse effects were generally mild and reversible, with the highest number in the 1200 mg cohort (17 [81%] patients with at least one adverse event). The most frequently reported adverse effect was an asymptomatic increase in plasma creatine phosphokinase concentration (200 mg, n=5; 400 mg, n=3; 800 mg, n=1; 1200 mg, n=3). INTERPRETATION: Artefenomel is a new synthetic antimalarial peroxide with a good safety profile that clears parasitaemia rapidly in both P falciparum and P vivax malaria. Its long half-life suggests a possible use in a single-dose treatment in combination with other drugs. FUNDING: Bill & Melinda Gates Foundation, Wellcome Trust, and UK Department for International Development.

Nguyen TD, Olliaro P, Dondorp AM, Baird JK, Lam HM, Farrar J, Thwaites GE, White NJ, Boni MF. 2015. Optimum population-level use of artemisinin combination therapies: a modelling study. Lancet Glob Health, 3 (12), pp. e758-e766. | Show Abstract | Read more

BACKGROUND: Artemisinin combination therapies (ACTs) are used worldwide as first-line treatment against confirmed or suspected Plasmodium falciparum malaria. Despite the success of ACTs at reducing the global burden of malaria, emerging resistance to artemisinin threatens these gains. Countering onset of resistance might need deliberate tactics aimed at slowing the reduction in ACT effectiveness. We assessed optimum use of ACTs at the population level, specifically focusing on a strategy of multiple first-line therapies (MFT), and comparing it with strategies of cycling or sequential use of single first-line ACTs. METHODS: With an individual-based microsimulation of regional malaria transmission, we looked at how to apply a therapy as widely as possible without accelerating reduction of efficacy by drug resistance. We compared simultaneous distribution of artemether-lumefantrine, artesunate-amodiaquine, and dihydroartemisinin-piperaquine (ie, MFT) against strategies in which these ACTs would be cycled or used sequentially, either on a fixed schedule or when population-level efficacy reaches the WHO threshold of 10% treatment failure. The main assessment criterion was total number of treatment failures per 100 people per year. Additionally, we analysed the benefits of including a single non-ACT therapy in an MFT strategy, and did sensitivity analyses in which we varied transmission setting, treatment coverage, partner-drug half-life, fitness cost of drug resistance, and the relation between drug concentration and resistance evolution. FINDINGS: Use of MFT was predicted to reduce the long-term number of treatment failures compared with strategies in which a single first-line ACT is recommended. This result was robust to various epidemiological, pharmacological, and evolutionary features of malaria transmission. Inclusion of a single non-ACT therapy in an MFT strategy would have substantial benefits in reduction of pressure on artemisinin resistance evolution, delaying its emergence and slowing its spread. INTERPRETATION: Adjusting national antimalarial treatment guidelines to encourage simultaneous use of MFT is likely to extend the useful therapeutic life of available antimalarial drugs, resulting in long-term beneficial outcomes for patients. FUNDING: Wellcome Trust, UK Medical Research Council, Li Ka Shing Foundation.

White NJ, Hien TT, Nosten FH. 2015. A Brief History of Qinghaosu. Trends Parasitol, 31 (12), pp. 607-610. | Show Abstract | Read more

The 2015 Nobel Prize for Medicine or Physiology was awarded to William C. Campbell and Satoshi Ōmura for their discovery of avermectins, and to Tu You You for her contribution to the discovery of artemisinin. The discovery and development of qinghaosu (artemisinin) as an antimalarial drug is a remarkable and convoluted tale.

White NJ. 2015. Declining Malaria Transmission and Pregnancy Outcomes in Southern Mozambique. N Engl J Med, 373 (17), pp. 1670-1671. | Read more

Andolina C, Landier J, Carrara V, Chu CS, Franetich JF, Roth A, Rénia L, Roucher C, White NJ, Snounou G, Nosten F. 2015. The suitability of laboratory-bred Anopheles cracens for the production of Plasmodium vivax sporozoites. Malar J, 14 (1), pp. 312. | Show Abstract | Read more

BACKGROUND: A stenogamous colony of Anopheles cracens (A. dirus B) established 20 years ago in a Thai insectary proved susceptible to Plasmodium vivax. However, routine sporozoite production by feeding on field-collected blood samples has not been described. The setting-up of an A. cracens colony in an insectary on the Thai-Myanmar border and the process of using P. vivax field samples for the production of infectious sporozoites are described. METHODS: The colony was started in 2012 from egg batches that were sent from the Department of Parasitology, Faculty of Medicine, University of Chiang Mai, to the Shoklo Malaria Research Unit (SMRU), on wet filter paper in sealed Petri dishes. From May 2013 to December 2014, P. vivax-infected blood samples collected from patients seeking care at SMRU clinics were used for membrane feeding assays and sporozoite production. RESULTS: Mosquitoes were fed on blood samples from 55 patients, and for 38 (69 %) this led to the production sporozoites. The average number of sporozoites obtained per mosquito was 26,112 (range 328-79,310). Gametocytaemia was not correlated with mosquito infectiousness (p = 0.82), or with the number of the sporozoites produced (Spearman's ρ = -0.016, p = 0.905). Infectiousness did not vary with the date of collection or the age of the patient. Mosquito survival was not correlated with sporozoite load (Spearman's ρ = 0.179, p = 0.282). CONCLUSION: Consistent and routine P. vivax sporozoites production confirms that A. cracens is highly susceptible to P. vivax infection. Laboratory-bred colonies of this vector are suitable for experimental transmission protocols and thus constitute a valuable resource.

Lwin KM, Imwong M, Suangkanarat P, Jeeyapant A, Vihokhern B, Wongsaen K, Snounou G, Keereecharoen L, White NJ, Nosten F. 2015. Elimination of Plasmodium falciparum in an area of multi-drug resistance. Malar J, 14 (1), pp. 319. | Show Abstract | Read more

BACKGROUND: Resistance to the artemisinin derivatives in Plasmodium falciparum has emerged in Cambodia and is now spreading throughout South-East Asia. The rapid elimination of P. falciparum seems to be the only viable option to avoid a public health disaster but this is difficult because even in low transmission settings many residents have asymptomatic parasitaemias. METHODS: In response to a large number of malaria cases reported in three remote villages on the Thai-Myanmar border where malaria is endemic and the disease is seasonal, surveys were conducted using an ultra-sensitive qPCR assay (LOD 22 parasites per mL). In one of the villages where it was feasible, mass anti-malarial drug administration was proposed to the population as a potential solution, and this was adopted. RESULTS: In the three villages 204/356 (57.3 %), 212/385 (55.1 %) and 195/286 (68.2 %) of the resident populations were positive by qPCR (approximately one-third P. falciparum and two-thirds P. vivax). Of those positive for P. falciparum 62 % carried single point mutations in the P. falciparum kelch protein (a marker of artemisinin resistance). In one of the villages 217 of 674 inhabitants received at least one dose of dihydroartemisinin-piperaquine chemoprevention in June 2012, 155 (71.4 %) received two consecutive months, and 98 (45.2 %) received three treatment doses. The chemoprevention was generally well tolerated. The sub-microscopic reservoir of P. falciparum malaria was eliminated during the six-month follow-up period (prevalence fell from 7 to 0 %); P. vivax malaria persisted (prevalence fell from 35 to 8 %). From June to October 2012 (rainy season) the number of clinical episodes of P. falciparum was six times lower (46), than during the same period in the previous year (290). CONCLUSION: Mass drug administration with dihydroartemisinin-piperaquine may be an effective strategy to eliminate P. falciparum rapidly where multi-drug resistance is present.

Worldwide Antimalarial Resistance Network (WWARN) AL Dose Impact Study Group. 2015. The effect of dose on the antimalarial efficacy of artemether-lumefantrine: a systematic review and pooled analysis of individual patient data. Lancet Infect Dis, 15 (6), pp. 692-702. | Show Abstract | Read more

BACKGROUND: Artemether-lumefantrine is the most widely used artemisinin-based combination therapy for malaria, although treatment failures occur in some regions. We investigated the effect of dosing strategy on efficacy in a pooled analysis from trials done in a wide range of malaria-endemic settings. METHODS: We searched PubMed for clinical trials that enrolled and treated patients with artemether-lumefantrine and were published from 1960 to December, 2012. We merged individual patient data from these trials by use of standardised methods. The primary endpoint was the PCR-adjusted risk of Plasmodium falciparum recrudescence by day 28. Secondary endpoints consisted of the PCR-adjusted risk of P falciparum recurrence by day 42, PCR-unadjusted risk of P falciparum recurrence by day 42, early parasite clearance, and gametocyte carriage. Risk factors for PCR-adjusted recrudescence were identified using Cox's regression model with frailty shared across the study sites. FINDINGS: We included 61 studies done between January, 1998, and December, 2012, and included 14,327 patients in our analyses. The PCR-adjusted therapeutic efficacy was 97·6% (95% CI 97·4-97·9) at day 28 and 96·0% (95·6-96·5) at day 42. After controlling for age and parasitaemia, patients prescribed a higher dose of artemether had a lower risk of having parasitaemia on day 1 (adjusted odds ratio [OR] 0·92, 95% CI 0·86-0·99 for every 1 mg/kg increase in daily artemether dose; p=0·024), but not on day 2 (p=0·69) or day 3 (0·087). In Asia, children weighing 10-15 kg who received a total lumefantrine dose less than 60 mg/kg had the lowest PCR-adjusted efficacy (91·7%, 95% CI 86·5-96·9). In Africa, the risk of treatment failure was greatest in malnourished children aged 1-3 years (PCR-adjusted efficacy 94·3%, 95% CI 92·3-96·3). A higher artemether dose was associated with a lower gametocyte presence within 14 days of treatment (adjusted OR 0·92, 95% CI 0·85-0·99; p=0·037 for every 1 mg/kg increase in total artemether dose). INTERPRETATION: The recommended dose of artemether-lumefantrine provides reliable efficacy in most patients with uncomplicated malaria. However, therapeutic efficacy was lowest in young children from Asia and young underweight children from Africa; a higher dose regimen should be assessed in these groups. FUNDING: Bill & Melinda Gates Foundation.

Hanson J, Phu NH, Hasan MU, Charunwatthana P, Plewes K, Maude RJ, Prapansilp P, Kingston HW et al. 2015. The clinical implications of thrombocytopenia in adults with severe falciparum malaria: a retrospective analysis. BMC Med, 13 (1), pp. 97. | Show Abstract | Read more

BACKGROUND: Thrombocytopenia is a common finding in adults with severe falciparum malaria, but its clinical and prognostic utility is incompletely defined. METHODS: Clinical and laboratory data from 647 adults with severe falciparum malaria were analysed retrospectively to determine the relationship between a patient's platelet count on admission to hospital and their subsequent clinical course. RESULTS: On admission, 614 patients (94.9%) were thrombocytopenic (platelet count <150 × 10(9)/L) and 328 (50.7%) had a platelet count <50 × 10(9)/L. The admission platelet count was inversely correlated with parasite biomass (estimated from plasma PfHRP2 concentrations, rs = -0.28, P = 0.003), the degree of microvascular sequestration (measured with orthogonal polarizing spectral imaging, rs = -0.31, P = 0.001) and disease severity (the number of World Health Organization severity criteria satisfied by the patient, rs = -0.21, P <0.001). Platelet counts were lower on admission in the patients who died (median: 30 (interquartile range 22 to 52) × 10(9)/L versus 50 (34 to 78) × 10(9)/L in survivors; P <0.001), but did not predict outcome independently from other established laboratory and clinical prognostic indices. The 39 patients (6%) with profound thrombocytopenia (platelet count <20 × 10(9)/L) were more likely to die (odds ratio: 5.00, 95% confidence interval: 2.56 to 9.75) than patients with higher platelet counts, but these high-risk patients could be identified more rapidly with simple bedside clinical assessment. The admission platelet count did not reliably identify the 50 patients (7.7%) with major bleeding during the study. CONCLUSIONS: Thrombocytopenia is a marker of disease severity in adults with falciparum malaria, but has limited utility in prognostication, triage and management.

Newton PN, Schellenberg D, Ashley EA, Ravinetto R, Green MD, ter Kuile FO, Tabernero P, White NJ, Guerin PJ. 2015. Quality assurance of drugs used in clinical trials: proposal for adapting guidelines. BMJ, 350 (feb25 10), pp. h602. | Read more

Bancone G, Chu CS, Somsakchaicharoen R, Chowwiwat N, Parker DM, Charunwatthana P, White NJ, Nosten FH. 2014. Characterization of G6PD genotypes and phenotypes on the northwestern PLoS ONE, 9 (12), | Show Abstract | Read more

© 2014 Bancone et al.Mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene result in red blood cells with increased susceptibility to oxidative damage. Significant haemolysis can be caused by primaquine and other 8-aminoquinoline antimalarials used for the radical treatment of Plasmodium vivax malaria. The distribution and phenotypes of mutations causing G6PD deficiency in the male population of migrants and refugees in a malaria endemic region on the Thailand-Myanmar border were characterized. Blood samples for G6PD fluorescent spot test (FST), G6PD genotyping, and malaria testing were taken from 504 unrelated males of Karen and Burman ethnicities presenting to the outpatient clinics. The overall frequency of G6PD deficiency by the FST was 13.7%. Among the deficient subjects, almost 90% had the Mahidol variant (487G>A) genotype. The remaining subjects had Chinese-4 (392G>T), Viangchan (871G>A), Açores (595A>G), Seattle (844G>C) and Mediterranean (563C>T) variants. Quantification of G6PD activity was performed using a modification of the standard spectrophotometric assay on a subset of 24 samples with Mahidol, Viangchan, Seattle and Chinese-4 mutations; all samples showed a residual enzymatic activity below 10% of normal and were diagnosed correctly by the FST. Further studies are needed to characterise the haemolytic risk of using 8-aminoquinolines in patients with these genotypes.

Mok S, Ashley EA, Ferreira PE, Zhu L, Lin Z, Yeo T, Chotivanich K, Imwong M et al. 2015. Drug resistance. Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance. Science, 347 (6220), pp. 431-435. | Show Abstract | Read more

Artemisinin resistance in Plasmodium falciparum threatens global efforts to control and eliminate malaria. Polymorphisms in the kelch domain-carrying protein K13 are associated with artemisinin resistance, but the underlying molecular mechanisms are unknown. We analyzed the in vivo transcriptomes of 1043 P. falciparum isolates from patients with acute malaria and found that artemisinin resistance is associated with increased expression of unfolded protein response (UPR) pathways involving the major PROSC and TRiC chaperone complexes. Artemisinin-resistant parasites also exhibit decelerated progression through the first part of the asexual intraerythrocytic development cycle. These findings suggest that artemisinin-resistant parasites remain in a state of decelerated development at the young ring stage, whereas their up-regulated UPR pathways mitigate protein damage caused by artemisinin. The expression profiles of UPR-related genes also associate with the geographical origin of parasite isolates, further suggesting their role in emerging artemisinin resistance in the Greater Mekong Subregion.

White NJ, Ashley EA, Recht J, Delves MJ, Ruecker A, Smithuis FM, Eziefula AC, Bousema T et al. 2014. Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria. Malar J, 13 (1), pp. 483. | Show Abstract | Read more

Indirect clinical measures assessing anti-malarial drug transmission-blocking activity in falciparum malaria include measurement of the duration of gametocytaemia, the rate of gametocyte clearance or the area under the gametocytaemia-time curve (AUC). These may provide useful comparative information, but they underestimate dose-response relationships for transmission-blocking activity. Following 8-aminoquinoline administration P. falciparum gametocytes are sterilized within hours, whereas clearance from blood takes days. Gametocytaemia AUC and clearance times are determined predominantly by the more numerous female gametocytes, which are generally less drug sensitive than the minority male gametocytes, whereas transmission-blocking activity and thus infectivity is determined by the more sensitive male forms. In choosing doses of transmission-blocking drugs there is no substitute yet for mosquito-feeding studies.

Hanson J, Anstey NM, Bihari D, White NJ, Day NP, Dondorp AM. 2014. The fluid management of adults with severe malaria. Crit Care, 18 (6), pp. 642. | Show Abstract | Read more

Fluid resuscitation has long been considered a key intervention in the treatment of adults with severe falciparum malaria. Profound hypovolemia is common in these patients and has the potential to exacerbate the acidosis and acute kidney injury that are independent predictors of death. However, new microvascular imaging techniques have shown that disease severity correlates more strongly with obstruction of the microcirculation by parasitized erythrocytes--a process termed sequestration. Fluid loading has little effect on sequestration and increases the risk of complications, particularly pulmonary edema, a condition that can develop suddenly and unpredictably and that is frequently fatal in this population. Accordingly, even if a patient is clinically hypovolemic, if there is an adequate blood pressure and urine output, there may be little advantage in infusing intravenous fluid beyond a maintenance rate of 1 to 2 mL/kg per hour. The optimal agent for fluid resuscitation remains uncertain; significant anemia requires blood transfusion, but colloid solutions may be associated with harm and should be avoided. The preferred crystalloid is unclear, although the use of balanced solutions requires investigation. There are fewer data to guide the fluid management of severe vivax and knowlesi malaria, although a similar conservative strategy would appear prudent.

Jittamala P, Pukrittayakamee S, Ashley EA, Nosten F, Hanboonkunupakarn B, Lee SJ, Thana P, Chairat K et al. 2015. Pharmacokinetic interactions between primaquine and pyronaridine-artesunate in healthy adult Thai subjects. Antimicrob Agents Chemother, 59 (1), pp. 505-513. | Show Abstract | Read more

Pyronaridine-artesunate is a newly introduced artemisinin-based combination treatment which may be deployed together with primaquine. A single-dose, randomized, three-sequence crossover study was conducted in healthy Thai volunteers to characterize potential pharmacokinetic interactions between these drugs. Seventeen healthy adults received a single oral dose of primaquine alone (30 mg base) and were then randomized to receive pyronaridine-artesunate alone (540-180 mg) or pyronaridine-artesunate plus primaquine in combination, with intervening washout periods between all treatments. The pharmacokinetic properties of primaquine, its metabolite carboxyprimaquine, artesunate, its metabolite dihydroartemisinin, and pyronaridine were assessed in 15 subjects using a noncompartmental approach followed by a bioequivalence evaluation. All drugs were well tolerated. The single oral dose of primaquine did not result in any clinically relevant pharmacokinetic alterations to pyronaridine, artesunate, or dihydroartemisinin exposures. There were significantly higher primaquine maximum plasma drug concentrations (geometric mean ratio, 30%; 90% confidence interval [CI], 17% to 46%) and total exposures (15%; 6.4% to 24%) during coadministration with pyronaridine-artesunate than when primaquine was given alone. Pyronaridine, like chloroquine and piperaquine, increases plasma primaquine concentrations. (This study has been registered at ClinicalTrials.gov under registration no. NCT01552330.).

McGready R, Prakash JA, Benjamin SJ, Watthanaworawit W, Anantatat T, Tanganuchitcharnchai A, Ling CL, Tan SO et al. 2014. Pregnancy outcome in relation to treatment of murine typhus and scrub typhus infection: a fever cohort and a case series analysis. PLoS Negl Trop Dis, 8 (11), pp. e3327. | Show Abstract | Read more

BACKGROUND: There is a paucity of published reports on pregnancy outcome following scrub and murine typhus despite these infections being leading causes of undifferentiated fever in Asia. This study aimed to relate pregnancy outcome with treatment of typhus. METHODOLOGY/PRINCIPAL FINDINGS: Data were analyzed from: i) pregnant women with a diagnosis of scrub and/or murine typhus from a fever cohort studies; ii) case series of published studies in PubMed using the search terms "scrub typhus" (ST), "murine typhus" (MT), "Orientia tsutsugamushi", "Rickettsia tsutsugamushi", "Rickettsia typhi", "rickettsiae", "typhus", or "rickettsiosis"; and "pregnancy", until February 2014 and iii) an unpublished case series. Fever clearance time (FCT) and pregnancy outcome (miscarriage and delivery) were compared to treatment. Poor neonatal outcome was a composite measure for pregnancies sustained to 28 weeks or more of gestation ending in stillbirth, preterm birth, or delivery of a growth restricted or low birth weight newborn. RESULTS: There were 26 women in the fever cohort. MT and ST were clinically indistinguishable apart from two ST patients with eschars. FCTs (median [range] hours) were 25 [16-42] for azithromycin (n=5), 34 [20-53] for antimalarials (n=5) and 92 [6-260] for other antibiotics/supportive therapy (n=16). There were 36.4% (8/22) with a poor neonatal outcome. In 18 years, 97 pregnancies were collated, 82 with known outcomes, including two maternal deaths. Proportions of miscarriage 17.3% (14/81) and poor neonatal outcomes 41.8% (28/67) were high, increasing with longer FCTs (p=0.050, linear trend). Use of azithromycin was not significantly associated with improved neonatal outcomes (p=0.610). CONCLUSION: The published ST and MT world literature amounts to less than 100 pregnancies due to under recognition and under diagnosis. Evidence supporting the most commonly used treatment, azithromycin, is weak. Collaborative, prospective clinical trials in pregnant women are urgently required to reduce the burden of adverse maternal and newborn outcomes and to determine the safety and efficacy of antimicrobial treatment.

Bergstrand M, Nosten F, Lwin KM, Karlsson MO, White NJ, Tarning J. 2014. Characterization of an in vivo concentration-effect relationship for piperaquine in malaria chemoprevention. Sci Transl Med, 6 (260), pp. 260ra147. | Show Abstract | Read more

A randomized, placebo-controlled trial conducted on the northwest border of Thailand compared malaria chemoprevention with monthly or bimonthly standard 3-day treatment regimens of dihydroartemisinin-piperaquine. Healthy adult male subjects (N = 1000) were followed weekly during 9 months of treatment. Using nonlinear mixed-effects modeling, the concentration-effect relationship for the malaria-preventive effect of piperaquine was best characterized with a sigmoidal Emax relationship, where plasma concentrations of 6.7 ng/ml [relative standard error (RSE), 23%] and 20 ng/ml were found to reduce the hazard of acquiring a malaria infection by 50% [that is, median inhibitory concentration (IC50)] and 95% (IC95), respectively. Simulations of monthly dosing, based on the final model and published pharmacokinetic data, suggested that the incidence of malaria infections over 1 year could be reduced by 70% with a recently suggested dosing regimen compared to the current manufacturer's recommendations for small children (8 to 12 kg). This model provides a rational framework for piperaquine dose optimization in different patient groups.

Imwong M, Woodrow CJ, Hendriksen IC, Veenemans J, Verhoef H, Faiz MA, Mohanty S, Mishra S et al. 2015. Plasma concentration of parasite DNA as a measure of disease severity in falciparum malaria. J Infect Dis, 211 (7), pp. 1128-1133. | Show Abstract | Read more

In malaria-endemic areas, Plasmodium falciparum parasitemia is common in apparently healthy children and severe malaria is commonly misdiagnosed in patients with incidental parasitemia. We assessed whether the plasma Plasmodium falciparum DNA concentration is a useful datum for distinguishing uncomplicated from severe malaria in African children and Asian adults. P. falciparum DNA concentrations were measured by real-time polymerase chain reaction (PCR) in 224 African children (111 with uncomplicated malaria and 113 with severe malaria) and 211 Asian adults (100 with uncomplicated malaria and 111 with severe malaria) presenting with acute falciparum malaria. The diagnostic accuracy of plasma P. falciparum DNA concentrations in identifying severe malaria was 0.834 for children and 0.788 for adults, similar to that of plasma P. falciparum HRP2 levels and substantially superior to that of parasite densities (P < .0001). The diagnostic accuracy of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations was significantly greater than that of plasma P. falciparum HRP2 concentrations alone (0.904 for children [P = .004] and 0.847 for adults [P = .003]). Quantitative real-time PCR measurement of parasite DNA in plasma is a useful method for diagnosing severe falciparum malaria on fresh or archived plasma samples.

Hoglund RM, Byakika-Kibwika P, Lamorde M, Merry C, Ashton M, Hanpithakpong W, Day NP, White NJ, Äbelö A, Tarning J. 2015. Artemether-lumefantrine co-administration with antiretrovirals: population pharmacokinetics and dosing implications. Br J Clin Pharmacol, 79 (4), pp. 636-649. | Show Abstract | Read more

AIM: Drug-drug interactions between antimalarial and antiretroviral drugs may influence antimalarial treatment outcomes. The aim of this study was to investigate the potential drug-drug interactions between the antimalarial drugs, lumefantrine, artemether and their respective metabolites desbutyl-lumefantrine and dihydroartemisinin, and the HIV drugs efavirenz, nevirapine and lopinavir/ritonavir. METHOD: Data from two clinical studies, investigating the influence of the HIV drugs efavirenz, nevirapine and lopinavir/ritonavir on the pharmacokinetics of the antimalarial drugs lumefantrine, artemether and their respective metabolites, in HIV infected patients were pooled and analyzed using a non-linear mixed effects modelling approach. RESULTS: Efavirenz and nevirapine significantly decreased the terminal exposure to lumefantrine (decrease of 69.9% and 25.2%, respectively) while lopinavir/ritonavir substantially increased the exposure (increase of 439%). All antiretroviral drugs decreased the total exposure to dihydroartemisinin (decrease of 71.7%, 41.3% and 59.7% for efavirenz, nevirapine and ritonavir/lopinavir, respectively). Simulations suggest that a substantially increased artemether-lumefantrine dose is required to achieve equivalent exposures when co-administered with efavirenz (250% increase) and nevirapine (75% increase). When co-administered with lopinavir/ritonavir it is unclear if the increased lumefantrine exposure compensates adequately for the reduced dihydroartemisinin exposure and thus whether dose adjustment is required. CONCLUSION: There are substantial drug interactions between artemether-lumefantrine and efavirenz, nevirapine and ritonavir/lopinavir. Given the readily saturable absorption of lumefantrine, the dose adjustments predicted to be necessary will need to be evaluated prospectively in malaria-HIV co-infected patients.

Maude RJ, Nguon C, Ly P, Bunkea T, Ngor P, Canavati de la Torre SE, White NJ, Dondorp AM, Day NP, White LJ, Chuor CM. 2014. Spatial and temporal epidemiology of clinical malaria in Cambodia 2004-2013. Malar J, 13 (1), pp. 385. | Show Abstract | Read more

BACKGROUND: Artemisinin-resistant Plasmodium falciparum malaria has recently been identified on the Thailand-Cambodia border and more recently in parts of Thailand, Myanmar and Vietnam. There is concern that if this resistance were to spread, it would severely hamper malaria control and elimination efforts worldwide. Efforts are currently underway to intensify malaria control activities and ultimately eliminate malaria from Cambodia. To support these efforts, it is crucial to have a detailed picture of disease burden and its major determinants over time. METHODS: An analysis of spatial and temporal data on clinical malaria in Cambodia collected by the National Centre for Parasitology, Entomology and Malaria Control (CNM) and the Department of Planning and Health Information, Ministry of Health Cambodia from 2004 to 2013 is presented. RESULTS: There has been a marked decrease of 81% in annual cases due to P. falciparum since 2009 coinciding with a rapid scale-up in village malaria workers (VMWs) and insecticide-treated bed nets (ITNs). Concurrently, the number of cases with Plasmodium vivax has greatly increased. It is estimated that there were around 112,000 total cases in 2012, 2.8 times greater than the WHO estimate for that year, and 68,000 in 2013 (an annual parasite incidence (API) of 4.6/1000). With the scale-up of VMWs, numbers of patients presenting to government facilities did not fall and it appears likely that those who saw VMWs had previously accessed healthcare in the private sector. Malaria mortality has decreased, particularly in areas with VMWs. There has been a marked decrease in cases in parts of western Cambodia, especially in Pailin and Battambang Provinces. In the northeast, the fall in malaria burden has been more modest, this area having the highest API in 2013. CONCLUSION: The clinical burden of falciparum malaria in most areas of Cambodia has greatly decreased from 2009 to 2013, associated with roll-out of ITNs and VMWs. Numbers of cases with P. vivax have increased. Possible reasons for these trends are discussed and areas requiring further study are highlighted. Although malaria surveillance data are prone to collection bias and tend to underestimate disease burden, the finding of similar trends in two independent datasets in this study greatly increased the robustness of the findings.

Hanboonkunupakarn B, Ashley EA, Jittamala P, Tarning J, Pukrittayakamee S, Hanpithakpong W, Chotsiri P, Wattanakul T et al. 2014. Open-label crossover study of primaquine and dihydroartemisinin-piperaquine pharmacokinetics in healthy adult thai subjects. Antimicrob Agents Chemother, 58 (12), pp. 7340-7346. | Show Abstract | Read more

Dihydroartemisinin-piperaquine is an artemisinin-based combination treatment (ACT) recommended by the WHO for uncomplicated Plasmodium falciparum malaria, and it is being used increasingly for resistant vivax malaria where combination with primaquine is required for radical cure. The WHO recently reinforced its recommendations to add a single dose of primaquine to ACTs to reduce P. falciparum transmission in low-transmission settings. The pharmacokinetics of primaquine and dihydroartemisinin-piperaquine were evaluated in 16 healthy Thai adult volunteers in a randomized crossover study. Volunteers were randomized to two groups of three sequential hospital admissions to receive 30 mg (base) primaquine, 3 tablets of dihydroartemisinin-piperaquine (120/960 mg), and the drugs together at the same doses. Blood sampling was performed over 3 days following primaquine and 36 days following dihydroartemisinin-piperaquine dosing. Pharmacokinetic assessment was done with a noncompartmental approach. The drugs were well tolerated. There were no statistically significant differences in dihydroartemisinin and piperaquine pharmacokinetics with or without primaquine. Dihydroartemisinin-piperaquine coadministration significantly increased plasma primaquine levels; geometric mean ratios (90% confidence interval [CI]) of primaquine combined versus primaquine alone for maximum concentration (Cmax), area under the concentration-time curve from 0 h to the end of the study (AUC0-last), and area under the concentration-time curve from 0 h to infinity (AUC0-∞) were 148% (117 to 187%), 129% (103 to 163%), and 128% (102 to 161%), respectively. This interaction is similar to that described recently with chloroquine and may result in an enhanced radical curative effect. (This study has been registered at ClinicalTrials.gov under registration no. NCT01525511.).

Price RN, von Seidlein L, Valecha N, Nosten F, Baird JK, White NJ. 2014. Global extent of chloroquine-resistant Plasmodium vivax: a systematic review and meta-analysis. Lancet Infect Dis, 14 (10), pp. 982-991. | Show Abstract | Read more

BACKGROUND: Chloroquine is the first-line treatment for Plasmodium vivax malaria in most endemic countries, but resistance is increasing. Monitoring of antimalarial efficacy is essential, but in P. vivax infections the assessment of treatment efficacy is confounded by relapse from the dormant liver stages. We systematically reviewed P. vivax malaria treatment efficacy studies to establish the global extent of chloroquine resistance. METHODS: We searched Medline, Web of Science, Embase, and the Cochrane Database of Systematic Reviews to identify studies published in English between Jan 1, 1960, and April 30, 2014, which investigated antimalarial treatment efficacy in P. vivax malaria. We excluded studies that did not include supervised schizonticidal treatment without primaquine. We determined rates of chloroquine resistance according to P. vivax malaria recurrence rates by day 28 whole-blood chloroquine concentrations at the time of recurrence and study enrolment criteria. FINDINGS: We identified 129 eligible clinical trials involving 21,694 patients at 179 study sites and 26 case reports describing 54 patients. Chloroquine resistance was present in 58 (53%) of 113 assessable study sites, spread across most countries that are endemic for P. vivax. Clearance of parasitaemia assessed by microscopy in 95% of patients by day 2, or all patients by day 3, was 100% predictive of chloroquine sensitivity. INTERPRETATION: Heterogeneity of study design and analysis has confounded global surveillance of chloroquine-resistant P. vivax, which is now present across most countries endemic for P. vivax. Improved methods for monitoring of drug resistance are needed to inform antimalarial policy in these regions. FUNDING: Wellcome Trust (UK).

Newton PN, Tabernero P, Dwivedi P, Culzoni MJ, Monge ME, Swamidoss I, Mildenhall D, Green MD et al. 2014. Falsified medicines in Africa: all talk, no action. Lancet Glob Health, 2 (9), pp. e509-e510. | Read more

Takala-Harrison S, Jacob CG, Arze C, Cummings MP, Silva JC, Dondorp AM, Fukuda MM, Hien TT et al. 2015. Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia. J Infect Dis, 211 (5), pp. 670-679. | Show Abstract | Read more

BACKGROUND: The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia. METHODS: P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently. RESULTS: The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas. CONCLUSIONS: K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.

Lubell Y, White L, Varadan S, Drake T, Yeung S, Cheah PY, Maude RJ, Dondorp A, Day NP, White NJ, Parker M. 2014. Ethics, economics, and the use of primaquine to reduce falciparum malaria transmission in asymptomatic populations. PLoS Med, 11 (8), pp. e1001704. | Show Abstract | Read more

Yoel Lubell and colleagues consider ethical and economic perspectives on mass drug administration of primaquine to limit transmission of P. falciparum malaria. Please see later in the article for the Editors' Summary.

Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, Sreng S, Anderson JM et al. 2014. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med, 371 (5), pp. 411-423. | Show Abstract | Read more

BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. METHODS: Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. RESULTS: The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. CONCLUSIONS: Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; ClinicalTrials.gov number, NCT01350856.).

White NJ, Pukrittayakamee S, Phyo AP, Rueangweerayut R, Nosten F, Jittamala P, Jeeyapant A, Jain JP et al. 2014. Spiroindolone KAE609 for falciparum and vivax malaria. N Engl J Med, 371 (5), pp. 403-410. | Show Abstract | Read more

BACKGROUND: KAE609 (cipargamin; formerly NITD609, Novartis Institute for Tropical Diseases) is a new synthetic antimalarial spiroindolone analogue with potent, dose-dependent antimalarial activity against asexual and sexual stages of Plasmodium falciparum. METHODS: We conducted a phase 2, open-label study at three centers in Thailand to assess the antimalarial efficacy, safety, and adverse-event profile of KAE609, at a dose of 30 mg per day for 3 days, in two sequential cohorts of adults with uncomplicated P. vivax malaria (10 patients) or P. falciparum malaria (11). The primary end point was the parasite clearance time. RESULTS: The median parasite clearance time was 12 hours in each cohort (interquartile range, 8 to 16 hours in patients with P. vivax malaria and 10 to 16 hours in those with P. falciparum malaria). The median half-lives for parasite clearance were 0.95 hours (range, 0.68 to 2.01; interquartile range, 0.85 to 1.14) in the patients with P. vivax malaria and 0.90 hours (range, 0.68 to 1.64; interquartile range, 0.78 to 1.07) in those with P. falciparum malaria. By comparison, only 19 of 5076 patients with P. falciparum malaria (<1%) who were treated with oral artesunate in Southeast Asia had a parasite clearance half-life of less than 1 hour. Adverse events were reported in 14 patients (67%), with nausea being the most common. The adverse events were generally mild and did not lead to any discontinuations of the drug. The mean terminal half-life for the elimination of KAE609 was 20.8 hours (range, 11.3 to 37.6), supporting a once-daily oral dosing regimen. CONCLUSIONS: KAE609, at dose of 30 mg daily for 3 days, cleared parasitemia rapidly in adults with uncomplicated P. vivax or P. falciparum malaria. (Funded by Novartis and others; ClinicalTrials.gov number, NCT01524341.).

Mayxay M, Khanthavong M, Cox L, Sichanthongthip O, Imwong M, Pongvongsa T, Hongvanthong B, Phompida S, Vanisaveth V, White NJ, Newton PN. 2014. Thiamin supplementation does not reduce the frequency of adverse events after anti-malarial therapy among patients with falciparum malaria in southern Laos. Malar J, 13 (1), pp. 275. | Show Abstract | Read more

BACKGROUND: In a recent study one third of Lao patients presenting with uncomplicated Plasmodium falciparum malaria had biochemical evidence of thiamin deficiency, which was associated with a higher incidence of adverse events. Thiamin supplementation might, therefore, reduce adverse events in this population. METHODS: An exploratory, double-blind, parallel group, placebo-controlled, superiority trial of thiamin supplementation in patients of all ages with uncomplicated and severe falciparum malaria was conducted in Xepon District, Savannakhet Province, southern Laos. Patients were randomly assigned to either oral thiamin 10 mg/day for 7 days immediately after standard anti-malarial treatment then 5 mg daily until day 42, or identical oral placebo. RESULTS: After interim analyses when 630 patients (314 in thiamin and 316 in placebo groups) had been recruited, the trial was discontinued on the grounds of futility. On admission biochemical thiamin deficiency (alpha ≥ 25%) was present in 27% of patients and 9% had severe deficiency (alpha > 31%). After 42 days of treatment, the frequency of thiamin deficiency was lower in the thiamin (2%, 1% severe) compared to the placebo (11%, 3% severe) groups (p < 0.001 and p = 0.05), respectively. Except for diarrhoea, 7% in the placebo compared to 3% in the thiamin group (p = 0.04), and dizziness on day 1 (33% vs 25%, p = 0.045), all adverse events were not significantly different between the groups (p > 0.05). Clinical, haematological, and parasitological responses to treatment did not differ significantly between the two groups. CONCLUSION: Thiamin supplementation reduced biochemical thiamin deficiency among Lao malaria patients following anti-malarial drug treatment, but it did not reduce the frequency of adverse events after anti-malarial therapy or have any detected clinical or parasitological impact. TRIAL REGISTRATION: ISRCTN 85411059.

Onyamboko MA, Fanello CI, Wongsaen K, Tarning J, Cheah PY, Tshefu KA, Dondorp AM, Nosten F, White NJ, Day NP. 2014. Randomized comparison of the efficacies and tolerabilities of three artemisinin-based combination treatments for children with acute Plasmodium falciparum malaria in the Democratic Republic of the Congo. Antimicrob Agents Chemother, 58 (9), pp. 5528-5536. | Show Abstract | Read more

An open-label, randomized controlled trial was carried out in 2011-2012 in the Democratic Republic of the Congo to test the efficacy, safety, and tolerability of the artemisinin-based combination treatments dihydroartemisinin-piperaquine, amodiaquine-artesunate, and artemether-lumefantrine. Six hundred eighty-four children aged 3 to 59 months with uncomplicated Plasmodium falciparum malaria were randomly allocated to each study arm. Children were hospitalized for 3 days, given supervised treatment, and followed up weekly for 42 days. All regimens were well tolerated and rapidly effective. The median parasitemia clearance half-life was 2.2 h, and half-lives were similar between arms (P=0.19). The PCR-uncorrected cure rates by day 42 were 73.0% for amodiaquine-artesunate, 70.2% for artemether-lumefantrine, and 86.3% for dihydroartemisinin-piperaquine (P=0.001). Early treatment failure occurred in three patients (0.5%), one in each arm. The PCR-corrected cure rates were 93.4% for amodiaquine-artesunate, 92.7% for artemether-lumefantrine, and 94.3% for dihydroartemisinin-piperaquine (P=0.78). The last provided a longer posttreatment prophylactic effect than did the other two treatments. The day 7 plasma concentration of piperaquine was below 30 ng/ml in 47% of the children treated with dihydroartemisinin-piperaquine, and the day 7 lumefantrine concentration was below 280 ng/ml in 37.0% of children who received artemether-lumefantrine. Thus, although cure rates were all satisfactory, they could be improved by increasing the dose. (This study has been registered with the International Standard Randomized Controlled Trial Number Register [www.isrctn.org] under registration no. ISRCTN20984426.).

Imwong M, Hanchana S, Malleret B, Rénia L, Day NP, Dondorp A, Nosten F, Snounou G, White NJ. 2014. High-throughput ultrasensitive molecular techniques for quantifying low-density malaria parasitemias. J Clin Microbiol, 52 (9), pp. 3303-3309. | Show Abstract | Read more

The epidemiology of malaria in "low-transmission" areas has been underestimated. Molecular detection methods have revealed higher prevalences of malaria than conventional microscopy or rapid diagnostic tests, but these typically evaluate finger-prick capillary blood samples (∼5 μl) and therefore cannot detect parasite densities of <200/ml. Their use underestimates true parasite carriage rates. To characterize the epidemiology of malaria in low-transmission settings and plan elimination strategies, more sensitive quantitative PCR (qPCR) is needed to identify and quantify low-density malaria parasitemias. A highly sensitive "high-volume" quantitative PCR (qPCR) method based on Plasmodium sp. 18S RNA was adapted for blood sample volumes of ≥250 μl and scaled for high throughput. The methods were validated by assessment of the analytical sensitivity and specificity, diagnostic sensitivity, and specificity, efficiency, precision, analytical and diagnostic accuracies, limit of detection, root cause analysis of false positives, and robustness. The high-volume qPCR method based on Plasmodium sp. 18S RNA gave high PCR efficiency of 90 to 105%. Concentrations of parasite DNA from large volumes of blood gave a consistent analytical detection limit (LOD) of 22 parasites/ml (95% CI, 21.79 to 74.9), which is some 2,500 times more sensitive than conventional microscopy and 50 times more sensitive than currently used PCR methods from filter paper blood spots. The diagnostic specificity was 99.75%. Using automated procedures it was possible to process 700 blood samples per week. A very sensitive and specific high-throughput high-volume qPCR method for the detection of low-density parasitemias (>20 parasites/ml) was developed and validated.

Kloprogge F, Jullien V, Piola P, Dhorda M, Muwanga S, Nosten F, Day NP, White NJ, Guerin PJ, Tarning J. 2014. Population pharmacokinetics of quinine in pregnant women with uncomplicated Plasmodium falciparum malaria in Uganda. J Antimicrob Chemother, 69 (11), pp. 3033-3040. | Show Abstract | Read more

OBJECTIVES: Oral quinine is used for the treatment of uncomplicated malaria during pregnancy, but few pharmacokinetic data are available for this population. Previous studies have reported a substantial effect of malaria on the pharmacokinetics of quinine resulting from increased α-1-acid glycoprotein levels and decreased cytochrome P450 3A4 activity. The aim of this study was to investigate the pharmacokinetic properties of oral quinine in pregnant women with uncomplicated malaria in Uganda using a population approach. METHODS: Data from 22 women in the second and third trimesters of pregnancy with uncomplicated Plasmodium falciparum malaria were analysed. Patients received quinine sulphate (10 mg of salt/kg) three times daily (0, 8 and 16 h) for 7 days. Plasma samples were collected daily and at frequent intervals after the first and last doses. A population pharmacokinetic model for quinine was developed accounting for different disposition, absorption, error and covariate models. RESULTS: Parasitaemia, as a time-varying covariate affecting relative bioavailability, and body temperature on admission as a covariate on elimination clearance, explained the higher exposure to quinine during acute malaria compared with the convalescent phase. Neither the estimated gestational age nor the trimester influenced the pharmacokinetic properties of quinine significantly. CONCLUSIONS: A population model was developed that adequately characterized quinine pharmacokinetics in pregnant Ugandan women with acute malaria. Quinine exposure was lower than previously reported in patients who were not pregnant. The measurement of free quinine concentration will be necessary to determine the therapeutic relevance of these observations.

Maude RJ, Kingston HW, Joshi S, Mohanty S, Mishra SK, White NJ, Dondorp AM. 2014. Reversibility of retinal microvascular changes in severe falciparum malaria. Am J Trop Med Hyg, 91 (3), pp. 493-495. | Show Abstract | Read more

Malarial retinopathy allows detailed study of central nervous system vascular pathology in living patients with severe malaria. An adult with cerebral malaria is described who had prominent retinal whitening with corresponding retinal microvascular obstruction, vessel dilatation, increased vascular tortuosity, and blood retinal barrier leakage with decreased visual acuity, all of which resolved on recovery. Additional study of these features and their potential role in elucidating the pathogenesis of cerebral malaria is warranted.

Das D, Cheah PY, Akter F, Paul D, Islam A, Sayeed AA, Samad R, Rahman R et al. 2014. Participants' perceptions and understanding of a malaria clinical trial in Bangladesh Malaria Journal, 13 (1), | Show Abstract | Read more

Background: Existing evidence suggests that there is often limited understanding among participants in clinical trials about the informed consent process, resulting in their providing consent without really understanding the purpose of the study, specific procedures, and their rights. The objective of the study was to determine the subjects' understanding of research, perceptions of voluntariness and motivations for participation in a malaria clinical trial. Methods. In this study semi-structured interviews of adult clinical trial participants with uncomplicated falciparum malaria were conducted in Ramu Upazila Health Complex, in Bangladesh. Results: Of 16 participants, the vast majority (81%) were illiterate. All subjects had a 'therapeutic misconception' i.e. the trial was perceived to be conducted primarily for the benefit of individual patients when in fact the main objective was to provide information to inform public health policy. From the patients' perspective, getting well from their illness was their major concern. Poor actual understanding of trial specific procedures was reported despite participants' satisfaction with treatment and nursing care. Conclusion: There is frequently a degree of overlap between research and provision of clinical care in malaria research studies. Patients may be motivated to participate to research without a good understanding of the principal objectives of the study despite a lengthy consent process. The findings suggest that use of a standard consent form following the current ICH-GCP guidelines does not result in achieving fully informed consent and the process should be revised, simplified and adapted to individual trial settings. © 2014 Das et al.; licensee BioMed Central Ltd.

McGready R, Wongsaen K, Chu CS, Tun NW, Chotivanich K, White NJ, Nosten F. 2014. Uncomplicated Plasmodium vivax malaria in pregnancy associated with mortality from acute respiratory distress syndrome Malaria Journal, 13 (1), | Show Abstract | Read more

The association between severe malaria and Plasmodium vivax species is contentious. On the Thai-Myanmar border, all pregnant women are followed systematically with active weekly malaria screening. Over a 27-year period of providing antenatal care, 48,983 have been prospectively followed until pregnancy outcome (miscarriage or delivery) and 4,298 women have had P. vivax detected at least once. Reported here is the first known P. vivax-associated death amongst these women. The initial patient presentation was of uncomplicated P. vivax (0.5% parasitaemia) in a term, multigravida woman who responded rapidly to oral artesunate and mefloquine treatment, clearing her blood stage parasites within 48 hours. The patient appeared well, was ambulatory and due to be discharged but became unwell with acute respiratory distress syndrome (ARDS) requiring ventilation three days (67 hours) into treatment. Despite induction and delivery of a stillborn foetus, ventilatory requirements increased and the patient died on day 7. The patient had a low body mass index. Sensitive detection with nested PCR confirmed only the presence of P. vivax species and concomitant infections such as tuberculosis and human immunodeficiency virus (HIV) were also ruled out. The contemporaneous treatment of acute uncomplicated P. vivax and the onset of ARDS on day 3 in this patient implies a possible but unconfirmed association with death in this patient. Assuming this death was caused by P. vivax, the risk of ARDS-related maternal mortality in this setting did not differ significantly between Plasmodium falciparum and P. vivax (0.24 per 1,000 (1/4,158) versus 0.23 per 1,000 (1/4,298), contrary to the increased risk of maternal mortality from P. falciparum compared to P. vivax, 2.89 per 1,000 (12/4,158) versus 0.23 per 1,000 (1/4,298), P = 0.003. © 2014 McGready et al.; licensee BioMed Central Ltd.

McGready R, Wongsaen K, Chu CS, Tun NW, Chotivanich K, White NJ, Nosten F. 2014. Uncomplicated Plasmodium vivax malaria in pregnancy associated with mortality from acute respiratory distress syndrome. Malar J, 13 (1), pp. 191. | Show Abstract | Read more

The association between severe malaria and Plasmodium vivax species is contentious. On the Thai-Myanmar border, all pregnant women are followed systematically with active weekly malaria screening. Over a 27-year period of providing antenatal care, 48,983 have been prospectively followed until pregnancy outcome (miscarriage or delivery) and 4,298 women have had P. vivax detected at least once. Reported here is the first known P. vivax-associated death amongst these women. The initial patient presentation was of uncomplicated P. vivax (0.5% parasitaemia) in a term, multigravida woman who responded rapidly to oral artesunate and mefloquine treatment, clearing her blood stage parasites within 48 hours. The patient appeared well, was ambulatory and due to be discharged but became unwell with acute respiratory distress syndrome (ARDS) requiring ventilation three days (67 hours) into treatment. Despite induction and delivery of a stillborn foetus, ventilatory requirements increased and the patient died on day 7. The patient had a low body mass index. Sensitive detection with nested PCR confirmed only the presence of P. vivax species and concomitant infections such as tuberculosis and human immunodeficiency virus (HIV) were also ruled out. The contemporaneous treatment of acute uncomplicated P. vivax and the onset of ARDS on day 3 in this patient implies a possible but unconfirmed association with death in this patient. Assuming this death was caused by P. vivax, the risk of ARDS-related maternal mortality in this setting did not differ significantly between Plasmodium falciparum and P. vivax (0.24 per 1,000 (1/4,158) versus 0.23 per 1,000 (1/4,298), contrary to the increased risk of maternal mortality from P. falciparum compared to P. vivax, 2.89 per 1,000 (12/4,158) versus 0.23 per 1,000 (1/4,298), P = 0.003.

Lourens C, Lindegardh N, Barnes KI, Guerin PJ, Sibley CH, White NJ, Tarning J. 2014. Benefits of a pharmacology antimalarial reference standard and proficiency testing program provided by the Worldwide Antimalarial Resistance Network (WWARN). Antimicrob Agents Chemother, 58 (7), pp. 3889-3894. | Show Abstract | Read more

Comprehensive assessment of antimalarial drug resistance should include measurements of antimalarial blood or plasma concentrations in clinical trials and in individual assessments of treatment failure so that true resistance can be differentiated from inadequate drug exposure. Pharmacometric modeling is necessary to assess pharmacokinetic-pharmacodynamic relationships in different populations to optimize dosing. To accomplish both effectively and to allow comparison of data from different laboratories, it is essential that drug concentration measurement is accurate. Proficiency testing (PT) of laboratory procedures is necessary for verification of assay results. Within the Worldwide Antimalarial Resistance Network (WWARN), the goal of the quality assurance/quality control (QA/QC) program is to facilitate and sustain high-quality antimalarial assays. The QA/QC program consists of an international PT program for pharmacology laboratories and a reference material (RM) program for the provision of antimalarial drug standards, metabolites, and internal standards for laboratory use. The RM program currently distributes accurately weighed quantities of antimalarial drug standards, metabolites, and internal standards to 44 pharmacology, in vitro, and drug quality testing laboratories. The pharmacology PT program has sent samples to eight laboratories in four rounds of testing. WWARN technical experts have provided advice for correcting identified problems to improve performance of subsequent analysis and ultimately improved the quality of data. Many participants have demonstrated substantial improvements over subsequent rounds of PT. The WWARN QA/QC program has improved the quality and value of antimalarial drug measurement in laboratories globally. It is a model that has potential to be applied to strengthening laboratories more widely and improving the therapeutics of other infectious diseases.

White NJ. 2014. Malaria: a molecular marker of artemisinin resistance. Lancet, 383 (9927), pp. 1439-1440. | Read more

Gaubert A, Kauss T, Marchivie M, Ba BB, Lembege M, Fawaz F, Boiron JM, Lafarge X et al. 2014. Preliminary pharmaceutical development of antimalarial-antibiotic cotherapy as a pre-referral paediatric treatment of fever in malaria endemic areas. Int J Pharm, 468 (1-2), pp. 55-63. | Show Abstract | Read more

Artemether (AM) plus azithromycin (AZ) rectal co-formulations were studied to provide pre-referral treatment for children with severe febrile illnesses in malaria-endemic areas. The target profile required that such product should be cheap, easy to administer by non-medically qualified persons, rapidly effective against both malaria and bacterial infections. Analytical and pharmacotechnical development, followed by in vitro and in vivo evaluation, were conducted for various AMAZ coformulations. Of the formulations tested, stability was highest for dry solid forms and bioavailability for hard gelatin capsules; AM release from AMAZ rectodispersible tablet was suboptimal due to a modification of its micro-crystalline structure.

Pukrittayakamee S, Tarning J, Jittamala P, Charunwatthana P, Lawpoolsri S, Lee SJ, Hanpithakpong W, Hanboonkunupakarn B, Day NP, Ashley EA, White NJ. 2014. Pharmacokinetic interactions between primaquine and chloroquine. Antimicrob Agents Chemother, 58 (6), pp. 3354-3359. | Show Abstract | Read more

Chloroquine combined with primaquine has been the standard radical curative regimen for Plasmodium vivax and Plasmodium ovale malaria for over half a century. In an open-label crossover pharmacokinetic study, 16 healthy volunteers (4 males and 12 females) aged 20 to 47 years were randomized into two groups of three sequential hospital admissions to receive a single oral dose of 30 mg (base) primaquine, 600 mg (base) chloroquine, and the two drugs together. The coadministration of the two drugs did not affect chloroquine or desethylchloroquine pharmacokinetics but increased plasma primaquine concentrations significantly (P ≤ 0.005); the geometric mean (90% confidence interval [CI]) increases were 63% (47 to 81%) in maximum concentration and 24% (13 to 35%) in total exposure. There were also corresponding increases in plasma carboxyprimaquine concentrations (P ≤ 0.020). There were no significant electrocardiographic changes following primaquine administration, but there was slight corrected QT (QTc) (Fridericia) interval lengthening following chloroquine administration (median [range] = 6.32 [-1.45 to 12.3] ms; P < 0.001), which was not affected by the addition of primaquine (5.58 [1.74 to 11.4] ms; P = 0.642). This pharmacokinetic interaction may explain previous observations of synergy in preventing P. vivax relapse. This trial was registered at ClinicalTrials.gov under reference number NCT01218932.

Chotivanich K, Tripura R, Das D, Yi P, Day NP, Pukrittayakamee S, Chuor CM, Socheat D, Dondorp AM, White NJ. 2014. Laboratory detection of artemisinin-resistant Plasmodium falciparum. Antimicrob Agents Chemother, 58 (6), pp. 3157-3161. | Show Abstract | Read more

Conventional 48-h in vitro susceptibility tests have low sensitivity in identifying artemisinin-resistant Plasmodium falciparum, defined phenotypically by low in vivo parasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistant P. falciparum is prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P = 0.001). TMI IC50s correlated significantly with the in vivo responses to artesunate (parasite clearance time [r = 0.44, P = 0.001] and parasite clearance half-life [r = 0.46, P = 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility.

Jaroensuk J, Stoesser N, Leimanis ML, Jittamala P, White NJ, Nosten FH, McGready R. 2014. Treatment of suspected hyper-reactive malarial splenomegaly (HMS) in pregnancy with mefloquine. Am J Trop Med Hyg, 90 (4), pp. 609-611. | Show Abstract | Read more

Malaria infections in pregnancy are associated with adverse outcomes for both mother and child. There are few data on hyper-reactive malarial splenomegaly, an aberrant immunological response to chronic or recurrent malaria in pregnancy. This retrospective assessment reviewed the impact of mefloquine treatment on pregnant women with suspected hyper-reactive malarial splenomegaly in an area of low malaria transmission in the 1990s, showing significant reductions in spleen size and anemia and anti-malarial antibody titers without any notable negative effect on treated women or their newborns.

Jittamala P, Pukrittayakamee S, Tarning J, Lindegardh N, Hanpithakpong W, Taylor WR, Lawpoolsri S, Charunwattana P, Panapipat S, White NJ, Day NP. 2014. Pharmacokinetics of orally administered oseltamivir in healthy obese and nonobese Thai subjects. Antimicrob Agents Chemother, 58 (3), pp. 1615-1621. | Show Abstract | Read more

Oseltamivir is the most widely used anti-influenza drug. In the 2009 H1N1 pandemic, in which the influenza viruses were oseltamivir sensitive, obesity was identified as a risk factor for severe disease and unfavorable outcomes. The aim of this study was to investigate the pharmacokinetic properties of oseltamivir and its active metabolite, oseltamivir carboxylate, in obese and nonobese healthy subjects. A single-dose, randomized, two-sequence crossover study was conducted in 12 obese and 12 nonobese healthy Thai volunteers. Each volunteer was given 75 mg and 150 mg oseltamivir orally with an intervening washout period of more than 3 days. The pharmacokinetic properties of oseltamivir and oseltamivir carboxylate were evaluated using a noncompartmental approach. The median (range) body mass indexes (BMIs) for obese subjects were 33.8 kg/m(2) (30.8 to 43.2) and 22.2 (18.8 to 24.2) for nonobese subjects. The pharmacokinetic parameters of oseltamivir carboxylate, the active metabolite of oseltamivir, were not significantly different between obese and nonobese subjects for both 75-mg and 150-mg doses. Both doses were well tolerated. Despite the lower dose per kilogram body weight in obese subjects, there was no significant difference in the exposure of oseltamivir carboxylate between the obese and nonobese groups. Standard dosing is appropriate for obese subjects. (The study was registered at ClinicalTrials.gov under registration no. NCT 01049763.).

Maung Lwin K, Cheah PY, Cheah PK, White NJ, Day NPJ, Nosten F, Parker M. 2014. Motivations and perceptions of community advisory boards in the ethics of medical research: The case of the Thai-Myanmar border BMC Medical Ethics, 15 (1), | Show Abstract | Read more

Background: Community engagement is increasingly promoted as a marker of good, ethical practice in the context of international collaborative research in low-income countries. There is, however, no widely agreed definition of community engagement or of approaches adopted. Justifications given for its use also vary. Community engagement is, for example, variously seen to be of value in: the development of more effective and appropriate consent processes; improved understanding of the aims and forms of research; higher recruitment rates; the identification of important ethical issues; the building of better relationships between the community and researchers; the obtaining of community permission to approach potential research participants; and, the provision of better health care. Despite these diverse and potentially competing claims made for the importance of community engagement, there is very little published evidence on effective models of engagement or their evaluation. Methods. In this paper, drawing upon interviews with the members of a Community Advisory Board on the Thai-Myanmar border, we describe and critically reflect upon an approach to community engagement which was developed in the context of international collaborative research in the border region. Results and conclusions. Drawing on our analysis, we identify a number of considerations relevant to the development of an approach to evaluating community engagement in this complex research setting. The paper also identifies a range of important ways in which the Community Advisory Board is in practice understood by its members (and perhaps by community members beyond this) to have morally significant roles and responsibilities beyond those usually associated with the successful and appropriate conduct of research. © 2014 Maung Lwin et al.; licensee BioMed Central Ltd.

Kondrashin A, Baranova AM, Ashley EA, Recht J, White NJ, Sergiev VP. 2014. Mass primaquine treatment to eliminate vivax malaria: Lessons from the past Malaria Journal, 13 (1), | Show Abstract | Read more

Recent successes in malaria control have put malaria eradication back on the public health agenda. A significant obstacle to malaria elimination in Asia is the large burden of Plasmodium vivax, which is more difficult to eliminate than Plasmodium falciparum. Persistent P. vivax liver stages can be eliminated only by radical treatment with a ≥ seven-day course of an 8-aminoquinoline, with the attendant risk of acute haemolytic anaemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Primaquine is the only generally available 8-aminoquinoline. Testing for G6PD deficiency is not widely available, and so whilst it is widely recommended, primaquine is often not prescribed. In the past, some countries aiming for vivax malaria eradication deployed mass treatments with primaquine on a massive scale, without G6PD testing. In Azerbaijan, Tajikistan (formerly USSR), North Afghanistan and DPR Korea 8,270,185 people received either a 14-day "standard" or a 17-day "interrupted" primaquine treatment to control post-eradication malaria epidemics. These mass primaquine preventive treatment campaigns were conducted by dedicated teams who administered the drugs under supervision and then monitored the population for adverse events. Despite estimated G6PD prevalences up to 38.7%, the reported frequency of severe adverse events related to primaquine was very low. This experience shows that with careful planning and implementation of mass treatment strategies using primaquine and adequate medical support to manage haemolytic toxicity, it is possible to achieve high population coverage, substantially reduce malaria transmission, and manage the risk of severe acute haemolytic anaemia in communities with a relatively high prevalence of G6PD deficiency safely. © 2014 Kondrashin et al.; licensee BioMed Central Ltd.

Vermeersch A, Libaud-Moal A, Rodrigues A, White NJ, Olliaro P, Gomes M, Ashley EA, Millet P. 2014. Introducing the concept of a new pre-referral treatment for severely ill febrile children at community level: a sociological approach in Guinea-Bissau. Malar J, 13 (1), pp. 50. | Show Abstract | Read more

BACKGROUND: Innovative strategies are needed to tackle childhood mortality in the rural tropics. Artesunate suppositories were developed to bring emergency treatment closer to severely ill children with malaria in rural areas where injectable treatment is not possible for several hours. Adding an antibacterial rectal drug would extend this strategy to treat non-malarial febrile illness as well. The objective of these studies was to assess acceptability of such a new pre-referral strategy by healthcare providers and likely uptake by the population. METHODS: Two qualitative studies were conducted between May and July 2009. Study 1 investigated the acceptability of introducing a combined antimalarial-antibacterial suppository by interviewing 27 representatives of the three administrative levels (central government, regional, local) of the health sector; Study 2 investigated treatment-seeking behaviour and acceptability of this intervention at community level by interviewing 74 mothers in 2 villages. RESULTS AND CONCLUSIONS: Up to 92% of health representatives were in favour of introducing a new pre-referral strategy to tackle both malaria and non-malaria related severe illnesses in Guinea-Bissau, provided it was endorsed by international health authorities. The main obstacles to implementation were the very limited human and financial resources. In the two villages surveyed, 44% of the mothers associated severe illness with fever only, or fever plus one additional symptom. Mothers' judgement of severity and ensuing decisions were not specific for serious illness, indicating that initial training to recognize signs of severe disease and treatment availability for non-severe, fever-associated symptoms will be required to prevent overuse of a new intervention designed as a pre-referral treatment for severely ill children. Level C health centres were the first resort in both villages (50% and 87% of respondents respectively). This information is encouraging for the implementation of a pre-referral treatment.

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Eziefula AC, Bousema T, Yeung S, Kamya M, Owaraganise A, Gabagaya G, Bradley J, Grignard L et al. 2014. Single dose primaquine for clearance of Plasmodium falaparum gametocytes in children with uncomplicated malaria in Uganda: a randomised, controlled, double-blind, dose-ranging trial LANCET INFECTIOUS DISEASES, 14 (2), pp. 130-139. | Read more

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Eziefula AC, Bousema T, Yeung S, Kamya M, Owaraganise A, Gabagaya G, Bradley J, Grignard L et al. 2014. Single dose primaquine for clearance of Plasmodium falciparum gametocytes in children with uncomplicated malaria in Uganda: A randomised, controlled, double-blind, dose-ranging trial The Lancet Infectious Diseases, 14 (2), pp. 130-139. | Show Abstract | Read more

Background: Primaquine is the only available drug that clears mature Plasmodium falciparum gametocytes in infected human hosts, thereby preventing transmission of malaria to mosquitoes. However, concerns about dose-dependent haemolysis in people with glucose-6-phosphate dehydrogenase (G6PD) deficiencies have limited its use. We assessed the dose-response association of single-dose primaquine for gametocyte clearance and for safety in P falciparum malaria. Methods: We undertook this randomised, double-blind, placebo-controlled trial with four parallel groups in Jinja district, eastern Uganda. We randomly allocated Ugandan children aged 1-10 years with uncomplicated falciparum malaria and normal G6PD enzyme function to receive artemether-lumefantrine, combined with either placebo or with 0·1 mg/kg, 0·4 mg/kg, or 0·75 mg/kg (WHO reference dose) primaquine base. Randomisation was done with computer-generated four-digit treatment assignment codes allocated to random dose groups in block sizes of 16. Study staff who provided care or assessed outcomes and the participants remained masked to the intervention group after assignment. The primary efficacy endpoint was the non-inferiority of the mean duration of gametocyte carriage in the test doses compared with the reference group of 0·75 mg primaquine per kg, with a non-inferiority margin of 2·5 days. The primary safety endpoint was the superiority of the arithmetic mean maximum decrease in haemoglobin concentration from enrolment to day 28 of follow-up in the primaquine treatment groups compared with placebo, with use of significance testing of pairwise comparisons with a cutoff of p=0·05. The trial is registered with ClinicalTrials.gov, number NCT01365598. Findings: We randomly allocated 468 participants to receive artemether-lumefantrine combined with placebo (119 children) or with 0·1 mg/kg (116), 0·4 mg/kg (116), or 0·75 mg/kg (117) primaquine base. The mean duration of gametocyte carriage was 6·6 days (95% CI 5·3-7·8) in the 0·75 mg/kg reference group, 6·3 days (5·1-7·5) in the 0·4 mg/kg primaquine group (p=0·74), 8·0 days (6·6-9·4) in the 0·1 mg/kg primaquine group (p=0·14), and 12·4 days (9·9-15·0) in the placebo group (p<0·0001). No children showed evidence of treatment-related haemolysis, and the mean maximum decrease in haemoglobin concentration was not associated with the dose of primaquine received-it did not differ significantly compared with placebo (10·7 g/L, SD 11·1) in the 0·1 mg/kg (11·4 g/L, 9·4; p=0·61), 0·4 mg/kg (11·3 g/L, 10·0; p=0·67), or 0·75 mg/kg (12·7 g/L, 8·2; p=0·11) primaquine groups. Interpretation: We conclude that 0·4 mg/kg primaquine has similar gametocytocidal efficacy to the reference 0·75 mg/kg primaquine dose, but a dose of 0·1 mg/kg was inconclusive for non-inferiority. Our findings call for the prioritisation of further trials into the efficacy and safety of doses of primaquine between 0·1 mg/kg and 0·4 mg/kg (including the dose of 0·25 mg/kg recently recommended by WHO), in view of the potential for widespread use of the drug to block malaria transmission. Funding: Wellcome Trust and the Bill & Melinda Gates Foundation. © 2014 Eziefula et al. Open Access article distributed under the terms of CC BY-NC-ND.

Tarning J, Lindegardh N, Lwin KM, Annerberg A, Kiricharoen L, Ashley E, White NJ, Nosten F, Day NP. 2014. Population pharmacokinetic assessment of the effect of food on piperaquine bioavailability in patients with uncomplicated malaria. Antimicrob Agents Chemother, 58 (4), pp. 2052-2058. | Show Abstract | Read more

Previously published literature reports various impacts of food on the oral bioavailability of piperaquine. The aim of this study was to use a population modeling approach to investigate the impact of concomitant intake of a small amount of food on piperaquine pharmacokinetics. This was an open, randomized comparison of piperaquine pharmacokinetics when administered as a fixed oral formulation once daily for 3 days with (n=15) and without (n=15) concomitant food to patients with uncomplicated Plasmodium falciparum malaria in Thailand. Nonlinear mixed-effects modeling was used to characterize the pharmacokinetics of piperaquine and the influence of concomitant food intake. A modified Monte Carlo mapped power approach was applied to evaluate the relationship between statistical power and various degrees of covariate effect sizes of the given study design. Piperaquine population pharmacokinetics were described well in fasting and fed patients by a three-compartment distribution model with flexible absorption. The final model showed a 25% increase in relative bioavailability per dose occasion during recovery from malaria but demonstrated no clinical impact of concomitant intake of a low-fat meal. Body weight and age were both significant covariates in the final model. The novel power approach concluded that the study was adequately powered to detect a food effect of at least 35%. This modified Monte Carlo mapped power approach may be a useful tool for evaluating the power to detect true covariate effects in mixed-effects modeling and a given study design. A small amount of food does not affect piperaquine absorption significantly in acute malaria.

Beaudry JT, Krause MA, Diakite SA, Fay MP, Joshi G, Diakite M, White NJ, Fairhurst RM. 2014. Ex-vivo cytoadherence phenotypes of Plasmodium falciparum strains from Malian children with hemoglobins A, S, and C. PLoS One, 9 (3), pp. e92185. | Show Abstract | Read more

Sickle hemoglobin (Hb) S and HbC may protect against malaria by reducing the expression of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of parasitized red blood cells (RBCs), thereby weakening their cytoadherence to microvascular endothelial cells (MVECs) and impairing their activation of MVECs to produce pathological responses. Therefore, we hypothesized that parasites causing malaria in HbAS or HbAC heterozygotes have overcome this protective mechanism by expressing PfEMP1 variants which mediate relatively strong binding to MVECs. To test this hypothesis, we performed 31 cytoadherence comparisons between parasites from HbAA and HbAS (or HbAC) Malian children with malaria. Ring-stage parasites from HbAA and HbAS (or HbAC) children were cultivated to trophozoites, purified, and then inoculated in parallel into the same wildtype uninfected RBCs. After one cycle of invasion and maturation to the trophozoite stage expressing PfEMP1, parasite strains were compared for binding to MVECs. In this assay, there were no significant differences in the binding of parasites from HbAS and HbAC children to MVECs compared to those from HbAA children (HbAS, fold-change  = 1.46, 95% CI 0.97-2.19, p = 0.07; HbAC, fold-change  = 1.19, 95% CI 0.77-1.84, p = 0.43). These data suggest that in-vitro reductions in cytoadherence by HbS and HbC may not be selecting for expression of high-avidity PfEMP1 variants in vivo. Future studies that identify PfEMP1 domains or amino-acid motifs which are selectively expressed in parasites from HbAS children may provide further insights into the mechanism of malaria protection by the sickle-cell trait.

Tanomsing N, Mayxay M, Newton PN, Nosten F, Dolecek C, Hien TT, White NJ, Day NP, Dondorp AM, Imwong M. 2014. Genetic variability of Plasmodium malariae dihydropteroate synthase (dhps) in four Asian countries. PLoS One, 9 (4), pp. e93942. | Show Abstract | Read more

The dihydropteroate synthase (dhps) genes of 44 P. malariae strains from four Asian countries were isolated. Only a limited number of polymorphisms were observed. Comparison with homologous mutations in other Plasmodium species showed that these polymorphisms are unlikely to be associated with sulfadoxine resistance.

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Scopus

White NJ, Pukrittayakamee S, Hien TT, Faiz MA, Mokuolu OA, Dondorp AM. 2014. Malaria The Lancet, 383 (9918), pp. 723-735. | Show Abstract | Read more

Although global morbidity and mortality have decreased substantially, malaria, a parasite infection of red blood cells, still kills roughly 2000 people per day, most of whom are children in Africa. Two factors largely account for these decreases; increased deployment of insecticide-treated bednets and increased availability of highly effective artemisinin combination treatments. In large trials, parenteral artesunate (an artemisinin derivative) reduced severe malaria mortality by 22·5% in Africa and 34·7% in Asia compared with quinine, whereas adjunctive interventions have been uniformly unsuccessful. Rapid tests have been an important addition to microscopy for malaria diagnosis. Chemopreventive strategies have been increasingly deployed in Africa, notably intermittent sulfadoxine-pyrimethamine treatment in pregnancy, and monthly amodiaquine-sulfadoxine-pyrimethamine during the rainy season months in children aged between 3 months and 5 years across the sub-Sahel. Enthusiasm for malaria elimination has resurfaced. This ambitious but laudable goal faces many challenges, including the worldwide economic downturn, difficulties in elimination of vivax malaria, development of pyrethroid resistance in some anopheline mosquitoes, and the emergence of artemisinin resistance in Plasmodium falciparum in southeast Asia. We review the epidemiology, clinical features, pathology, prevention, and treatment of malaria.

Kondrashin A, Baranova AM, Ashley EA, Recht J, White NJ, Sergiev VP. 2014. Mass primaquine treatment to eliminate vivax malaria: lessons from the past. Malar J, 13 (1), pp. 51. | Show Abstract | Read more

Recent successes in malaria control have put malaria eradication back on the public health agenda. A significant obstacle to malaria elimination in Asia is the large burden of Plasmodium vivax, which is more difficult to eliminate than Plasmodium falciparum. Persistent P. vivax liver stages can be eliminated only by radical treatment with a ≥ seven-day course of an 8-aminoquinoline, with the attendant risk of acute haemolytic anaemia in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Primaquine is the only generally available 8-aminoquinoline. Testing for G6PD deficiency is not widely available, and so whilst it is widely recommended, primaquine is often not prescribed. In the past, some countries aiming for vivax malaria eradication deployed mass treatments with primaquine on a massive scale, without G6PD testing. In Azerbaijan, Tajikistan (formerly USSR), North Afghanistan and DPR Korea 8,270,185 people received either a 14-day "standard" or a 17-day "interrupted" primaquine treatment to control post-eradication malaria epidemics. These mass primaquine preventive treatment campaigns were conducted by dedicated teams who administered the drugs under supervision and then monitored the population for adverse events. Despite estimated G6PD prevalences up to 38.7%, the reported frequency of severe adverse events related to primaquine was very low. This experience shows that with careful planning and implementation of mass treatment strategies using primaquine and adequate medical support to manage haemolytic toxicity, it is possible to achieve high population coverage, substantially reduce malaria transmission, and manage the risk of severe acute haemolytic anaemia in communities with a relatively high prevalence of G6PD deficiency safely.

Jamornthanyawat N, Awab GR, Tanomsing N, Pukrittayakamee S, Yamin F, Dondorp AM, Day NP, White NJ, Woodrow CJ, Imwong M. 2014. A population survey of the glucose-6-phosphate dehydrogenase (G6PD) 563C>T (Mediterranean) mutation in Afghanistan. PLoS One, 9 (2), pp. e88605. | Show Abstract | Read more

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzyme defect and an important problem in areas with Plasmodium vivax infection because of the risk of haemolysis following administration of primaquine to treat the liver forms of the parasite. We undertook a genotypic survey of 713 male individuals across nine provinces of Afghanistan in which malaria is found, four in the north and five in the east. RFLP typing at nucleotide position 563 detected 40 individuals with the Mediterranean mutation 563C>T, an overall prevalence of 5.6%. This varied according to self-reported ethnicity, with prevalence in the Pashtun/Pashai group of 33/369 (8.9%) compared to 7/344 individuals in the rest of the population (2.0%; p<0.001, Chi-squared test). Multivariate analysis of ethnicity and geographical location indicated an adjusted odds ratio of 3.50 (95% CI 1.36-9.02) for the Pashtun/Pashai group, while location showed only a trend towards higher prevalence in eastern provinces (adjusted odds ratio = 1.73, 0.73-4.13). Testing of known polymorphic markers (1311C>T in exon 11, and C93T in intron XI) in a subset of 82 individuals wild-type at C563 revealed a mixture of 3 haplotypes in the background population and was consistent with data from the 1000 Genomes Project and published studies. By comparison individuals with G6PD deficiency showed a highly skewed haplotype distribution, with 95% showing the CT haplotype, a finding consistent with relatively recent appearance and positive selection of the Mediterranean variant in Afghanistan. Overall, the data confirm that the Mediterranean variant of G6PD is common in many ethnic groups in Afghanistan, indicating that screening for G6PD deficiency is required in all individuals before radical treatment of P. vivax with primaquine.

Maung Lwin K, Cheah PY, Cheah PK, White NJ, Day NP, Nosten F, Parker M. 2014. Motivations and perceptions of community advisory boards in the ethics of medical research: the case of the Thai-Myanmar border. BMC Med Ethics, 15 (1), pp. 12. | Show Abstract | Read more

BACKGROUND: Community engagement is increasingly promoted as a marker of good, ethical practice in the context of international collaborative research in low-income countries. There is, however, no widely agreed definition of community engagement or of approaches adopted. Justifications given for its use also vary. Community engagement is, for example, variously seen to be of value in: the development of more effective and appropriate consent processes; improved understanding of the aims and forms of research; higher recruitment rates; the identification of important ethical issues; the building of better relationships between the community and researchers; the obtaining of community permission to approach potential research participants; and, the provision of better health care. Despite these diverse and potentially competing claims made for the importance of community engagement, there is very little published evidence on effective models of engagement or their evaluation. METHODS: In this paper, drawing upon interviews with the members of a Community Advisory Board on the Thai-Myanmar border, we describe and critically reflect upon an approach to community engagement which was developed in the context of international collaborative research in the border region. RESULTS AND CONCLUSIONS: Drawing on our analysis, we identify a number of considerations relevant to the development of an approach to evaluating community engagement in this complex research setting. The paper also identifies a range of important ways in which the Community Advisory Board is in practice understood by its members (and perhaps by community members beyond this) to have morally significant roles and responsibilities beyond those usually associated with the successful and appropriate conduct of research.

Hanson J, Lee SJ, Mohanty S, Faiz MA, Anstey NM, Price RN, Charunwatthana P, Yunus EB et al. 2014. Rapid clinical assessment to facilitate the triage of adults with falciparum malaria, a retrospective analysis. PLoS One, 9 (1), pp. e87020. | Show Abstract | Read more

BACKGROUND: Most adults dying from falciparum malaria will die within 48 hours of their hospitalisation. An essential component of early supportive care is the rapid identification of patients at greatest risk. In resource-poor settings, where most patients with falciparum malaria are managed, decisions regarding patient care must frequently be made using clinical evaluation alone. METHODS: We retrospectively analysed 4 studies of 1801 adults with severe falciparum malaria to determine whether the presence of simple clinical findings might assist patient triage. RESULTS: If present on admission, shock, oligo-anuria, hypo- or hyperglycaemia, an increased respiratory rate, a decreased Glasgow Coma Score and an absence of fever were independently predictive of death. The variables were used to construct a simple clinical algorithm. When applied to the 1801 patients, this algorithm's positive predictive value for survival to 48 hours was 99.4 (95% confidence interval (CI) 97.8-99.9) and for survival to discharge 96.9% (95% CI 94.3-98.5). In the 712 patients receiving artesunate, the algorithm's positive predictive value for survival to 48 hours was 100% (95% CI 97.3-100) and to discharge was 98.5% (95% CI 94.8-99.8). CONCLUSIONS: Simple clinical findings are closely linked to the pathophysiology of severe falciparum malaria in adults. A basic algorithm employing these indices can facilitate the triage of patients in settings where intensive care services are limited. Patients classified as low-risk by this algorithm can be safely managed initially on a general ward whilst awaiting senior clinical review and laboratory data.

Rahimi BA, Thakkinstian A, White NJ, Sirivichayakul C, Dondorp AM, Chokejindachai W. 2014. Severe vivax malaria: a systematic review and meta-analysis of clinical studies since 1900. Malar J, 13 (1), pp. 481. | Show Abstract | Read more

BACKGROUND: Malaria caused by Plasmodium vivax was long considered to have a low mortality, but recent reports from some geographical areas suggest that severe and complicated vivax malaria may be more common than previously thought. METHODS: The primary objective of this systematic review and meta-analysis was to describe the reported clinical characteristics and the geographical variation in prevalence of reported severe vivax malaria and its change over time derived from English-language articles published since 1900. Medline and Scopus databases were searched for original papers on severe vivax malaria, using as inclusion criteria modified 2010 WHO criteria for the diagnosis of severe falciparum malaria. Articles before 1949 were identified through reference lists in journals, textbooks, and personal collections of colleagues. RESULTS: A total of 77 studies with reported severe vivax malaria and 63 studies with no reported severe vivax malaria (totaling 46,411 and 6,753 vivax malaria patients, respectively) were included. The 77 studies with reported severe vivax malaria were mainly from India (n = 33), USA (n = 8), Indonesia (n = 6), and Pakistan (n = 6). Vivax endemic countries not reporting severe vivax malaria beyond individual case reports included: the Greater Mekong Sub-region, China, North Korea, Bangladesh, Afghanistan, Middle East (except Qatar), the horn of Africa, and Madagascar. Only 17/77 reports were from before 2000. Vivax mono-infection was confirmed by PCR in 14 studies and co-morbidities were ruled out in 23 studies. Among the 77 studies reporting severe vivax malaria, severe thrombocytopenia (<50,000/mm3) was the most common "severe" manifestation (888/45,775 with pooled prevalence of 8.6%). The case fatality was 0.3% (353/46,411). Severity syndromes varied widely between different geographical areas, with severe anaemia being most prominent in areas of high transmission and chloroquine resistance. CONCLUSION: Plasmodium vivax can cause severe and even fatal disease, but there is a recent increase in reports over the past 15 years with larger series restricted to a limited number of geographical areas. The biological basis of these variations is currently not known. More detailed epidemiological studies are needed which dissociate causation from association to refine the definition and estimate the prevalence of severe vivax malaria.

Ashley EA, White NJ. 2014. The duration of Plasmodium falciparum infections. Malar J, 13 (1), pp. 500. | Show Abstract | Read more

Plasmodium vivax and Plasmodium ovale are often considered the malaria parasites best adapted to long-term survival in the human host because of their latent exo-erythrocytic forms. The prevailing opinion until the middle of the last century was that the maximum duration of Plasmodium falciparum infections was less than two years. Case reports and series investigating blood donors following accidental malaria infection of blood transfusion recipients and other sporadic malaria cases in non-endemic countries have shown clearly that asymptomatic P. falciparum infections may persist for up to a decade or longer (maximum confirmed 13 years). Current policies in malaria-free countries of excluding blood donors who have lived in malarious areas are justified. Vigilance for longer than three years after declaring elimination in an area may be needed.

Bancone G, Chu CS, Somsakchaicharoen R, Chowwiwat N, Parker DM, Charunwatthana P, White NJ, Nosten FH. 2014. Characterization of G6PD genotypes and phenotypes on the northwestern Thailand-Myanmar border. PLoS One, 9 (12), pp. e116063. | Show Abstract | Read more

Mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene result in red blood cells with increased susceptibility to oxidative damage. Significant haemolysis can be caused by primaquine and other 8-aminoquinoline antimalarials used for the radical treatment of Plasmodium vivax malaria. The distribution and phenotypes of mutations causing G6PD deficiency in the male population of migrants and refugees in a malaria endemic region on the Thailand-Myanmar border were characterized. Blood samples for G6PD fluorescent spot test (FST), G6PD genotyping, and malaria testing were taken from 504 unrelated males of Karen and Burman ethnicities presenting to the outpatient clinics. The overall frequency of G6PD deficiency by the FST was 13.7%. Among the deficient subjects, almost 90% had the Mahidol variant (487G>A) genotype. The remaining subjects had Chinese-4 (392G>T), Viangchan (871G>A), Açores (595A>G), Seattle (844G>C) and Mediterranean (563C>T) variants. Quantification of G6PD activity was performed using a modification of the standard spectrophotometric assay on a subset of 24 samples with Mahidol, Viangchan, Seattle and Chinese-4 mutations; all samples showed a residual enzymatic activity below 10% of normal and were diagnosed correctly by the FST. Further studies are needed to characterise the haemolytic risk of using 8-aminoquinolines in patients with these genotypes.

Lubell Y, Dondorp A, Guérin PJ, Drake T, Meek S, Ashley E, Day NP, White NJ, White LJ. 2014. Artemisinin resistance--modelling the potential human and economic costs. Malar J, 13 (1), pp. 452. | Show Abstract | Read more

BACKGROUND: Artemisinin combination therapy is recommended as first-line treatment for falciparum malaria across the endemic world and is increasingly relied upon for treating vivax malaria where chloroquine is failing. Artemisinin resistance was first detected in western Cambodia in 2007, and is now confirmed in the Greater Mekong region, raising the spectre of a malaria resurgence that could undo a decade of progress in control, and threaten the feasibility of elimination. The magnitude of this threat has not been quantified. METHODS: This analysis compares the health and economic consequences of two future scenarios occurring once artemisinin-based treatments are available with high coverage. In the first scenario, artemisinin combination therapy (ACT) is largely effective in the management of uncomplicated malaria and severe malaria is treated with artesunate, while in the second scenario ACT are failing at a rate of 30%, and treatment of severe malaria reverts to quinine. The model is applied to all malaria-endemic countries using their specific estimates for malaria incidence, transmission intensity and GDP. The model describes the direct medical costs for repeated diagnosis and retreatment of clinical failures as well as admission costs for severe malaria. For productivity losses, the conservative friction costing method is used, which assumes a limited economic impact for individuals that are no longer economically active until they are replaced from the unemployment pool. RESULTS: Using conservative assumptions and parameter estimates, the model projects an excess of 116,000 deaths annually in the scenario of widespread artemisinin resistance. The predicted medical costs for retreatment of clinical failures and for management of severe malaria exceed US$32 million per year. Productivity losses resulting from excess morbidity and mortality were estimated at US$385 million for each year during which failing ACT remained in use as first-line treatment. CONCLUSIONS: These 'ballpark' figures for the magnitude of the health and economic threat posed by artemisinin resistance add weight to the call for urgent action to detect the emergence of resistance as early as possible and contain its spread from known locations in the Mekong region to elsewhere in the endemic world.

Ashley EA, Recht J, White NJ. 2014. Primaquine: the risks and the benefits. Malar J, 13 (1), pp. 418. | Show Abstract | Read more

Primaquine is the only generally available anti-malarial that prevents relapse in vivax and ovale malaria, and the only potent gametocytocide in falciparum malaria. Primaquine becomes increasingly important as malaria-endemic countries move towards elimination, and although it is widely recommended, it is commonly not given to malaria patients because of haemolytic toxicity in subjects who are glucose-6-phosphate dehydrogenase (G6PD) deficient (gene frequency typically 3-30% in malaria endemic areas; >180 different genetic variants). In six decades of primaquine use in approximately 200 million people, 14 deaths have been reported. Confining the estimate to reports with known denominators gives an estimated mortality of one in 621,428 (upper 95% CI: one in 407,807). All but one death followed multiple dosing to prevent vivax malaria relapse. Review of dose-response relationships and clinical trials of primaquine in G6PD deficiency suggests that the currently recommended WHO single low dose (0.25 mg base/kg) to block falciparum malaria transmission confers a very low risk of haemolytic toxicity.

Zaloumis SG, Tarning J, Krishna S, Price RN, White NJ, Davis TM, McCaw JM, Olliaro P et al. 2014. Population pharmacokinetics of intravenous artesunate: a pooled analysis of individual data from patients with severe malaria. CPT Pharmacometrics Syst Pharmacol, 3 (11), pp. e145. | Show Abstract | Read more

There are ~660,000 deaths from severe malaria each year. Intravenous artesunate (i.v. ARS) is the first-line treatment in adults and children. To optimize the dosing regimen of i.v. ARS, the largest pooled population pharmacokinetic study to date of the active metabolite dihydroartemisinin (DHA) was performed. The pooled dataset consisted of 71 adults and 195 children with severe malaria, with a mixture of sparse and rich sampling within the first 12 h after drug administration. A one-compartment model described the population pharmacokinetics of DHA adequately. Body weight had the greatest impact on DHA pharmacokinetics, resulting in lower DHA exposure for smaller children (6-10 kg) than adults. Post hoc estimates of DHA exposure were not significantly associated with parasitological outcomes. Comparable DHA exposure in smaller children and adults after i.v. ARS was achieved under a dose modification for intramuscular ARS proposed in a separate analysis of children.

Kloprogge F, Simpson JA, Day NP, White NJ, Tarning J. 2014. Statistical power calculations for mixed pharmacokinetic study designs using a population approach. AAPS J, 16 (5), pp. 1110-1118. | Show Abstract | Read more

Simultaneous modelling of dense and sparse pharmacokinetic data is possible with a population approach. To determine the number of individuals required to detect the effect of a covariate, simulation-based power calculation methodologies can be employed. The Monte Carlo Mapped Power method (a simulation-based power calculation methodology using the likelihood ratio test) was extended in the current study to perform sample size calculations for mixed pharmacokinetic studies (i.e. both sparse and dense data collection). A workflow guiding an easy and straightforward pharmacokinetic study design, considering also the cost-effectiveness of alternative study designs, was used in this analysis. Initially, data were simulated for a hypothetical drug and then for the anti-malarial drug, dihydroartemisinin. Two datasets (sampling design A: dense; sampling design B: sparse) were simulated using a pharmacokinetic model that included a binary covariate effect and subsequently re-estimated using (1) the same model and (2) a model not including the covariate effect in NONMEM 7.2. Power calculations were performed for varying numbers of patients with sampling designs A and B. Study designs with statistical power >80% were selected and further evaluated for cost-effectiveness. The simulation studies of the hypothetical drug and the anti-malarial drug dihydroartemisinin demonstrated that the simulation-based power calculation methodology, based on the Monte Carlo Mapped Power method, can be utilised to evaluate and determine the sample size of mixed (part sparsely and part densely sampled) study designs. The developed method can contribute to the design of robust and efficient pharmacokinetic studies.

Kyaw SS, Drake T, Ruangveerayuth R, Chierakul W, White NJ, Newton PN, Lubell Y. 2014. Cost of treating inpatient falciparum malaria on the Thai-Myanmar border. Malar J, 13 (1), pp. 416. | Show Abstract | Read more

BACKGROUND: Despite demonstrated benefits and World Health Organization (WHO) endorsement, parenteral artesunate is the recommended treatment for patients with severe Plasmodium falciparum malaria in only one fifth of endemic countries. One possible reason for this slow uptake is that a treatment course of parenteral artesunate is costlier than quinine and might, therefore, pose a substantial economic burden to health care systems. This analysis presents a detailed account of the resources used in treating falciparum malaria by either parenteral artesunate or quinine in a hospital on the Thai-Myanmar border. METHODS: The analysis used data from four studies, with random allocation of inpatients with falciparum malaria to treatment with parenteral artesunate or quinine, conducted in Mae Sot Hospital, Thailand from 1995 to 2001. Detailed resource use data were collected during admission and unit costs from the 2008 hospital price list were applied to these. Total admission costs were broken down into five categories: 1) medication; 2) intravenous fluids; 3) disposables; 4) laboratory tests; and 5) services. RESULTS: While the medication costs were higher for patients treated with artesunate, total admission costs were similar in those treated with quinine, US$ 243 (95% CI: 167.5-349.7) and in those treated with artesunate US$ 190 (95% CI: 131.0-263.2) (P=0.375). For cases classified as severe malaria (59%), the total cost of admission was US$ 298 (95% CI: 203.6-438.7) in the quinine group as compared with US$ 284 (95% CI: 181.3-407) in the artesunate group (P=0.869). CONCLUSION: This analysis finds no evidence for a difference in total admission costs for malaria inpatients treated with artesunate as compared with quinine. Assuming this is generalizable to other settings, the higher cost of a course of artesunate should not be considered a barrier for its implementation in the treatment of malaria.

Maude RJ, Nguon C, Dondorp AM, White LJ, White NJ. 2014. The diminishing returns of atovaquone-proguanil for elimination of Plasmodium falciparum malaria: modelling mass drug administration and treatment. Malar J, 13 (1), pp. 380. | Show Abstract | Read more

BACKGROUND: Artemisinin resistance is a major threat to current efforts to eliminate Plasmodium falciparum malaria which rely heavily on the continuing efficacy of artemisinin combination therapy (ACT). It has been suggested that ACT should not be used in mass drug administration (MDA) in areas where artemisinin-resistant P. falciparum is prevalent, and that atovaquone-proguanil (A-P) might be a preferable alternative. However, a single point mutation in the cytochrome b gene confers high level resistance to atovaquone, and such mutant parasites arise frequently during treatment making A-P a vulnerable tool for elimination. METHODS: A deterministic, population level, mathematical model was developed based on data from Cambodia to explore the possible effects of large-scale use of A-P compared to dihydroartemisinin-piperaquine ACT for mass drug administration and/or treatment of P. falciparum malaria, with and without adjunctive primaquine (PQ) and long-lasting insecticide-treated bed nets (LLIN). The aim was local elimination. RESULTS: The model showed the initial efficacy of ACT and A-P for MDA to be similar. However, each round of A-P MDA resulted in rapid acquisition and spread of atovaquone resistance. Even a single round of MDA could compromise efficacy sufficient to preclude its use for treatment or prophylaxis. A switch to A-P for treatment of symptomatic episodes resulted in a complete loss of efficacy in the population within four to five years of its introduction. The impact of MDA was temporary and a combination of maintained high coverage with ACT treatment for symptomatic individuals and LLIN was necessary for elimination. CONCLUSION: For malaria elimination, A-P for MDA or treatment of symptomatic cases should be avoided. A combined strategy of high coverage with ACT for treatment of symptomatic episodes, LLIN and ACT + P MDA would be preferable.

Tarning J, Thana P, Phyo AP, Lwin KM, Hanpithakpong W, Ashley EA, Day NP, Nosten F, White NJ. 2014. Population Pharmacokinetics and Antimalarial Pharmacodynamics of Piperaquine in Patients With Plasmodium vivax Malaria in Thailand. CPT Pharmacometrics Syst Pharmacol, 3 (8), pp. e132. | Show Abstract | Read more

Dihydroartemisinin-piperaquine is an effective drug in the treatment of Plasmodium falciparum and P. vivax malaria. The objective of this study was to evaluate the population pharmacokinetics and pharmacodynamics of piperaquine in patients with P. vivax malaria in Thailand after a standard regimen of dihydroartemisinin-piperaquine to determine whether residual piperaquine prevents or delays the emergence of P. vivax relapse. Sparse blood samples were collected from 116 patients. Piperaquine pharmacokinetics were described well by a three-compartment distribution model. Relapsing P. vivax malaria was accommodated by a constant baseline hazard (8.94 relapses/year) with the addition of a surge function in a fixed 3-week interval and a protective piperaquine effect. The results suggest that a large proportion of the first relapses were suppressed completely by residual piperaquine concentrations and that recurrences resulted mainly from emergence of the second or third relapse or from reinfection. This suggests a significant reduction in P. vivax morbidity when using dihydroartemisinin-piperaquine compared with other antimalarial drugs with shorter terminal postprophylactic effects.

Maude RJ, Barkhof F, Hassan MU, Ghose A, Hossain A, Abul Faiz M, Choudhury E, Rashid R et al. 2014. Magnetic resonance imaging of the brain in adults with severe falciparum malaria. Malar J, 13 (1), pp. 177. | Show Abstract | Read more

BACKGROUND: Magnetic resonance imaging (MRI) allows detailed study of structural and functional changes in the brain in patients with cerebral malaria. METHODS: In a prospective observational study in adult Bangladeshi patients with severe falciparum malaria, MRI findings in the brain were correlated with clinical and laboratory parameters, retinal photography and optic nerve sheath diameter (ONSD) ultrasound (a marker of intracranial pressure). RESULTS: Of 43 enrolled patients, 31 (72%) had coma and 12 (28%) died. MRI abnormalities were present in 79% overall with mostly mild changes in a wide range of anatomical sites. There were no differences in MRI findings between patients with cerebral and non-cerebral or fatal and non-fatal disease. Subtle diffuse cerebral swelling was common (n = 22/43), but mostly without vasogenic oedema or raised intracranial pressure (ONSD). Also seen were focal extracellular oedema (n = 11/43), cytotoxic oedema (n = 8/23) and mildly raised brain lactate on magnetic resonance spectroscopy (n = 5/14). Abnormalities were much less prominent than previously described in Malawian children. Retinal whitening was present in 36/43 (84%) patients and was more common and severe in patients with coma. CONCLUSION: Cerebral swelling is mild and not specific to coma or death in adult severe falciparum malaria. This differs markedly from African children. Retinal whitening, reflecting heterogeneous obstruction of the central nervous system microcirculation by sequestered parasites resulting in small patches of ischemia, is associated with coma and this process is likely important in the pathogenesis.

Das D, Cheah PY, Akter F, Paul D, Islam A, Sayeed AA, Samad R, Rahman R et al. 2014. Participants' perceptions and understanding of a malaria clinical trial in Bangladesh. Malar J, 13 (1), pp. 217. | Show Abstract | Read more

BACKGROUND: Existing evidence suggests that there is often limited understanding among participants in clinical trials about the informed consent process, resulting in their providing consent without really understanding the purpose of the study, specific procedures, and their rights. The objective of the study was to determine the subjects' understanding of research, perceptions of voluntariness and motivations for participation in a malaria clinical trial. METHODS: In this study semi-structured interviews of adult clinical trial participants with uncomplicated falciparum malaria were conducted in Ramu Upazila Health Complex, in Bangladesh. RESULTS: Of 16 participants, the vast majority (81%) were illiterate. All subjects had a 'therapeutic misconception' i.e. the trial was perceived to be conducted primarily for the benefit of individual patients when in fact the main objective was to provide information to inform public health policy. From the patients' perspective, getting well from their illness was their major concern. Poor actual understanding of trial specific procedures was reported despite participants' satisfaction with treatment and nursing care. CONCLUSION: There is frequently a degree of overlap between research and provision of clinical care in malaria research studies. Patients may be motivated to participate to research without a good understanding of the principal objectives of the study despite a lengthy consent process. The findings suggest that use of a standard consent form following the current ICH-GCP guidelines does not result in achieving fully informed consent and the process should be revised, simplified and adapted to individual trial settings.

Sriboonvorakul N, Leepipatpiboon N, Dondorp AM, Pouplin T, White NJ, Tarning J, Lindegardh N. 2013. Liquid chromatographic-mass spectrometric method for simultaneous determination of small organic acids potentially contributing to acidosis in severe malaria. J Chromatogr B Analyt Technol Biomed Life Sci, 941 pp. 116-122. | Show Abstract | Read more

Acidosis is an important cause of mortality in severe falciparum malaria. Lactic acid is a major contributor to metabolic acidosis, but accounts for only one-quarter of the strong anion gap. Other unidentified organic acids have an independent strong prognostic significance for a fatal outcome. In this study, a simultaneous bio-analytical method for qualitative and quantitative assessment in plasma and urine of eight small organic acids potentially contributing to acidosis in severe malaria was developed and validated. High-throughput strong anion exchange solid-phase extraction in a 96-well plate format was used for sample preparation. Hydrophilic interaction liquid chromatography (HILIC) coupled to negative mass spectroscopy was utilized for separation and detection. Eight possible small organic acids; l-lactic acid (LA), α-hydroxybutyric acid (aHBA), β-hydroxybutyric acid (bHBA), p-hydroxyphenyllactic acid (pHPLA), malonic acid (MA), methylmalonic acid (MMA), ethylmalonic acid (EMA) and α-ketoglutaric acid (aKGA) were analyzed simultaneously using a ZIC-HILIC column with an isocratic elution containing acetonitrile and ammonium acetate buffer. This method was validated according to U.S. Food and Drug Administration guidelines with additional validation procedures for endogenous substances. Accuracy for all eight acids ranged from 93.1% to 104.0%, and the within-day and between-day precisions (i.e. relative standard deviations) were lower than 5.5% at all tested concentrations. The calibration ranges were: 2.5-2500μg/mL for LA, 0.125-125μg/mL for aHBA, 7.5-375μg/mL for bHBA, 0.1-100μg/mL for pHPLA, 1-1000μg/mL for MA, 0.25-250μg/mL for MMA, 0.25-100μg/mL for EMA, and 30-1500μg/mL for aKGA. Clinical applicability was demonstrated by analyzing plasma and urine samples from five patients with severe falciparum malaria; five acids had increased concentrations in plasma (range LA=177-1169μg/mL, aHBA=4.70-38.4μg/mL, bHBA=7.70-38.0μg/mL, pHPLA=0.900-4.30μg/mL and aKGA=30.2-32.0) and seven in urine samples (range LA=11.2-513μg/mL, aHBA=1.50-69.5μg/mL, bHBA=8.10-111μg/mL, pHPLA=4.30-27.7μg/mL, MMA=0.300-13.3μg/mL, EMA=0.300-48.1μg/mL and aKGA=30.4-107μg/mL). In conclusion, a novel bioanalytical method was developed and validated which allows for simultaneous quantification of eight small organic acids in plasma and urine. This new method may be a useful tool for the assessment of acidosis in patients with severe malaria, and other conditions complicated by acidosis.

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Achan J, Adam I, Arinaitwe E, Ashley EA, Awab GR, Ba MS, Barnes KI, Bassat Q et al. 2013. The Effect of Dosing Regimens on the Antimalarial Efficacy of Dihydroartemisinin-Piperaquine: A Pooled Analysis of Individual Patient Data PLOS MEDICINE, 10 (12), pp. e1001564-e1001564. | Show Abstract | Read more

Background:Dihydroartemisinin-piperaquine (DP) is increasingly recommended for antimalarial treatment in many endemic countries; however, concerns have been raised over its potential under dosing in young children. We investigated the influence of different dosing schedules on DP's clinical efficacy.Methods and Findings:A systematic search of the literature was conducted to identify all studies published between 1960 and February 2013, in which patients were enrolled and treated with DP. Principal investigators were approached and invited to share individual patient data with the WorldWide Antimalarial Resistance Network (WWARN). Data were pooled using a standardised methodology. Univariable and multivariable risk factors for parasite recrudescence were identified using a Cox's regression model with shared frailty across the study sites. Twenty-four published and two unpublished studies (n = 7,072 patients) were included in the analysis. After correcting for reinfection by parasite genotyping, Kaplan-Meier survival estimates were 97.7% (95% CI 97.3%-98.1%) at day 42 and 97.2% (95% CI 96.7%-97.7%) at day 63. Overall 28.6% (979/3,429) of children aged 1 to 5 years received a total dose of piperaquine below 48 mg/kg (the lower limit recommended by WHO); this risk was 2.3-2.9-fold greater compared to that in the other age groups and was associated with reduced efficacy at day 63 (94.4% [95% CI 92.6%-96.2%], p<0.001). After adjusting for confounding factors, the mg/kg dose of piperaquine was found to be a significant predictor for recrudescence, the risk increasing by 13% (95% CI 5.0%-21%) for every 5 mg/kg decrease in dose; p = 0.002. In a multivariable model increasing the target minimum total dose of piperaquine in children aged 1 to 5 years old from 48 mg/kg to 59 mg/kg would halve the risk of treatment failure and cure at least 95% of patients; such an increment was not associated with gastrointestinal toxicity in the ten studies in which this could be assessed.Conclusions:DP demonstrates excellent efficacy in a wide range of transmission settings; however, treatment failure is associated with a lower dose of piperaquine, particularly in young children, suggesting potential for further dose optimisation.Please see later in the article for the Editors' Summary. © 2013 Price et al.

Turner P, Turner C, Green N, Ashton L, Lwe E, Jankhot A, Day NP, White NJ, Nosten F, Goldblatt D. 2013. Serum antibody responses to pneumococcal colonization in the first 2 years of life: Results from an SE Asian longitudinal cohort study Clinical Microbiology and Infection, 19 (12), | Show Abstract | Read more

Assessment of antibody responses to pneumococcal colonization in early childhood may aid our understanding of protection and inform vaccine antigen selection. Serum samples were collected from mother-infant pairs during a longitudinal pneumococcal colonization study in Burmese refugees. Maternal and cord sera were collected at birth and infants were bled monthly (1-24 months of age). Nasopharyngeal swabs were taken monthly to detect colonization. Serum IgG titres to 27 pneumococcal protein antigens were measured in 2624 sera and IgG to dominant serotypes (6B, 14, 19F, 19A and 23F) were quantified in 864 infant sera. Antibodies to all protein antigens were detectable in maternal sera. Titres to four proteins (LytB, PcpA, PhtD and PhtE) were significantly higher in mothers colonized by pneumococci at delivery. Maternally-derived antibodies to PiuA and Spr0096 were associated with delayed pneumococcal acquisition in infants in univariate, but not multivariate models. Controlling for infant age and previous homologous serotype exposure, nasopharyngeal acquisition of serotypes 19A, 23F, 14 or 19F was associated significantly with a ≥2-fold antibody response to the homologous capsule (OR 12.84, 7.52, 6.52, 5.33; p <0.05). Acquisition of pneumococcal serotypes in the nasopharynx of infants was not significantly associated with a ≥2-fold rise in antibodies to any of the protein antigens studied. In conclusion, nasopharyngeal colonization in young children resulted in demonstrable serum IgG responses to pneumococcal capsules and surface/virulence proteins. However, the relationship between serum IgG and the prevention of, or response to, pneumococcal nasopharyngeal colonization remains complex. Mechanisms other than serum IgG are likely to have a role but are currently poorly understood. ©2013 The Authors. Clinical Microbiology and Infection published by John Wiley & Sons Ltd on behalf of the European Society of Clinical Microbiology and Infectious Diseases.

Flegg JA, Guérin PJ, Nosten F, Ashley EA, Phyo AP, Dondorp AM, Fairhurst RM, Socheat D et al. 2013. Optimal sampling designs for estimation of Plasmodium falciparum clearance rates in patients treated with artemisinin derivatives. Malar J, 12 (1), pp. 411. | Show Abstract | Read more

BACKGROUND: The emergence of Plasmodium falciparum resistance to artemisinins in Southeast Asia threatens the control of malaria worldwide. The pharmacodynamic hallmark of artemisinin derivatives is rapid parasite clearance (a short parasite half-life), therefore, the in vivo phenotype of slow clearance defines the reduced susceptibility to the drug. Measurement of parasite counts every six hours during the first three days after treatment have been recommended to measure the parasite clearance half-life, but it remains unclear whether simpler sampling intervals and frequencies might also be sufficient to reliably estimate this parameter. METHODS: A total of 2,746 parasite density-time profiles were selected from 13 clinical trials in Thailand, Cambodia, Mali, Vietnam, and Kenya. In these studies, parasite densities were measured every six hours until negative after treatment with an artemisinin derivative (alone or in combination with a partner drug). The WWARN Parasite Clearance Estimator (PCE) tool was used to estimate "reference" half-lives from these six-hourly measurements. The effect of four alternative sampling schedules on half-life estimation was investigated, and compared to the reference half-life (time zero, 6, 12, 24 (A1); zero, 6, 18, 24 (A2); zero, 12, 18, 24 (A3) or zero, 12, 24 (A4) hours and then every 12 hours). Statistical bootstrap methods were used to estimate the sampling distribution of half-lives for parasite populations with different geometric mean half-lives. A simulation study was performed to investigate a suite of 16 potential alternative schedules and half-life estimates generated by each of the schedules were compared to the "true" half-life. The candidate schedules in the simulation study included (among others) six-hourly sampling, schedule A1, schedule A4, and a convenience sampling schedule at six, seven, 24, 25, 48 and 49 hours. RESULTS: The median (range) parasite half-life for all clinical studies combined was 3.1 (0.7-12.9) hours. Schedule A1 consistently performed the best, and schedule A4 the worst, both for the individual patient estimates and for the populations generated with the bootstrapping algorithm. In both cases, the differences between the reference and alternative schedules decreased as half-life increased. In the simulation study, 24-hourly sampling performed the worst, and six-hourly sampling the best. The simulation study confirmed that more dense parasite sampling schedules are required to accurately estimate half-life for profiles with short half-life (≤ three hours) and/or low initial parasite density (≤ 10,000 per μL). Among schedules in the simulation study with six or fewer measurements in the first 48 hours, a schedule with measurements at times (time windows) of 0 (0-2), 6 (4-8), 12 (10-14), 24 (22-26), 36 (34-36) and 48 (46-50) hours, or at times 6, 7 (two samples in time window 5-8), 24, 25 (two samples during time 23-26), and 48, 49 (two samples during time 47-50) hours, until negative most accurately estimated the "true" half-life. For a given schedule, continuing sampling after two days had little effect on the estimation of half-life, provided that adequate sampling was performed in the first two days and the half-life was less than three hours. If the measured parasitaemia at two days exceeded 1,000 per μL, continued sampling for at least once a day was needed for accurate half-life estimates. CONCLUSIONS: This study has revealed important insights on sampling schedules for accurate and reliable estimation of Plasmodium falciparum half-life following treatment with an artemisinin derivative (alone or in combination with a partner drug). Accurate measurement of short half-lives (rapid clearance) requires more dense sampling schedules (with more than twice daily sampling). A more intensive sampling schedule is, therefore, recommended in locations where P. falciparum susceptibility to artemisinins is not known and the necessary resources are available. Counting parasite density at six hours is important, and less frequent sampling is satisfactory for estimating long parasite half-lives in areas where artemisinin resistance is present.

Smithuis FM, Kyaw MK, Phe UO, van der Broek I, Katterman N, Rogers C, Almeida P, Kager PA et al. 2013. The effect of insecticide-treated bed nets on the incidence and prevalence of malaria in children in an area of unstable seasonal transmission in western Myanmar. Malar J, 12 (1), pp. 363. | Show Abstract | Read more

BACKGROUND: Insecticide-treated bed nets (ITN) reduce malaria morbidity and mortality consistently in Africa, but their benefits have been less consistent in Asia. This study's objective was to evaluate the malaria protective efficacy of village-wide usage of ITN in Western Myanmar and estimate the cost-effectiveness of ITN compared with extending early diagnosis and treatment services. METHODS: A cluster-randomized controlled trial was conducted in Rakhine State to assess the efficacy of ITNs in preventing malaria and anaemia in children and their secondary effects on nutrition and development. The data were aggregated for each village to obtain cluster-level infection rates. In total 8,175 children under 10 years of age were followed up for 10 months, which included the main malaria transmission period. The incidence and prevalence of Plasmodium falciparum and Plasmodium vivax infections, and the biting behaviour of Anopheles mosquitoes in the area were studied concurrently. The trial data along with costs for current recommended treatment practices were modelled to estimate the cost-effectiveness of ITNs compared with, or in addition to extending the coverage of early diagnosis and treatment services. RESULTS: In aggregate, malaria infections, spleen rates, haemoglobin concentrations, and weight for height, did not differ significantly during the study period between villages with and without ITNs, with a weighted mean difference of -2.6 P. falciparum episodes per 1,000 weeks at risk (95% Confidence Interval -7 to 1.8). In areas with a higher incidence of malaria there was some evidence ITN protective efficacy. The economic analysis indicated that, despite the uncertainty and variability in their protective efficacy in the different study sites, ITN could still be cost-effective, but not if they displaced funding for early diagnosis and effective treatment which is substantially more cost-effective. CONCLUSION: In Western Myanmar deployment of ITNs did not provide consistent protection against malaria in children living in malaria endemic villages. Early diagnosis and effective treatment is a more cost effective malaria control strategy than deployment of ITNs in this area where the main vector bites early in the evening, often before people are protected by an ITN.

Smithuis FM, Kyaw MK, Phe UO, van der Broek I, Katterman N, Rogers C, Almeida P, Kager PA et al. 2013. Entomological determinants of insecticide-treated bed net effectiveness in Western Myanmar. Malar J, 12 (1), pp. 364. | Show Abstract | Read more

BACKGROUND: In a large cluster randomized control trial of insecticide-treated bed nets (ITN) in Western Myanmar the malaria protective effect of ITN was found to be highly variable and, in aggregate, the effect was not statistically significant. A coincident entomological investigation measured malaria vector abundance and biting behaviour and the human population sleeping habits, factors relevant to ITN effectiveness. METHODS: Entomological surveys were carried out using different catching methods to identify potential malaria vector species and characterise their biting habits. The salivary glands were dissected from all female anophelines caught to identify sporozoites by microscopy. FINDINGS: Between 1995 and 2000 a total of 4,824 female anopheline mosquitoes were caught with various catching methods. A total of 916 person nights yielded 3,009 female anopheline mosquitoes between 6 pm and 6 am. Except for Anopheles annularis, which showed no apparent preference (51% outdoor biting), all major species showed a strong preference for outdoor biting; Anopheles epiroticus (79%), Anopheles subpictus (72%), Anopheles maculatus (92%), Anopheles aconitus (85%) and Anopheles vagus (72%). Most human biting occurred in the early evening with the peak biting time between 6 pm and 7 pm (35%). Overall 51% (1447/2837) of all bites recorded were between 6 pm and 8 pm. A large proportion of children were not sleeping under an ITN during peak biting times. Only one An. annularis mosquito (0.02%) had malaria sporozoites identified in the salivary glands. CONCLUSIONS: Peak vector biting occurred early in the evening and mainly occurred outdoors. The limited efficacy of ITN in this area of Western Myanmar may be explained by the biting behaviour of the prevalent Anopheles mosquito vectors in this area.

Tanomsing N, Imwong M, Sutherland CJ, Dolecek C, Hien TT, Nosten F, Day NP, White NJ, Snounou G. 2013. Genetic marker suitable for identification and genotyping of Plasmodium ovale curtisi and Plasmodium ovale wallikeri. J Clin Microbiol, 51 (12), pp. 4213-4216. | Show Abstract | Read more

We present a seminested PCR method that specifically discriminates between Plasmodium ovale curtisi and P. ovale wallikeri with high sensitivity. The test is based on species-specific amplification of a size-polymorphic fragment of the tryptophan-rich antigen gene, potra, which also permits discrimination of intraspecific sequence variants at this locus.

White NJ. 2013. Pharmacokinetic and pharmacodynamic considerations in antimalarial dose optimization. Antimicrob Agents Chemother, 57 (12), pp. 5792-5807. | Show Abstract | Read more

Antimalarial drugs have usually been first deployed in areas of malaria endemicity at doses which were too low, particularly for high-risk groups such as young children and pregnant women. This may accelerate the emergence and spread of resistance, thereby shortening the useful life of the drug, but it is an inevitable consequence of the current imprecise method of dose finding. An alternative approach to dose finding is suggested in which phase 2 studies concentrate initially on pharmacokinetic-pharmacodynamic (PK-PD) characterization and in vivo calibration of in vitro susceptibility information. PD assessment is facilitated in malaria because serial parasite densities are readily assessed by microscopy, and at low densities by quantitative PCR, so that initial therapeutic responses can be quantitated accurately. If the in vivo MIC could be characterized early in phase 2 studies, it would provide a sound basis for the choice of dose in all target populations in subsequent combination treatments. Population PK assessments in phase 2b and phase 3 studies which characterize PK differences between different age groups, clinical disease states, and human populations can then be combined with the PK-PD observations to provide a sound evidence base for dose recommendations in different target groups.

Maude RJ, Silamut K, Plewes K, Charunwatthana P, Ho M, Abul Faiz M, Rahman R, Hossain MA et al. 2014. Randomized controlled trial of levamisole hydrochloride as adjunctive therapy in severe falciparum malaria with high parasitemia. J Infect Dis, 209 (1), pp. 120-129. | Show Abstract | Read more

BACKGROUND: Cytoadherence and sequestration of erythrocytes containing mature stages of Plasmodium falciparum are central to the pathogenesis of severe malaria. The oral anthelminthic drug levamisole inhibits cytoadherence in vitro and reduces sequestration of late-stage parasites in uncomplicated falciparum malaria treated with quinine. METHODS: Fifty-six adult patients with severe malaria and high parasitemia admitted to a referral hospital in Bangladesh were randomized to receive a single dose of levamisole hydrochloride (150 mg) or no adjuvant to antimalarial treatment with intravenous artesunate. RESULTS: Circulating late-stage parasites measured as the median area under the parasite clearance curves were 2150 (interquartile range [IQR], 0-28 025) parasites/µL × hour in patients treated with levamisole and 5489 (IQR, 192-25 848) parasites/µL × hour in controls (P = .25). The "sequestration ratios" at 6 and 12 hours for all parasite stages and changes in microvascular blood flow did not differ between treatment groups (all P > .40). The median time to normalization of plasma lactate (<2 mmol/L) was 24 (IQR, 12-30) hours with levamisole vs 28 (IQR, 12-36) hours without levamisole (P = .15). CONCLUSIONS: There was no benefit of a single-dose of levamisole hydrochloride as adjuvant to intravenous artesunate in the treatment of adults with severe falciparum malaria. Rapid parasite killing by intravenous artesunate might obscure the effects of levamisole.

Lim P, Dek D, Try V, Eastman RT, Chy S, Sreng S, Suon S, Mao S et al. 2013. Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in western, northern, and eastern Cambodia, 2011-2012: association with molecular markers. Antimicrob Agents Chemother, 57 (11), pp. 5277-5283. | Show Abstract | Read more

In 2008, dihydroartemisinin (DHA)-piperaquine (PPQ) became the first-line treatment for uncomplicated Plasmodium falciparum malaria in western Cambodia. Recent reports of increased treatment failure rates after DHA-PPQ therapy in this region suggest that parasite resistance to DHA, PPQ, or both is now adversely affecting treatment. While artemisinin (ART) resistance is established in western Cambodia, there is no evidence of PPQ resistance. To monitor for resistance to PPQ and other antimalarials, we measured drug susceptibilities for parasites collected in 2011 and 2012 from Pursat, Preah Vihear, and Ratanakiri, in western, northern, and eastern Cambodia, respectively. Using a SYBR green I fluorescence assay, we calculated the ex vivo 50% inhibitory concentrations (IC50s) of 310 parasites to six antimalarials: chloroquine (CQ), mefloquine (MQ), quinine (QN), PPQ, artesunate (ATS), and DHA. Geometric mean IC50s (GMIC50s) for all drugs (except PPQ) were significantly higher in Pursat and Preah Vihear than in Ratanakiri (P ≤ 0.001). An increased copy number of P. falciparum mdr1 (pfmdr1), an MQ resistance marker, was more prevalent in Pursat and Preah Vihear than in Ratanakiri and was associated with higher GMIC50s for MQ, QN, ATS, and DHA. An increased copy number of a chromosome 5 region (X5r), a candidate PPQ resistance marker, was detected in Pursat but was not associated with reduced susceptibility to PPQ. The ex vivo IC50 and pfmdr1 copy number are important tools in the surveillance of multidrug-resistant (MDR) parasites in Cambodia. While MDR P. falciparum is prevalent in western and northern Cambodia, there is no evidence for PPQ resistance, suggesting that DHA-PPQ treatment failures result mainly from ART resistance.

Thanapongpichat S, McGready R, Luxemburger C, Day NP, White NJ, Nosten F, Snounou G, Imwong M. 2013. Microsatellite genotyping of Plasmodium vivax infections and their relapses in pregnant and non-pregnant patients on the Thai-Myanmar border. Malar J, 12 (1), pp. 275. | Show Abstract | Read more

BACKGROUND: Plasmodium vivax infections in pregnancy are associated with low birth weight and anaemia. This parasites species is also characterised by relapses, erythrocytic infections initiated by the activation of the dormant liver stages, the hypnozoites, to mature. Genotyping of P. vivax using microsatellite markers has opened the way to comparative investigations of parasite populations. The aim of the study was to assess whether there were any differences between the parasites found in pregnant and non-pregnant patients, and/or between the admission infections and recurrent episodes during follow-up. METHODS: Blood samples were collected from 18 pregnant and 18 non-pregnant patients, who had at least two recurrent episodes during follow-up, that were recruited in two previous trials on the efficacy of chloroquine treatment of P. vivax infections on the Thai-Myanmar border. DNA was purified and the P. vivax populations genotyped with respect to eight polymorphic microsatellite markers. Analyses of the genetic diversity, multiplicity of infection (MOI), and a comparison of the genotypes in the samples from each patient were conducted. RESULTS: The P. vivax parasites present in the samples exhibited high genetic diversity (6 to 15 distinct allelic variants found for the 8 loci). Similar expected heterozygosity (He) values were obtained for isolates from pregnant (0.837) and non-pregnant patients (0.852). There were modest differences between the MOI values calculated for both admission and recurrence samples from the pregnant patients (2.00 and 2.05, respectively) and the equivalent samples from the non-pregnant patients (1.67 and 1.64, respectively). Furthermore, the mean number of distinct alleles enumerated in the admission samples from the pregnant (6.88) and non-pregnant (7.63) patients were significantly lower than that found in the corresponding recurrent episodes samples (9.25 and 9.63, respectively). CONCLUSIONS: The P. vivax populations circulating in inhabitants along the Thai-Myanmar border, an area of low malaria transmission, displayed high genetic diversity. A subtle increase in the multiplicity of P. vivax infections in pregnant patients suggests a higher susceptibility to infection. The higher allelic diversity in the relapse as compared to the admission samples in both patient groups is consistent with the hypothesis that a febrile episode promotes the activation of hypnozoites.

Pasaribu AP, Chokejindachai W, Sirivichayakul C, Tanomsing N, Chavez I, Tjitra E, Pasaribu S, Imwong M, White NJ, Dondorp AM. 2013. A randomized comparison of dihydroartemisinin-piperaquine and artesunate-amodiaquine combined with primaquine for radical treatment of vivax malaria in Sumatera, Indonesia. J Infect Dis, 208 (11), pp. 1906-1913. | Show Abstract | Read more

BACKGROUND: A high prevalence of chloroquine-resistant Plasmodium vivax in Indonesia has shifted first-line treatment to artemisinin-based combination therapies, combined with primaquine (PQ) for radical cure. Which combination is most effective and safe remains to be established. METHODS: We conducted a prospective open-label randomized comparison of 14 days of PQ (0.25 mg base/kg) plus either artesunate-amodiaquine (AAQ + PQ) or dihydroartemisinin-piperaquine (DHP + PQ) for the treatment of uncomplicated monoinfection P. vivax malaria in North Sumatera, Indonesia. Patients were randomized and treatments were given without prior testing for G6PD status. The primary outcome was parasitological failure at day 42. Patients were followed up to 1 year. RESULTS: Between December 2010 and April 2012, 331 patients were included. After treatment with AAQ + PQ, recurrent infection occurred in 0 of 167 patients within 42 days and in 15 of 130 (11.5%; 95% confidence interval [CI], 6.6%-18.3%) within a year. With DHP + PQ, this was 1 of 164 (0.6%; 95% CI, 0.01%-3.4%) and 13 of 143 (9.1%; 95% CI, 4.9%-15.0%), respectively (P > .2). Intravascular hemolysis occurred in 5 patients, of which 3 males were hemizygous for the G6PD-Mahidol mutation. Minor adverse events were more frequent with AAQ + PQ. CONCLUSIONS: In North Sumatera, Indonesia, AAQ and DHP, both combined with PQ, were effective for blood-stage parasite clearance of uncomplicated P. vivax malaria. Both treatments were safe, but DHP + PQ was better tolerated. CLINICAL TRIALS REGISTRATION: NCT01288820.

Tarning J, Kloprogge F, Dhorda M, Jullien V, Nosten F, White NJ, Guerin PJ, Piola P. 2013. Pharmacokinetic properties of artemether, dihydroartemisinin, lumefantrine, and quinine in pregnant women with uncomplicated plasmodium falciparum malaria in Uganda. Antimicrob Agents Chemother, 57 (10), pp. 5096-5103. | Show Abstract | Read more

Pregnancy alters the pharmacokinetic properties of many drugs used in the treatment of malaria, usually resulting in lower drug exposures. This increases the risks of treatment failure, adverse outcomes for the fetus, and the development of resistance. The pharmacokinetic properties of artemether and its principal metabolite dihydroartemisinin (n = 21), quinine (n = 21), and lumefantrine (n = 26) in pregnant Ugandan women were studied. Lumefantrine pharmacokinetics in a nonpregnant control group (n = 17) were also studied. Frequently sampled patient data were evaluated with noncompartmental analysis. No significant correlation was observed between estimated gestational age and artemether, dihydroartemisinin, lumefantrine, or quinine exposures. Artemether/dihydroartemisinin and quinine exposures were generally low in these pregnant women compared to values reported previously for nonpregnant patients. Median day 7 lumefantrine concentrations were 488 (range, 30.7 to 3,550) ng/ml in pregnant women compared to 720 (339 to 2,150) ng/ml in nonpregnant women (P = 0.128). There was no statistical difference in total lumefantrine exposure or maximum concentration. More studies with appropriate control groups in larger series are needed to characterize the degree to which pregnant women are underdosed with current antimalarial dosing regimens.

Newton PN, Stepniewska K, Dondorp A, Silamut K, Chierakul W, Krishna S, Davis TM, Suputtamongkol Y et al. 2013. Prognostic indicators in adults hospitalized with falciparum malaria in Western Thailand. Malar J, 12 (1), pp. 229. | Show Abstract | Read more

BACKGROUND: Severe malaria remains a major cause of death and morbidity amongst adults in the Asiatic tropics. METHODS: A retrospective analysis of the clinical and laboratory data of 988 adult patients, hospitalized with Plasmodium falciparum malaria and prospectively recruited to malaria studies in western Thailand between 1986 and 2002, was performed to assess the factors associated with a fatal outcome. Different severity scores and classifications for defining severe malaria were compared and, using multiple logistic regression, simple models for predicting mortality developed. RESULTS: The proportion of patients fulfilling the WHO 2000 definition of severe malaria was 78.1%, and their mortality was 10%. Mortality in patients given parenteral artesunate or artemether (16/317, 5%) was lower than in those given parenteral quinine (59/442, 13%) (P < 0.001). Models using parameter sets based on WHO 1990, 2000 and Adapted AQ criteria plus blood smear parasite-stage assessment gave the best mortality prediction. A malaria prognostic index (MPI), derived from the dataset using five clinical or laboratory variables gave similar prognostic accuracy. CONCLUSIONS: The mortality of severe malaria in adults has fallen and the switch from quinine to artesunate has probably been an important contributor. Prognostic indices based on WHO 2000 definitions, and other simpler indices based on fewer variables, provide clinically useful predictions of outcome in Asian adults with severe malaria.

Douglas NM, Simpson JA, Phyo AP, Siswantoro H, Hasugian AR, Kenangalem E, Poespoprodjo JR, Singhasivanon P et al. 2013. Gametocyte dynamics and the role of drugs in reducing the transmission potential of Plasmodium vivax. J Infect Dis, 208 (5), pp. 801-812. | Show Abstract | Read more

BACKGROUND: Designing interventions that will reduce transmission of vivax malaria requires knowledge of Plasmodium vivax gametocyte dynamics. METHODS: We analyzed data from a randomized controlled trial in northwestern Thailand and 2 trials in Papua, Indonesia, to identify and compare risk factors for vivax gametocytemia at enrollment and following treatment. RESULTS: A total of 492 patients with P. vivax infections from Thailand and 476 patients (162 with concurrent falciparum parasitemia) from Indonesia were evaluable. Also, 84.3% (415/492) and 66.6% (209/314) of patients with monoinfection were gametocytemic at enrollment, respectively. The ratio of gametocytemia to asexual parasitemia did not differ between acute and recurrent infections (P = .48 in Thailand, P = .08 in Indonesia). High asexual parasitemia was associated with an increased risk of gametocytemia during follow-up in both locations. In Thailand, the cumulative incidence of gametocytemia between day 7 and day 42 following dihydroartemisinin + piperaquine (DHA + PIP) was 6.92% vs 29.1% following chloroquine (P < .001). In Indonesia, the incidence of gametocytemia was 33.6% following artesunate + amodiaquine (AS + AQ), 7.42% following artemether + lumefantrine, and 6.80% following DHA + PIP (P < .001 for DHA + PIP vs AS + AQ). CONCLUSIONS: P. vivax gametocyte carriage mirrors asexual-stage infection. Prevention of relapses, particularly in those with high asexual parasitemia, is likely the most important strategy for interrupting P. vivax transmission.

Turner P, Turner C, Green N, Ashton L, Lwe E, Jankhot A, Day NP, White NJ, Nosten F, Goldblatt D. 2013. Serum antibody responses to pneumococcal colonisation in the first two years of life: results from a SE Asian longitudinal cohort study. Clin Microbiol Infect, 19 (12), pp. E551-E558. | Show Abstract | Read more

Assessment of antibody responses to pneumococcal colonisation in early childhood may aid our understanding of protection and inform vaccine antigen selection. Serum samples were collected from mother-infant pairs during a longitudinal pneumococcal colonisation study in Burmese refugees. Maternal and cord sera were collected at birth and infants were bled monthly (1-24 months of age). Nasopharyngeal swabs were taken monthly to detect colonisation. Serum IgG titres to 27 pneumococcal protein antigens were measured in 2,624 sera and IgG to dominant serotypes (6B,14,19F,19A,23F) were quantified in 864 infant sera. Antibodies to all protein antigens were detectable in maternal sera. Titres to four proteins (LytB,PcpA,PhtD,PhtE) were significantly higher in mothers colonised by pneumococci at delivery. Maternally-derived antibodies to PiuA and Spr0096 were associated with delayed pneumococcal acquisition in infants in univariate, but not multivariate models. Controlling for infant age and previous homologous serotype exposure, nasopharyngeal acquisition of serotypes 19A, 23F, 14, or 19F were associated significantly with a ≥2-fold antibody response to the homologous capsule (OR 12.84, 7.52, 6.52, 5.33; p<0.05). Acquisition of pneumococcal serotypes in the nasopharynx of infants was not significantly associated with a ≥2-fold rise in antibodies to any of the protein antigens studied. In conclusion, nasopharyngeal colonisation in young children resulted in demonstrable serum IgG responses to pneumococcal capsules and surface/virulence proteins. However, the relationship between serum IgG and the prevention of, or response to, pneumococcal nasopharyngeal colonisation remains complex. Mechanisms other than serum IgG are likely to have a role but are currently poorly understood. This article is protected by copyright. All rights reserved.

Bright AT, Alenazi T, Shokoples S, Tarning J, Paganotti GM, White NJ, Houston S, Winzeler EA, Yanow SK. 2013. Genetic analysis of primaquine tolerance in a patient with relapsing vivax malaria. Emerg Infect Dis, 19 (5), pp. 802-805. | Show Abstract | Read more

Patients with Plasmodium vivax malaria are treated with primaquine to prevent relapse infections. We report primaquine failure in a patient with 3 relapses without any possibility of re-infection. Using whole genome sequencing of the relapsing parasite isolates, we identified single nucleotide variants as candidate molecular markers of resistance.

Hendriksen IC, Mtove G, Kent A, Gesase S, Reyburn H, Lemnge MM, Lindegardh N, Day NP et al. 2013. Population pharmacokinetics of intramuscular artesunate in African children with severe malaria: implications for a practical dosing regimen. Clin Pharmacol Ther, 93 (5), pp. 443-450. | Show Abstract | Read more

Parenteral artesunate (ARS) is the drug of choice for the treatment of severe malaria. Pharmacokinetics data on intramuscular ARS are limited with respect to the main treatment group that carries the highest mortality, namely, critically ill children with severe malaria. A population pharmacokinetic study of ARS and dihydroartemisinin (DHA) was conducted from sparse sampling in 70 Tanzanian children of ages 6 months to 11 years. All the children had been admitted with severe falciparum malaria and were treated with intramuscular ARS (2.4 mg/kg at 0, 12, and 24 h). Venous plasma concentration-time profiles were characterized using nonlinear mixed-effects modeling (NONMEM). A one-compartment disposition model accurately described first-dose population pharmacokinetics of ARS and DHA. Body weight significantly affected clearance and apparent volume of distribution (P < 0.001), resulting in lower ARS and DHA exposure levels in smaller children. An adapted dosing regimen including a practical dosing table per weight band is proposed for young children based on the pharmacokinetic model.

Miotto O, Almagro-Garcia J, Manske M, Macinnis B, Campino S, Rockett KA, Amaratunga C, Lim P et al. 2013. Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia. Nat Genet, 45 (6), pp. 648-655. | Show Abstract | Read more

We describe an analysis of genome variation in 825 P. falciparum samples from Asia and Africa that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia. Within this relatively small geographic area, we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalog of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in transporter proteins and DNA mismatch repair proteins. These data provide a population-level genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist in its elimination.

White NJ, Turner GD, Day NP, Dondorp AM. 2013. Lethal malaria: Marchiafava and Bignami were right. J Infect Dis, 208 (2), pp. 192-198. | Show Abstract | Read more

One hundred and twenty years ago, the Italian malariologists Marchiafava and Bignami proposed that the fundamental pathological process underlying lethal falciparum malaria was microvascular obstruction. Since then, several alternative hypotheses have been proposed. These formed the basis for adjunctive interventions, which have either been ineffective or harmful. Recent evidence strongly suggests that Marchiafava and Bignami were right.

Turner C, Thein NAM, Turner P, Nosten F, White NJ. 2013. Erratum to Rectal pH in well and unwell infants [J Trop Pediatr, 58 (2012) 311-313] Journal of Tropical Pediatrics, 59 (2), pp. 162. | Read more

Turner C, Mya Thein NA, Turner P, Nosten F, White NJ. 2013. Rectal pH in Well and Unwell Infants. J Trop Pediatr, 59 (2), pp. 162. | Read more

Instiaty I, Lindegardh N, Jittmala P, Hanpithakpong W, Blessborn D, Pukrittayakamee S, White NJ, Tarning J. 2013. Comparison of oseltamivir and oseltamivir carboxylate concentrations in venous plasma, venous blood, and capillary blood in healthy volunteers. Antimicrob Agents Chemother, 57 (6), pp. 2858-2862. | Show Abstract | Read more

Oseltamivir and oseltamivir carboxylate concentrations were measured in venous plasma, venous blood, and capillary blood taken simultaneously from 24 healthy volunteers. Median (range) venous-blood-to-plasma ratios were 1.42 (0.920 to 1.97) for oseltamivir and 0.673 (0.564 to 0.814) for oseltamivir carboxylate. Capillary blood/venous plasma ratios were 1.32 (0.737 to 3.16) for oseltamivir and 0.685 (0.502 to 1.34) for oseltamivir carboxylate. Oseltamivir concentrations in venous and capillary blood were similar. Oseltamivir carboxylate showed a time-dependent distribution between venous and capillary blood.

Chairat K, Tarning J, White NJ, Lindegardh N. 2013. Pharmacokinetic properties of anti-influenza neuraminidase inhibitors. J Clin Pharmacol, 53 (2), pp. 119-139. | Show Abstract | Read more

Neuraminidase inhibitors are the mainstay of anti-influenza treatment. Oseltamivir is the most widely used drug but is currently available only as an oral formulation. Resistance spreads rapidly in seasonal H1N1 influenza A viruses, which were universally resistant in 2008, because of the H275Y mutation in the neuraminidase (NA) gene. Oseltamivir is a prodrug for the active carboxylate metabolite. Ex vivo conversion in blood samples may have confounded early pharmacokinetic studies. Oseltamivir shows dose linear kinetics, and oseltamivir carboxylate has an elimination half-life (t(1/2) β) after oral administration in healthy individuals of approximately 7.7 hours. Oseltamivir carboxylate is eliminated primarily by tubular secretion, and both clearance and tissue distribution are reduced by probenecid. The H275Y mutation in NA confers high-level oseltamivir resistance and intermediate peramivir resistance but does not alter zanamivir susceptibility. Zanamivir is available as a powder for inhalation, and a parenteral form is under development. Zanamivir distributes in an apparent volume of distribution approximating that of extracellular water and is rapidly eliminated (t(1/2) β of approximately 3.0 hours). Peramivir is slowly eliminated (t(1/2) β of 7.7-20.8 hours) and is prescribed as either a once-daily injection or as a single infusion. Laninamivir is a recently developed slowly eliminated compound for administration by inhalation.

Hanson JP, Lam SW, Mohanty S, Alam S, Pattnaik R, Mahanta KC, Hasan MU, Charunwatthana P et al. 2013. Fluid resuscitation of adults with severe falciparum malaria: effects on Acid-base status, renal function, and extravascular lung water. Crit Care Med, 41 (4), pp. 972-981. | Show Abstract | Read more

OBJECTIVE: To evaluate the efficacy and safety of liberal fluid resuscitation of adults with severe malaria. DESIGN, SETTING, PATIENTS, AND METHODS: Twenty-eight Bangladeshi and Indian adults with severe falciparum malaria received crystalloid resuscitation guided by transpulmonary thermodilution (PiCCO) in an intensive care setting. Systemic hemodynamics, microvascular indices and measures of acidosis, renal function, and pulmonary edema were followed prospectively. RESULTS: All patients were hypovolemic (global end-diastolic volume index<680 mL/m) on enrollment. Patients received a median (range) 3230 mL (390-7300) of isotonic saline in the first 6 hours and 5450 mL (710-13,720) in the first 24 hours. With resuscitation, acid-base status deteriorated in 19 of 28 (68%), and there was no significant improvement in renal function. Extravascular lung water increased in 17 of 22 liberally resuscitated patients (77%); eight of these patients developed pulmonary edema, five of whom died. All other patients survived. All patients with pulmonary edema during the study were hypovolemic or euvolemic at the time pulmonary edema developed. Plasma lactate was lower in hypovolemic patients before (rs=0.38; p=0.05) and after (rs=0.49; p=0.01) resuscitation but was the strongest predictor of mortality before (chi-square=9.9; p=0.002) and after resuscitation (chi-square=11.1; p<0.001) and correlated with the degree of visualized microvascular sequestration of parasitized erythrocytes at both time points (rs=0.55; p=0.003 and rs=0.43; p=0.03, respectively). Persisting sequestration was evident in 7 of 15 patients (47%) 48 hours after enrollment. CONCLUSIONS: Lactic acidosis--the strongest prognostic indicator in adults with severe falciparum malaria--results from sequestration of parasitized erythrocytes in the microcirculation, not from hypovolemia. Liberal fluid resuscitation has little effect on this sequestration and does not improve acid-base status or renal function. Pulmonary edema--secondary to increased pulmonary vascular permeability--is common, unpredictable, and exacerbated by fluid loading. Liberal fluid replacement of adults with severe malaria should be avoided.

Eziefula AC, Bousema T, Yeung S, Kamya M, Owaraganise A, Gabagaya G, Bradley J, Grignard L et al. 2014. Single dose primaquine for clearance of Plasmodium falciparum gametocytes in children with uncomplicated malaria in Uganda: a randomised, controlled, double-blind, dose-ranging trial. Lancet Infect Dis, 14 (2), pp. 130-139. | Show Abstract | Read more

BACKGROUND: Primaquine is the only available drug that clears mature Plasmodium falciparum gametocytes in infected human hosts, thereby preventing transmission of malaria to mosquitoes. However, concerns about dose-dependent haemolysis in people with glucose-6-phosphate dehydrogenase (G6PD) deficiencies have limited its use. We assessed the dose-response association of single-dose primaquine for gametocyte clearance and for safety in P falciparum malaria. METHODS: We undertook this randomised, double-blind, placebo-controlled trial with four parallel groups in Jinja district, eastern Uganda. We randomly allocated Ugandan children aged 1-10 years with uncomplicated falciparum malaria and normal G6PD enzyme function to receive artemether-lumefantrine, combined with either placebo or with 0.1 mg/kg, 0.4 mg/kg, or 0.75 mg/kg (WHO reference dose) primaquine base. Randomisation was done with computer-generated four-digit treatment assignment codes allocated to random dose groups in block sizes of 16. Study staff who provided care or assessed outcomes and the participants remained masked to the intervention group after assignment. The primary efficacy endpoint was the non-inferiority of the mean duration of gametocyte carriage in the test doses compared with the reference group of 0.75 mg primaquine per kg, with a non-inferiority margin of 2.5 days. The primary safety endpoint was the superiority of the arithmetic mean maximum decrease in haemoglobin concentration from enrolment to day 28 of follow-up in the primaquine treatment groups compared with placebo, with use of significance testing of pairwise comparisons with a cutoff of p=0.05. The trial is registered with ClinicalTrials.gov, number NCT01365598. FINDINGS: We randomly allocated 468 participants to receive artemether-lumefantrine combined with placebo (119 children) or with 0.1 mg/kg (116), 0.4 mg/kg (116), or 0.75 mg/kg (117) primaquine base. The mean duration of gametocyte carriage was 6.6 days (95% CI 5.3-7.8) in the 0.75 mg/kg reference group, 6.3 days (5.1-7.5) in the 0.4 mg/kg primaquine group (p=0.74), 8.0 days (6.6-9.4) in the 0.1 mg/kg primaquine group (p=0.14), and 12.4 days (9.9-15.0) in the placebo group (p<0.0001). No children showed evidence of treatment-related haemolysis, and the mean maximum decrease in haemoglobin concentration was not associated with the dose of primaquine received-it did not differ significantly compared with placebo (10.7 g/L, SD 11.1) in the 0.1 mg/kg (11.4 g/L, 9.4; p=0.61), 0.4 mg/kg (11.3 g/L, 10.0; p=0.67), or 0.75 mg/kg (12.7 g/L, 8.2; p=0.11) primaquine groups. INTERPRETATION: We conclude that 0.4 mg/kg primaquine has similar gametocytocidal efficacy to the reference 0.75 mg/kg primaquine dose, but a dose of 0.1 mg/kg was inconclusive for non-inferiority. Our findings call for the prioritisation of further trials into the efficacy and safety of doses of primaquine between 0.1 mg/kg and 0.4 mg/kg (including the dose of 0.25 mg/kg recently recommended by WHO), in view of the potential for widespread use of the drug to block malaria transmission. FUNDING: Wellcome Trust and the Bill & Melinda Gates Foundation.

Kloprogge F, Piola P, Dhorda M, Muwanga S, Turyakira E, Apinan S, Lindegårdh N, Nosten F et al. 2013. Population Pharmacokinetics of Lumefantrine in Pregnant and Nonpregnant Women With Uncomplicated Plasmodium falciparum Malaria in Uganda. CPT Pharmacometrics Syst Pharmacol, 2 (11), pp. e83. | Show Abstract | Read more

Pregnancy alters the pharmacokinetic properties of many antimalarial compounds. The objective of this study was to evaluate the pharmacokinetic properties of lumefantrine in pregnant and nonpregnant women with uncomplicated Plasmodium falciparum malaria in Uganda after a standard fixed oral artemether-lumefantrine treatment. Dense venous (n = 26) and sparse capillary (n = 90) lumefantrine samples were drawn from pregnant patients. A total of 17 nonpregnant women contributed with dense venous lumefantrine samples. Lumefantrine pharmacokinetics was best described by a flexible absorption model with multiphasic disposition. Pregnancy and body temperature had a significant impact on the pharmacokinetic properties of lumefantrine. Simulations from the final model indicated 27% lower day 7 concentrations in pregnant women compared with nonpregnant women and a decreased median time of 0.92 and 0.42 days above previously defined critical concentration cutoff values (280 and 175 ng/ml, respectively). The standard artemether-lumefantrine dose regimen in P. falciparum malaria may need reevaluation in nonimmune pregnant women.CPT: Pharmacometrics & Systems Pharmacology (2013) 2, e83; doi:10.1038/psp.2013.59; advance online publication 13 November 2013.

Orth H, Jensen BO, Holtfreter MC, Kocheril SJ, Mallach S, MacKenzie C, Müller-Stöver I, Henrich B et al. 2013. Plasmodium knowlesi infection imported to Germany, January 2013. Euro Surveill, 18 (40), pp. 4-6. | Show Abstract

Plasmodium knowlesi was known as a plasmodium of macaques until P. knowlesi transmission to humans was recognised in Borneo and later throughout South-East Asia. We describe here a case of a P. knowlesi infection imported to Germany from Thailand. The patient had not taken antimalarial chemoprophylaxis and suffered from daily fever attacks. Microscopy revealed trophozoites and gametocytes resembling P. malariae. P. knowlesi malaria was confirmed by PCR.

Nantakomol D, Paul R, Palasuwan A, Day NP, White NJ, Imwong M. 2013. Evaluation of the phenotypic test and genetic analysis in the detection of glucose-6-phosphate dehydrogenase deficiency. Malar J, 12 (1), pp. 289. | Show Abstract | Read more

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is particularly prevalent in historically malaria-endemic countries. Although most individuals with G6PD deficiency are asymptomatic, deficiency can result in acute haemolytic anaemia after exposure to oxidative agents. A reliable test is necessary for diagnosing the deficiency to prevent an acute haemolytic crisis following, for example, anti-malarial treatment. The aim of this study was to investigate which method was the best predictor of this disorder. METHODS: The present study investigated four G6PD activity detections (fluorescence spot (FS), methaemoglobin reduction (MR), biochemical and cytochemical test). These methods accompanied with mutation analysis of blood samples were taken from 295 apparently healthy individuals with unknown G6PD deficiency status. RESULTS: Molecular characterization of 295 Thai adults revealed an overall prevalence of 14.2%. The G6PD Viangchan (871 G>A) was the most common (83.3%), followed by G6PD Mahidol (487G>A) (11.9%), and G6PD Union (1360 C>T) (4.8%). There were two cases of G6PD deficiency carrying the double mutations of Viangchan (871G > A)-Mahidol (487G > A) and Viangchan (871G > A)-Union (1360C > T). In comparison, the prevalence of G6PD deficiency was 6.1% by FS test and 7.1% by MR test. G6PD activity was 11 ± 2.5 IU/gHb in non-deficient females (mean ± SD), and 10.9 ± 0.6 IU/gHb in non-deficient males. The upper and lower limit cut-off points for partial and severe deficiency in adults were 5.7 IU/gHb (60% of the normal mean) and 0.95 IU/gHb (10% of the normal mean), respectively. All hemizygote, homozygote and double mutations were associated with severe enzyme deficiency (the residual enzyme activity <10% of the normal mean), whereas only 14.3% of the heterozygote mutations showed severe enzyme deficiency. Based on the cut-off value <5.7 IU/gHb, the quantitative G6PD assay diagnosed 83% of cases as G6PD-deficient. Using a cut-off number of negative cell >20% in the cytochemical assay to define G6PD deficiency, the prevalence of G6PD deficiency was closest to the molecular analysis (12.9% G6PD-deficient) compared to the others methods. CONCLUSION: The cytochemical method is a significant predictor of this disease, while FS and MR test are recommended for the detection of severe G6PD deficiency in developing countries.

Turner C, Carrara V, Aye Mya Thein N, Chit Mo Mo Win N, Turner P, Bancone G, White NJ, McGready R, Nosten F. 2013. Neonatal intensive care in a Karen refugee camp: a 4 year descriptive study. PLoS One, 8 (8), pp. e72721. | Show Abstract | Read more

BACKGROUND: A third of all deaths in children aged <5 years occur in the neonatal period. Neonatal intensive care is often considered too complex and expensive to be implemented in resource poor settings. Consequently the reductions that have been made in infant mortality in the poorest countries have not been made in the neonatal period. This manuscript describes the activities surrounding the introduction of special care baby unit (SCBU) in a refugee setting and the resulting population impact. METHODS: A SCBU was developed in Maela refugee camp on the Thailand-Myanmar border. This unit comprised of a dedicated area, basic equipment, drugs and staff training. Training was built around neonatal guidelines, comprising six clinical steps: recognition, resuscitation, examination, supportive medical care, specialised medical care, and counselling of parents with sick newborns. RESULTS: From January 2008 until December 2011, 952 infants were admitted to SCBU. The main admission diagnoses were early onset neonatal sepsis, jaundice and prematurity. Early prematurity (<34 weeks) carried the highest risk of mortality (OR 9.5, 95% CI 5.4-16.5, p<0.001). There was a significant decrease in mortality from 19.3% (2008) to 4.8% (2011) among the infants admitted for prematurity (p=0.03). The neonatal mortality in Maela camp as a whole declined by 51% from 21.8 to 10.7 deaths per 1000 live births over the corresponding period (p=0.04). Staff expressed more confidence in their ability to take care of neonates and there was a more positive attitude towards premature infants. CONCLUSION: Neonatal mortality can be reduced in a resource poor setting by introduction of a simple low cost unit specialising in care of sick neonates and run by local health workers following adequate training. Training in recognition and provision of simple interventions at a high standard can increase staff confidence and reduce fatalistic attitudes towards premature neonates.

Simpson JA, Jamsen KM, Anderson TJ, Zaloumis S, Nair S, Woodrow C, White NJ, Nosten F, Price RN. 2013. Nonlinear mixed-effects modelling of in vitro drug susceptibility and molecular correlates of multidrug resistant Plasmodium falciparum. PLoS One, 8 (7), pp. e69505. | Show Abstract | Read more

The analysis of in vitro anti-malarial drug susceptibility testing is vulnerable to the effects of different statistical approaches and selection biases. These confounding factors were assessed with respect to pfmdr1 gene mutation and amplification in 490 clinical isolates. Two statistical approaches for estimating the drug concentration associated with 50% effect (EC50 ) were compared: the commonly used standard two-stage (STS) method, and nonlinear mixed-effects modelling. The in vitro concentration-effect relationships for, chloroquine, mefloquine, lumefantrine and artesunate, were derived from clinical isolates obtained from patients on the western border of Thailand. All isolates were genotyped for polymorphisms in the pfmdr1 gene. The EC50 estimates were similar for the two statistical approaches but 15-28% of isolates in the STS method had a high coefficient of variation (>15%) for individual estimates of EC50 and these isolates had EC50 values that were 32 to 66% higher than isolates derived with more precision. In total 41% (202/490) of isolates had amplification of pfmdr1 and single nucleotide polymorphisms were found in 50 (10%). Pfmdr1 amplification was associated with an increase in EC50 for mefloquine (139% relative increase in EC50 for 2 copies, 188% for 3+ copies), lumefantrine (82% and 75% for 2 and 3+ copies respectively) and artesunate (63% and 127% for 2 and 3+ copies respectively). In contrast pfmdr1 mutation at codons 86 or 1042 were associated with an increase in chloroquine EC50 (44-48%). Sample size calculations showed that to demonstrate an EC50 shift of 50% or more with 80% power if the prevalence was 10% would require 430 isolates and 245 isolates if the prevalence was 20%. In conclusion, although nonlinear mixed-effects modelling did not demonstrate any major advantage for determining estimates of anti-malarial drug susceptibility, the method includes all isolates, thereby, potentially improving confirmation of candidate molecular markers of anti-malarial drug susceptibility.

Shanks GD, White NJ. 2013. The activation of vivax malaria hypnozoites by infectious diseases. Lancet Infect Dis, 13 (10), pp. 900-906. | Show Abstract | Read more

The periodicity of vivax malaria relapses may be explained by the activation of latent hypnozoites acquired from a previous malarial infection. The activation stimulus could be the febrile illness associated with acute malaria or a different febrile infection. We review historical records to examine the association between relapses of Plasmodium vivax and febrile infectious diseases. In data from British soldiers in Palestine, epidemic falciparum malaria triggered a smaller epidemic of P vivax relapses only in those who had been extensively exposed to malaria previously. Relapses did not follow pandemic influenza infection. Evidence from three simultaneous typhoid and malaria epidemics suggest that typhoid fever might activate P vivax hypnozoites. Some data lend support to the notion that vivax malaria relapse followed febrile illness caused by relapsing fever, trench fever, epidemic typhus, and Malta fever (brucellosis). These observations suggest that systemic parasitic and bacterial infections, but not viral infections, can activate P vivax hypnozoites. Specific components of the host's acute febrile inflammatory response, and not fever alone, are probably important factors in the provocation of a relapse of vivax malaria.

Eziefula AC, Staedke SG, Yeung S, Webb E, Kamya M, White NJ, Bousema T, Drakeley C. 2013. Study protocol for a randomised controlled double-blinded trial of the dose-dependent efficacy and safety of primaquine for clearance of gametocytes in children with uncomplicated falciparum malaria in Uganda BMJ OPEN, 3 (3), pp. e002759-e002759. | Show Abstract | Read more

Objectives: For the purpose of blocking transmission of Plasmodium falciparum malaria from humans to mosquitoes, a single dose of primaquine is recommended by theWHO as an addition to artemisinin combination therapy. Primaquine clears gametocytes but causes dose-dependent haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Evidence is needed to inform the optimal dosing of primaquine for malaria elimination programmes and for the purpose of interrupting the spread of artemisininresistant malaria. This study investigates the efficacy and safety of reducing doses of primaquine for clearance of gametocytes in participants with normal G6PD status. Methods and analysis: In this prospective, fourarmed randomised placebo-controlled double-blinded trial, children aged 1-10 years, weighing over 10 kg, with haemoglobin ≥8 g/dl and uncomplicated P falciparum malaria are treated with artemether lumefantrine and randomised to receive a dose of primaquine (0.1, 0.4 or 0.75 mg base/kg) or placebo on the third day of treatment. Participants are followed up for 28 days. Gametocytaemia is measured by quantitative nucleic acid sequence-based analysis on days 0, 2, 3, 7, 10 and 14 with a primary endpoint of the number of days to gametocyte clearance in each treatment arm and secondarily the area under the curve of gametocyte density over time. Analysis is for non-inferiority of efficacy compared to the reference dose, 0.75 mg base/kg. Safety is assessed by pair-wise comparisons of the arithmetic mean (±SD) change in haemoglobin concentration per treatment arm and analysed for superiority to placebo and incidence of adverse events. Ethics and dissemination Approval was obtained from the ethical committees of Makerere University School of Medicine, the Ugandan National Council of Science and Technology and the London School of Hygiene and Tropical Medicine. Results: These will be disseminated to inform malaria elimination policy, through peer-reviewed publication and academic presentations.

White NJ. 2013. Primaquine to prevent transmission of falciparum malaria. The Lancet infectious diseases, 13 (2), pp. 175-181. | Show Abstract

Falciparum malaria is transmitted by anopheline mosquitoes that have fed on blood containing gametocytes of Plasmodium falciparum. In areas of low malaria transmission, where symptomatic infections contribute substantially to malaria transmission, the use of gametocytocidal drugs reduces the incidence of malaria. Artemisinin-based combination therapies provide high cure rates and substantially reduce gametocyte carriage. Artemisinin resistance in P falciparum lessens overall gametocytocidal activity, which provides a selective pressure to the spread of these resistant parasites. The 8-aminoquinoline compounds possess unique gametocytocidal properties and rapidly sterilise the mature transmissible stages of P falciparum. The addition of one dose of primaquine to artemisinin-based combination regimens could help to counter the spread of artemisinin resistance. Although primaquine is commonly recommended for falciparum and vivax malaria, concerns about drug-related haemolysis frequently prevent its administration. The limited available evidence on transmission-blocking effects of primaquine and its forerunner plasmoquine suggests that doses lower than currently recommended (0.50-0.75 mg base per kg), which would be safer, might still be very effective. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hanson JP, Lam SWK, Mohanty S, Alam S, Pattnaik R, Mahanta KC, Hasan MU, Charunwatthana P et al. 2013. Fluid resuscitation of adults with severe falciparum malaria: Effects on acid-base status, renal function, and extravascular lung water Critical Care Medicine, 41 (4), pp. 972-981. | Show Abstract | Read more

Objective: To evaluate the efficacy and safety of liberal fluid resuscitation of adults with severe malaria. DESIGN, Setting, Patients, and Methods:: Twenty-eight Bangladeshi and Indian adults with severe falciparum malaria received crystalloid resuscitation guided by transpulmonary thermodilution (PiCCO) in an intensive care setting. Systemic hemodynamics, microvascular indices and measures of acidosis, renal function, and pulmonary edema were followed prospectively. Results: All patients were hypovolemic (global end-diastolic volume index < 680 mL/m) on enrollment. Patients received a median (range) 3230 mL (390-7300) of isotonic saline in the first 6 hours and 5450 mL (710-13,720) in the first 24 hours. With resuscitation, acid-base status deteriorated in 19 of 28 (68%), and there was no significant improvement in renal function. Extravascular lung water increased in 17 of 22 liberally resuscitated patients (77%); eight of these patients developed pulmonary edema, five of whom died. All other patients survived. All patients with pulmonary edema during the study were hypovolemic or euvolemic at the time pulmonary edema developed. Plasma lactate was lower in hypovolemic patients before (rs = 0.38; p = 0.05) and after (rs = 0.49; p = 0.01) resuscitation but was the strongest predictor of mortality before (chi-square = 9.9; p = 0.002) and after resuscitation (chi-square = 11.1; p < 0.001) and correlated with the degree of visualized microvascular sequestration of parasitized erythrocytes at both time points (rs = 0.55; p = 0.003 and rs = 0.43; p = 0.03, respectively). Persisting sequestration was evident in 7 of 15 patients (47%) 48 hours after enrollment. Conclusions: Lactic acidosis-the strongest prognostic indicator in adults with severe falciparum malaria-results from sequestration of parasitized erythrocytes in the microcirculation, not from hypovolemia. Liberal fluid resuscitation has little effect on this sequestration and does not improve acid-base status or renal function. Pulmonary edema-secondary to increased pulmonary vascular permeability-is common, unpredictable, and exacerbated by fluid loading. Liberal fluid replacement of adults with severe malaria should be avoided. © 2013 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins.

Boel ME, Rijken MJ, Leenstra T, Phyo AP, Pimanpanarak M, Keereecharoen NL, Proux S, Laochan N et al. 2013. Malaria in the post-partum period; a prospective cohort study. PLoS One, 8 (3), pp. e57890. | Show Abstract | Read more

BACKGROUND: Several studies have shown a prolonged or increased susceptibility to malaria in the post-partum period. A matched cohort study was conducted to evaluate prospectively the susceptibility to malaria of post-partum women in an area where P.falciparum and P.vivax are prevalent. METHODS: In an area of low seasonal malaria transmission on the Thai-Myanmar border pregnant women attending antenatal clinics were matched to a non-pregnant, non-post-partum control and followed up prospectively until 12 weeks after delivery. RESULTS: Post-partum women (n = 744) experienced significantly less P.falciparum episodes than controls (hazard ratio (HR) 0.39 (95%CI 0.21-0.72) p = 0.003) but significantly more P.vivax (HR 1.34 (1.05-1.72) p = 0.018). The reduced risk of falciparum malaria was accounted for by reduced exposure, whereas a history of P.vivax infection during pregnancy was a strong risk factor for P.vivax in post-partum women (HR 13.98 (9.13-21.41), p<0.001). After controlling for effect modification by history of P.vivax, post-partum women were not more susceptible to P.vivax than controls (HR: 0.33 (0.21-0.51), p<0.001). Genotyping of pre-and post-partum infections [Formula in text] showed that each post-partum P.falciparum was a newly acquired infection. CONCLUSIONS: In this area of low seasonal malaria transmission post-partum women were less likely to develop falciparum malaria but more likely to develop vivax malaria than controls. This was explained by reduced risk of exposure and increased risk of relapse, respectively. There was no evidence for altered susceptibility to malaria in the post-partum period. The treatment of vivax malaria during and immediately after pregnancy needs to be improved.

Awab GR, Pukrittayakamee S, Jamornthanyawat N, Yamin F, Dondorp AM, Day NP, White NJ, Woodrow CJ, Imwong M. 2013. Prevalence of antifolate resistance mutations in Plasmodium falciparum isolates in Afghanistan. Malar J, 12 (1), pp. 96. | Show Abstract | Read more

BACKGROUND: Artesunate plus sulphadoxine-pyrimethamine (AS+SP) is now first-line treatment for Plasmodium falciparum infection in several south Asian countries, including Afghanistan. Molecular studies provide a sensitive means to investigate the current state of drug susceptibility to the SP component, and can also provide information on the likely efficacy of other potential forms of artemisinin-combination therapy. METHODS: During the years 2007 to 2010, 120 blood spots from patients with P. falciparum malaria were obtained in four provinces of Afghanistan. PCR-based methods were used to detect drug-resistance mutations in dhfr, dhps, pfcrt and pfmdr1, as well as to determine copy number of pfmdr1. RESULTS: The majority (95.5%) of infections had a double mutation in the dhfr gene (C59R, S108N); no mutations at dhfr positions 16, 51 or 164 were seen. Most isolates were wild type across the dhps gene, but five isolates from the provinces of Kunar and Nangarhar in eastern Afghanistan had the triple mutation A437G / K540E / A581G; all five cases were successfully treated with three receiving AS+SP and two receiving dihydroartemisinin-piperaquine. All isolates showed the pfcrt SVNMT chloroquine resistance haplotype. Five of 79 isolates had the pfmdr1 N86Y mutation, while 52 had pfmdr1 Y184F; positions 1034, 1042 and 1246 were wild type in all isolates. The pfmdr1 gene was not amplified in any sample. CONCLUSIONS: This study indicates that shortly after the adoption of AS+SP as first-line treatment in Afghanistan, most parasites had a double mutation haplotype in dhfr, and a small number of isolates from eastern Afghanistan harboured a triple mutation haplotype in dhps. The impact of these mutations on the efficacy of AS+SP remains to be assessed in significant numbers of patients, but these results are clearly concerning since they suggest a higher degree of SP resistance than previously detected. Further focused molecular and clinical studies in this region are urgently required.

Prapansilp P, Medana I, Mai NT, Day NP, Phu NH, Yeo TW, Hien TT, White NJ, Anstey NM, Turner GD. 2013. A clinicopathological correlation of the expression of the angiopoietin-Tie-2 receptor pathway in the brain of adults with Plasmodium falciparum malaria. Malar J, 12 (1), pp. 50. | Show Abstract | Read more

BACKGROUND: Plasma angiopoietin (Ang)-2 is associated with disease severity and mortality in adults and children with falciparum malaria. However the mechanism of action of the angiopoietins in fatal malaria is unclear. This study aimed to determine whether the expression of Ang-1 and Ang-2 and their receptor Tie-2 in cerebral endothelial or parenchymal cells was specific to cerebral malaria (CM), correlated with coma or other severe clinical features, and whether plasma and CSF levels of these markers correlated with the clinical and neuropathological features of severe and fatal malaria in Vietnamese adults. METHODS: Immunohistochemistry was performed for Ang-1, Ang-2 and Tie-2 on post-mortem brain tissue from fatal malaria cases and controls. Quantitative ELISA for plasma and cerebrospinal fluid levels of Ang-1, Ang-2 and Tie-2 was done to compare fatal cases with surviving patients from the same study. RESULTS: Immunohistochemistry revealed significant differences in expression in endothelial and parenchymal cells compared to controls. However there was no significant difference in expression of these markers on endothelial cells, astroglial cells or neurons between CM and non-cerebral malaria cases. Immunostaining of Ang-1, Ang-2 and Tie-2 was also not associated with Plasmodium falciparum-infected erythrocyte sequestration in the brain. However Ang-1 and Ang-2 expression in neurons was significantly correlated with the incidence of microscopic haemorrhages. Plasma levels of Ang-2 and Ang-2/Ang-1 ratio were associated with the number of severe malaria complications and were significant and independent predictors of metabolic acidosis and fatal outcome. CONCLUSIONS: The independent prognostic significance of Ang-2 and the Ang-2/Ang-1 ratio in severe malaria was confirmed, although immunohistochemistry in fatal cases did not reveal increased expression on brain endothelium in cerebral versus non-cerebral cases. Activation of the Ang-Tie-2 pathway in severe malaria is therefore related to acidosis, number of severity criteria and outcome, but is not a specific event in the brain during cerebral malaria.

Deen J, White NJ, Lubell Y. 2013. Bloodstream infections in south and southeast Asia - authors' reply. Lancet Infect Dis, 13 (1), pp. 15. | Read more

Turner C, Turner P, Carrara V, Burgoine K, Tha Ler Htoo S, Watthanaworawit W, Day NP, White NJ, Goldblatt D, Nosten F. 2013. High rates of pneumonia in children under two years of age in a South East Asian refugee population. PLoS One, 8 (1), pp. e54026. | Show Abstract | Read more

BACKGROUND: There are an estimated 150 million episodes of childhood pneumonia per year, with 11-20 million hospital admissions and 1.575 million deaths. Refugee children are particularly vulnerable, with poorly defined pneumonia epidemiology. METHODS: We followed a birth cohort of 955 refugee infants, born over a one-year period, until two years of age. Clinical and radiographic pneumonia were diagnosed according to WHO criteria. Detailed characteristics were collected to determine risk factors for clinical, radiological and multiple episodes of pneumonia. Investigations were taken during a pneumonia episode to help determine or to infer an aetiological diagnosis. FINDINGS: The incidence of clinical pneumonia was 0.73 (95% CI 0.70-0.75) episodes per child year (/CY) and of radiological primary endpoint pneumonia (PEP) was 0.22/CY (95% CI 0.20-0.24). The incidence of pneumonia without severe signs was 0.50/CY (95% CI 0.48-0.53), severe pneumonia 0.15/CY (95% CI 0.13-0.17) and very severe pneumonia 0.06/CY (0.05-0.07). Virus was detected, from a nasopharyngeal aspirate, in 61.3% of episodes. A reduced volume of living space per person (IRR 0.99, 95% CI 0.99-1.0, p = 0.003) and young maternal age (IRR 1.59, 95% CI 1.12-2.27, p = 0.01) were risk factors for developing pneumonia. The risk of a child having >1 episode of pneumonia was increased by having a shorter distance to the next house (IRR 0.86, 95% CI 0.74-1.00, p = 0.04). Infants were at risk of having an episode of PEP if there was a shorter distance from stove to bed (IRR 0.89, 95% CI 0.80-0.99, p = 0.03). Raised CRP and neutrophil values were associated with PEP. CONCLUSIONS: There was a high incidence of pneumonia in young children in this SE Asian refugee population. Viral infections were important, however CXR and non-specific marker findings suggested that bacteria may be involved in up to a third of cases.

Kauss T, Gaubert A, Boyer C, Ba BB, Manse M, Massip S, Léger JM, Fawaz F et al. 2013. Pharmaceutical development and optimization of azithromycin suppository for paediatric use International Journal of Pharmaceutics, 441 (1-2), pp. 218-226. | Show Abstract | Read more

Pharmaceutical development and manufacturing process optimization work was undertaken in order to propose a potential paediatric rectal formulation of azithromycin as an alternative to existing oral or injectable formulations. The target product profile was to be easy-to-use, cheap and stable in tropical conditions, with bioavailability comparable to oral forms, rapidly achieving and maintaining bactericidal concentrations. PEG solid solution suppositories were characterized in vitro using visual, HPLC, DSC, FTIR and XRD analyses. In vitro drug release and in vivo bioavailability were assessed; a study in rabbits compared the bioavailability of the optimized solid solution suppository to rectal solution and intra-venous product (as reference) and to the previous, non-optimized formulation (suspended azithromycin suppository). The bioavailability of azithromycin administered as solid solution suppositories relative to intra-venous was 43%, which compared well to the target of 38% (oral product in humans). The results of 3-month preliminary stability and feasibility studies were consistent with industrial production scale-up. This product has potential both as a classical antibiotic and as a product for use in severely ill children in rural areas. Industrial partners for further development are being sought. © 2012 Elsevier B.V. All rights reserved.

Anekthananon T, Pukrittayakamee S, Ratanasuwan W, Jittamala P, Werarak P, Charunwatthana P, Suwanagool S, Lawpoolsri S et al. 2013. Oseltamivir and inhaled zanamivir as influenza prophylaxis in Thai health workers: a randomized, double-blind, placebo-controlled safety trial over 16 weeks. J Antimicrob Chemother, 68 (3), pp. 697-707. | Show Abstract | Read more

OBJECTIVES: Long-term chemoprophylaxis using neuraminidase inhibitors may be needed during influenza epidemics but safety data are limited to several weeks. We sought to assess the tolerability of oseltamivir and zanamivir as primary prophylaxis over 16 weeks. METHODS: We conducted a parallel group, double blind, 2 (active drug) :1 (placebo) randomized trial of oral oseltamivir/placebo or inhaled zanamivir/placebo over 16 weeks in healthy, Thai hospital professionals at two Bangkok hospitals. The primary endpoint was study withdrawal due to drug-related (possibly, probably, definitely) serious or adverse events (AEs) graded ≥ 2. RESULTS: Recruited subjects numbered 129 oseltamivir/65 placebo and 131 zanamivir/65 placebo. A total of 102 grade ≥ 2 AEs were reported or detected in 69 subjects: 23/129 (17.8%) versus 15/65 (23.1%) (P=0.26), and 23/131 (17.6%) versus 8/65 (12.3%) (P=0.28). Intercurrent infections/fevers [26/102 (25.5%)], abnormal biochemistry [25/102 (24.5%)] and gastrointestinal symptoms [18/102 (17.6%)] were the most frequently reported AEs. There were no drug-related study withdrawals. Eight serious AEs were all due to intercurrent illnesses. Laboratory, lung function and ECG parameters were similar between drugs and placebos. CONCLUSIONS: Oseltamivir and zanamivir were well tolerated in healthy hospital professionals. Both drugs can be recommended for primary influenza prophylaxis for up to 16 weeks.

Lwin KM, Peto TJ, White NJ, Day NPJ, Nosten F, Parker M, Cheah PY. 2013. The practicality and sustainability of a community advisory board at a large medical research unit on the Thai-Myanmar border Health, 05 (02), pp. 229-236. | Read more

Takala-Harrison S, Clark TG, Jacob CG, Cummings MP, Miotto O, Dondorp AM, Fukuda MM, Nosten F et al. 2013. Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia. Proc Natl Acad Sci U S A, 110 (1), pp. 240-245. | Show Abstract | Read more

The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.

Hoglund RM, Adam I, Hanpithakpong W, Ashton M, Lindegardh N, Day NP, White NJ, Nosten F, Tarning J. 2012. A population pharmacokinetic model of piperaquine in pregnant and non-pregnant women with uncomplicated Plasmodium falciparum malaria in Sudan. Malar J, 11 (1), pp. 398. | Show Abstract | Read more

BACKGROUND: Pregnancy is associated with an increased risk of developing a malaria infection and a higher risk of developing severe malaria. The pharmacokinetic properties of many anti-malarials are also altered during pregnancy, often resulting in a decreased drug exposure. Piperaquine is a promising anti-malarial partner drug used in a fixed-dose combination with dihydroartemisinin. The aim of this study was to investigate the population pharmacokinetics of piperaquine in pregnant and non-pregnant Sudanese women with uncomplicated Plasmodium falciparum malaria. METHOD: Symptomatic patients received a standard dose regimen of the fixed dose oral piperaquine-dihydroartemisinin combination treatment. Densely sampled plasma aliquots were collected and analysed using a previously described LC-MS/MS method. Data from 12 pregnant and 12 non-pregnant women were analysed using nonlinear mixed-effects modelling. A Monte Carlo Mapped Power (MCMP) analysis was conducted based on a previously published study to evaluate the power of detecting covariates in this relatively small study. RESULTS: A three-compartment disposition model with a transit-absorption model described the observed data well. Body weight was added as an allometric function on all clearance and volume parameters. A statistically significant decrease in estimated terminal piperaquine half-life in pregnant compared with non-pregnant women was found, but there were no differences in post-hoc estimates of total piperaquine exposure. The MCMP analysis indicated a minimum of 13 pregnant and 13 non-pregnant women were required to identify pregnancy as a covariate on relevant pharmacokinetic parameters (80% power and p=0.05). Pregnancy was, therefore, evaluated as a categorical and continuous covariate (i.e. estimate gestational age) in a full covariate approach. Using this approach pregnancy was not associated with any major change in piperaquine elimination clearance. However, a trend of increasing elimination clearance with increasing gestational age could be seen. CONCLUSIONS: The population pharmacokinetic properties of piperaquine were well described by a three-compartment disposition model in pregnant and non-pregnant women with uncomplicated malaria. The modelling approach showed no major difference in piperaquine exposure between the two groups and data presented here do not warrant a dose adjustment in pregnancy in this vulnerable population.

Hendriksen IC, Maiga D, Lemnge MM, Mtove G, Gesase S, Reyburn H, Lindegardh N, Day NP et al. 2013. Population pharmacokinetic and pharmacodynamic properties of intramuscular quinine in Tanzanian children with severe Falciparum malaria. Antimicrob Agents Chemother, 57 (2), pp. 775-783. | Show Abstract | Read more

Although artesunate is clearly superior, parenteral quinine is still used widely for the treatment of severe malaria. A loading-dose regimen has been recommended for 30 years but is still often not used. A population pharmacokinetic study was conducted with 75 Tanzanian children aged 4 months to 8 years with severe malaria who received quinine intramuscularly; 69 patients received a loading dose of 20 mg quinine dihydrochloride (salt)/kg of body weight. Twenty-one patients had plasma quinine concentrations detectable at baseline. A zero-order absorption model with one-compartment disposition pharmacokinetics described the data adequately. Body weight was the only significant covariate and was implemented as an allometric function on clearance and volume parameters. Population pharmacokinetic parameter estimates (and percent relative standard errors [%RSE]) of elimination clearance, central volume of distribution, and duration of zero-order absorption were 0.977 liters/h (6.50%), 16.7 liters (6.39%), and 1.42 h (21.5%), respectively, for a typical patient weighing 11 kg. Quinine exposure was reduced at lower body weights after standard weight-based dosing; there was 18% less exposure over 24 h in patients weighing 5 kg than in those weighing 25 kg. Maximum plasma concentrations after the loading dose were unaffected by body weight. There was no evidence of dose-related drug toxicity with the loading dosing regimen. Intramuscular quinine is rapidly and reliably absorbed in children with severe falciparum malaria. Based on these pharmacokinetic data, a loading dose of 20 mg salt/kg is recommended, provided that no loading dose was administered within 24 h and no routine dose was administered within 12 h of admission. (This study has been registered with Current Controlled Trials under registration number ISRCTN 50258054.).

Das D, Tripura R, Phyo AP, Lwin KM, Tarning J, Lee SJ, Hanpithakpong W, Stepniewska K et al. 2013. Effect of high-dose or split-dose artesunate on parasite clearance in artemisinin-resistant falciparum malaria. Clin Infect Dis, 56 (5), pp. e48-e58. | Show Abstract | Read more

BACKGROUND: The emergence of Plasmodium falciparum resistance to artemisinins on the Cambodian and Myanmar-Thai borders poses severe threats to malaria control. We investigated whether increasing or splitting the dose of the short-half-life drug artesunate improves parasite clearance in falciparum malaria in the 2 regions. METHODS: In Pailin, western Cambodia (from 2008 to 2010), and Wang Pha, northwestern Thailand (2009-2010), patients with uncomplicated falciparum malaria were randomized to oral artesunate 6 mg/kg/d as a once-daily or twice-daily dose for 7 days, or artesunate 8 mg/kg/d as a once-daily or twice-daily dose for 3 days, followed by mefloquine. Parasite clearance and recrudescence for up to 63 days of follow-up were assessed. RESULTS: A total of 159 patients were enrolled. Overall median (interquartile range [IQR]) parasitemia half-life (half-life) was 6.03 (4.89-7.28) hours in Pailin versus 3.42 (2.20-4.85) hours in Wang Pha (P = .0001). Splitting or increasing the artesunate dose did not shorten half-life in either site. Pharmacokinetic profiles of artesunate and dihydroartemisinin were similar between sites and did not correlate with half-life. Recrudescent infections occurred in 4 of 79 patients in Pailin and 5 of 80 in Wang Pha and was not different between treatment arms (P = .68). CONCLUSIONS: Increasing the artesunate treatment dose up to 8 mg/kg/d or splitting the dose does not improve parasite clearance in either artemisinin resistant or more sensitive infections with P. falciparum. Clinical Trials Registration. ISRCTN15351875.

Hendriksen IC, White LJ, Veenemans J, Mtove G, Woodrow C, Amos B, Saiwaew S, Gesase S et al. 2013. Defining falciparum-malaria-attributable severe febrile illness in moderate-to-high transmission settings on the basis of plasma PfHRP2 concentration. J Infect Dis, 207 (2), pp. 351-361. | Show Abstract | Read more

BACKGROUND: In malaria-endemic settings, asymptomatic parasitemia complicates the diagnosis of malaria. Histidine-rich protein 2 (HRP2) is produced by Plasmodium falciparum, and its plasma concentration reflects the total body parasite burden. We aimed to define the malaria-attributable fraction of severe febrile illness, using the distributions of plasma P. falciparum HRP2 (PfHRP2) concentrations from parasitemic children with different clinical presentations. METHODS: Plasma samples were collected from and peripheral blood slides prepared for 1435 children aged 6-60 months in communities and a nearby hospital in northeastern Tanzania. The study population included children with severe or uncomplicated malaria, asymptomatic carriers, and healthy control subjects who had negative results of rapid diagnostic tests. The distributions of plasma PfHRP2 concentrations among the different groups were used to model severe malaria-attributable disease. RESULTS: The plasma PfHRP2 concentration showed a close correlation with the severity of infection. PfHRP2 concentrations of >1000 ng/mL denoted a malaria-attributable fraction of severe disease of 99% (95% credible interval [CI], 96%-100%), with a sensitivity of 74% (95% CI, 72%-77%), whereas a concentration of <200 ng/mL denoted severe febrile illness of an alternative diagnosis in >10% (95% CI, 3%-27%) of patients. Bacteremia was more common among patients in the lowest and highest PfHRP2 concentration quintiles. CONCLUSIONS: The plasma PfHRP2 concentration defines malaria-attributable disease and distinguishes severe malaria from coincidental parasitemia in African children in a moderate-to-high transmission setting.

Hanson J, Dondorp AM, Day NP, White NJ. 2013. Reply to Cunnington et al. J Infect Dis, 207 (2), pp. 370-371. | Read more

White NJ. 2012. Counter perspective: artemisinin resistance: facts, fears, and fables. Am J Trop Med Hyg, 87 (5), pp. 785. | Read more

White NJ. 2012. Melioidosis and Glanders pp. 580-583. | Read more

Wuthiekanun V, Amornchai P, Paris DH, Langla S, Thaipadunpanit J, Chierakul W, Smythe LD, White NJ, Day NP, Limmathurotsakul D, Peacock SJ. 2013. Rapid isolation and susceptibility testing of Leptospira spp. using a new solid medium, LVW agar. Antimicrob Agents Chemother, 57 (1), pp. 297-302. | Show Abstract | Read more

Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO(2) incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO(2) for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC(90) values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 μg/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research.

Hien TT, Thuy-Nhien NT, Phu NH, Boni MF, Thanh NV, Nha-Ca NT, Thai LEH, Thai CQ et al. 2012. In vivo susceptibility of Plasmodium falciparum to artesunate in Binh Phuoc Province, Vietnam. Malar J, 11 (1), pp. 355. | Show Abstract | Read more

BACKGROUND: By 2009, there were worrying signs from western Cambodia that parasitological responses to artesunate-containing treatment regimens for uncomplicated Plasmodium falciparum malaria were slower than elsewhere which suggested the emergence of artemisinin resistance. Vietnam shares a long land border with Cambodia with a large number of migrants crossing it on a daily basis. Therefore, there is an urgent need to investigate whether there is any evidence of a change in the parasitological response to the artemisinin derivatives in Vietnam. METHODS: From August 2010 to May 2011, a randomized controlled clinical trial in uncomplicated falciparum malaria was conducted to compare two doses of artesunate (AS) (2mg/kg/day versus 4 mg/kg/day for three days) followed by dihydroartemisinin-piperaquine (DHA-PPQ) and a control arm of DHA-PPQ. The goal was characterization of the current efficacy of artesunate in southern Vietnam. The primary endpoint of this study was the parasite clearance half-life; secondary endpoints included the parasite reduction ratios at 24 and 48 hours and the parasite clearance time. RESULTS: 166 patients were recruited into the study. The median parasite clearance half-lives were 3.54 (AS 2mg/kg), 2.72 (AS 4mg/kg), and 2.98 hours (DHA-PPQ) (p=0.19). The median parasite-reduction ratio at 24 hours was 48 in the AS 2mg/kg group compared with 212 and 113 in the other two groups, respectively (p=0.02). The proportions of patients with a parasite clearance time of >72 hours for AS 2mg/kg, AS 4mg/kg and DHA-PPQ were 27%, 27%, and 22%, respectively. Early treatment failure occurred in two (4%) and late clinical failure occurred in one (2%) of the 55 patients in the AS 2mg/kg group, as compared with none in the other two study arms. The PCR-corrected adequate clinical and parasitological response (APCR) rates in the three groups were 94%, 100%, and 100% (p=0.04). CONCLUSIONS: This study demonstrated faster P. falciparum parasite clearance in southern Vietnam than in western Cambodia but slower clearance in comparison with historical data from Vietnam. Further studies to determine whether this represents the emergence of artemisinin resistance in this area are needed. Currently, the therapeutic response to DHA-PPQ remains satisfactory in southern Vietnam. TRIAL REGISTRATION: NTC01165372.

Maude RJ, Hasan MU, Hossain MA, Sayeed AA, Kanti Paul S, Rahman W, Maude RR, Vaid N et al. 2012. Temporal trends in severe malaria in Chittagong, Bangladesh. Malar J, 11 (1), pp. 323. | Show Abstract | Read more

BACKGROUND: Epidemiological data on malaria in Bangladesh are sparse, particularly on severe and fatal malaria. This hampers the allocation of healthcare provision in this resource-poor setting. Over 85% of the estimated 150,000-250,000 annual malaria cases in Bangladesh occur in Chittagong Division with 80% in the Chittagong Hill Tracts (CHT). Chittagong Medical College Hospital (CMCH) is the major tertiary referral hospital for severe malaria in Chittagong Division. METHODS: Malaria screening data from 22,785 inpatients in CMCH from 1999-2011 were analysed to investigate the patterns of referral, temporal trends and geographical distribution of severe malaria in Chittagong Division, Bangladesh. RESULTS: From 1999 till 2011, 2,394 malaria cases were admitted, of which 96% harboured Plasmodium falciparum and 4% Plasmodium vivax. Infection was commonest in males (67%) between 15 and 34 years of age. Seasonality of malaria incidence was marked with a single peak in P. falciparum transmission from June to August coinciding with peak rainfall, whereas P. vivax showed an additional peak in February-March possibly representing relapse infections. Since 2007 there has been a substantial decrease in the absolute number of admitted malaria cases. Case fatality in severe malaria was 18% from 2008-2011, remaining steady during this period.A travel history obtained in 226 malaria patients revealed only 33% had been to the CHT in the preceding three weeks. Of all admitted malaria patients, only 9% lived in the CHT, and none in the more remote malaria endemic regions near the Indian border. CONCLUSIONS: The overall decline in admitted malaria cases to CMCH suggests recent control measures are successful. However, there are no reliable data on the incidence of severe malaria in the CHT, the most endemic area of Bangladesh, and most of these patients do not reach tertiary health facilities. Improvement of early treatment and simple supportive care for severe malaria in remote areas and implementation of a referral system for cases requiring additional supportive care could be important contributors to further reducing malaria-attributable disease and death in Bangladesh.

Amaratunga C, Sreng S, Suon S, Phelps ES, Stepniewska K, Lim P, Zhou C, Mao S et al. 2012. Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study. Lancet Infect Dis, 12 (11), pp. 851-858. | Show Abstract | Read more

BACKGROUND: Artemisinin-resistant Plasmodium falciparum has been reported in Pailin, western Cambodia, detected as a slow parasite clearance rate in vivo. Emergence of this phenotype in western Thailand and possibly elsewhere threatens to compromise the effectiveness of all artemisinin-based combination therapies. Parasite genetics is associated with parasite clearance rate but does not account for all variation. We investigated contributions of both parasite genetics and host factors to the artemisinin-resistance phenotype in Pursat, western Cambodia. METHODS: Between June 19 and Nov 28, 2009, and June 26 and Dec 6, 2010, we enrolled patients aged 10 years or older with uncomplicated falciparum malaria, a density of asexual parasites of at least 10,000 per μL of whole blood, no symptoms or signs of severe malaria, no other cause of febrile illness, and no chronic illness. We gave participants 4 mg/kg artesunate at 0, 24, and 48 h, 15 mg/kg mefloquine at 72 h, and 10 mg/kg mefloquine at 96 h. We assessed parasite density on thick blood films every 6 h until undetectable. The parasite clearance half-life was calculated from the parasite clearance curve. We genotyped parasites with 18 microsatellite markers and patients for haemoglobin E, α-thalassaemia, and a mutation of G6PD, which encodes glucose-6-phosphate dehydrogenase. To account for the possible effects of acquired immunity on half-life, we used three surrogates for increased likelihood of exposure to P falciparum: age, sex, and place of residence. This study is registered with ClinicalTrials.gov, number NCT00341003. FINDINGS: We assessed 3504 individuals from all six districts of Pursat province seeking treatment for malaria symptoms. We enrolled 168 patients with falciparum malaria who met inclusion criteria. The geometric mean half-life was 5·85 h (95% CI 5·54-6·18) in Pursat, similar to that reported in Pailin (p=0·109). We identified two genetically different parasite clone groups: parasite group 1 (PG1) and parasite group 2 (PG2). Non-significant increases in parasite clearance half-life were seen in patients with haemoglobin E (0·55 h; p=0·078), those of male sex (0·96 h; p=0·064), and in 2010 (0·68 h; p=0·068); PG1 was associated with a significant increase (0·79 h; p=0·033). The mean parasite heritability of half-life was 0·40 (SD 0·17). INTERPRETATION: Heritable artemisinin resistance is established in a second Cambodian province. To accurately identify parasites that are intrinsically susceptible or resistant to artemisinins, future studies should explore the effect of erythrocyte polymorphisms and specific immune responses on half-life variation. FUNDING: Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Tarning J, Chotsiri P, Jullien V, Rijken MJ, Bergstrand M, Cammas M, McGready R, Singhasivanon P et al. 2012. Population pharmacokinetic and pharmacodynamic modeling of amodiaquine and desethylamodiaquine in women with Plasmodium vivax malaria during and after pregnancy. Antimicrob Agents Chemother, 56 (11), pp. 5764-5773. | Show Abstract | Read more

Amodiaquine is effective for the treatment of Plasmodium vivax malaria, but there is little information on the pharmacokinetic and pharmacodynamic properties of amodiaquine in pregnant women with malaria. This study evaluated the population pharmacokinetic and pharmacodynamic properties of amodiaquine and its biologically active metabolite, desethylamodiaquine, in pregnant women with P. vivax infection and again after delivery. Twenty-seven pregnant women infected with P. vivax malaria on the Thai-Myanmar border were treated with amodiaquine monotherapy (10 mg/kg/day) once daily for 3 days. Nineteen women, with and without P. vivax infections, returned to receive the same amodiaquine dose postpartum. Nonlinear mixed-effects modeling was used to evaluate the population pharmacokinetic and pharmacodynamic properties of amodiaquine and desethylamodiaquine. Amodiaquine plasma concentrations were described accurately by lagged first-order absorption with a two-compartment disposition model followed by a three-compartment disposition of desethylamodiaquine under the assumption of complete in vivo conversion. Body weight was implemented as an allometric function on all clearance and volume parameters. Amodiaquine clearance decreased linearly with age, and absorption lag time was reduced in pregnant patients. Recurrent malaria infections in pregnant women were modeled with a time-to-event model consisting of a constant-hazard function with an inhibitory effect of desethylamodiaquine. Amodiaquine treatment reduced the risk of recurrent infections from 22.2% to 7.4% at day 35. In conclusion, pregnancy did not have a clinically relevant impact on the pharmacokinetic properties of amodiaquine or desethylamodiaquine. No dose adjustments are required in pregnancy.

Tarning J, Kloprogge F, Piola P, Dhorda M, Muwanga S, Turyakira E, Nuengchamnong N, Nosten F et al. 2012. Population pharmacokinetics of Artemether and dihydroartemisinin in pregnant women with uncomplicated Plasmodium falciparum malaria in Uganda. Malar J, 11 (1), pp. 293. | Show Abstract | Read more

BACKGROUND: Malaria in pregnancy increases the risk of maternal anemia, abortion and low birth weight. Approximately 85.3 million pregnancies occur annually in areas with Plasmodium falciparum transmission. Pregnancy has been reported to alter the pharmacokinetic properties of many anti-malarial drugs. Reduced drug exposure increases the risk of treatment failure. The objective of this study was to evaluate the population pharmacokinetic properties of artemether and its active metabolite dihydroartemisinin in pregnant women with uncomplicated P. falciparum malaria in Uganda. METHODS: Twenty-one women with uncomplicated P. falciparum malaria in the second and third trimesters of pregnancy received the fixed oral combination of 80 mg artemether and 480 mg lumefantrine twice daily for three days. Artemether and dihydroartemisinin plasma concentrations after the last dose administration were quantified using liquid chromatography coupled to tandem mass-spectroscopy. A simultaneous drug-metabolite population pharmacokinetic model for artemether and dihydroartemisinin was developed taking into account different disposition, absorption, error and covariate models. A separate modeling approach and a non-compartmental analysis (NCA) were also performed to enable a comparison with literature values and different modeling strategies. RESULTS: The treatment was well tolerated and there were no cases of recurrent malaria. A flexible absorption model with sequential zero-order and transit-compartment absorption followed by a simultaneous one-compartment disposition model for both artemether and dihydroartemisinin provided the best fit to the data. Artemether and dihydroartemisinin exposure was lower than that reported in non-pregnant populations. An approximately four-fold higher apparent volume of distribution for dihydroartemisinin was obtained by non-compartmental analysis and separate modeling compared to that from simultaneous modeling of the drug and metabolite. This highlights a potential pitfall when analyzing drug/metabolite data with traditional approaches. CONCLUSION: The population pharmacokinetic properties of artemether and dihydroartemisinin, in pregnant women with uncomplicated P. falciparum malaria in Uganda, were described satisfactorily by a simultaneous drug-metabolite model without covariates. Concentrations of artemether and its metabolite dihydroartemisinin were relatively low in pregnancy compared to literature data. However, this should be interpreted with caution considered the limited literature available. Further studies in larger series are urgently needed for this vulnerable group.

John GK, Douglas NM, von Seidlein L, Nosten F, Baird JK, White NJ, Price RN. 2012. Primaquine radical cure of Plasmodium vivax: a critical review of the literature. Malar J, 11 (1), pp. 280. | Show Abstract | Read more

BACKGROUND: Primaquine has been the only widely available hypnozoitocidal anti-malarial drug for half a century. Despite this its clinical efficacy is poorly characterized resulting in a lack of consensus over the optimal regimen for the radical cure of Plasmodium vivax. METHODS: Published studies since 1950 of the use of primaquine regimens for preventing P. vivax relapse were reviewed. Data were extracted systematically from available papers. Primaquine regimens were categorized according to the total dose administered: very low (≤2.5 mg/kg), low (>2.5 mg/kg- < 5.0 mg/kg) and high (≥ 5.0 mg/kg). The risk of recurrent infection were summarized across geographical regions and the odds ratios between treatment regimens calculated after stratifying by total treatment dose and duration of study follow up. RESULTS: Data could be retrieved from 87 clinical trials presenting data in 59,735 patients enrolled into 156 treatment arms, conducted in 20 countries. There was marked heterogeneity in study design, particularly primaquine dosing and duration of follow up. The median rate of recurrence following very low dose of primaquine (n = 44) was 25% (range 0-90%) at 4-6 months, compared to 6.7 % (range 0-59%) following low dose primaquine (n = 82). High dose primaquine regimens were assessed in 28 treatment arms, and were associated with a median recurrence rate of 0% (Range: 0-15%) at one month. In 18 studies with control arms, the effectiveness of a very low dose primaquine regimen was no different from patients who did not receive primaquine (OR = 0.60, 95%CI 0.33-1.09, p = 0.09), whereas for the low dose regimens a significant difference was reported in 50% (6/12) of studies (overall OR = 0.14, 95%CI: 0.06-0.35, p < 0.001). Two studies enrolling 171 patients demonstrated high effectiveness of high dose primaquine compared to a control arm (OR = 0.03 (95%CI: 0.01-0.13); p < 0.0001). CONCLUSIONS: Low dose regimens retain adequate efficacy in some areas, but this is not uniform. The efficacy and safety of pragmatic high dose primaquine regimens needs to be assessed in a range of endemic and geographical locations. Such studies will require a prolonged period of follow up and comparison with control arms to account for confounding factors.

Ramutton T, Hendriksen IC, Mwanga-Amumpaire J, Mtove G, Olaosebikan R, Tshefu AK, Onyamboko MA, Karema C et al. 2012. Sequence variation does not confound the measurement of plasma PfHRP2 concentration in African children presenting with severe malaria. Malar J, 11 (1), pp. 276. | Show Abstract | Read more

BACKGROUND: Plasmodium falciparum histidine-rich protein PFHRP2 measurement is used widely for diagnosis, and more recently for severity assessment in falciparum malaria. The Pfhrp2 gene is highly polymorphic, with deletion of the entire gene reported in both laboratory and field isolates. These issues potentially confound the interpretation of PFHRP2 measurements. METHODS: Studies designed to detect deletion of Pfhrp2 and its paralog Pfhrp3 were undertaken with samples from patients in seven countries contributing to the largest hospital-based severe malaria trial (AQUAMAT). The quantitative relationship between sequence polymorphism and PFHRP2 plasma concentration was examined in samples from selected sites in Mozambique and Tanzania. RESULTS: There was no evidence for deletion of either Pfhrp2 or Pfhrp3 in the 77 samples with lowest PFHRP2 plasma concentrations across the seven countries. Pfhrp2 sequence diversity was very high with no haplotypes shared among 66 samples sequenced. There was no correlation between Pfhrp2 sequence length or repeat type and PFHRP2 plasma concentration. CONCLUSIONS: These findings indicate that sequence polymorphism is not a significant cause of variation in PFHRP2 concentration in plasma samples from African children. This justifies the further development of plasma PFHRP2 concentration as a method for assessing African children who may have severe falciparum malaria. The data also add to the existing evidence base supporting the use of rapid diagnostic tests based on PFHRP2 detection.

White NJ, Dondorp AM, Faiz A, Mishra S, Hien TT. 2012. New global estimates of malaria deaths. Lancet, 380 (9841), pp. 559-560. | Read more

Hendriksen IC, Mwanga-Amumpaire J, von Seidlein L, Mtove G, White LJ, Olaosebikan R, Lee SJ, Tshefu AK et al. 2012. Diagnosing severe falciparum malaria in parasitaemic African children: a prospective evaluation of plasma PfHRP2 measurement. PLoS Med, 9 (8), pp. e1001297. | Show Abstract | Read more

BACKGROUND: In African children, distinguishing severe falciparum malaria from other severe febrile illnesses with coincidental Plasmodium falciparum parasitaemia is a major challenge. P. falciparum histidine-rich protein 2 (PfHRP2) is released by mature sequestered parasites and can be used to estimate the total parasite burden. We investigated the prognostic significance of plasma PfHRP2 and used it to estimate the malaria-attributable fraction in African children diagnosed with severe malaria. METHODS AND FINDINGS: Admission plasma PfHRP2 was measured prospectively in African children (from Mozambique, The Gambia, Kenya, Tanzania, Uganda, Rwanda, and the Democratic Republic of the Congo) aged 1 month to 15 years with severe febrile illness and a positive P. falciparum lactate dehydrogenase (pLDH)-based rapid test in a clinical trial comparing parenteral artesunate versus quinine (the AQUAMAT trial, ISRCTN 50258054). In 3,826 severely ill children, Plasmadium falciparum PfHRP2 was higher in patients with coma (p = 0.0209), acidosis (p<0.0001), and severe anaemia (p<0.0001). Admission geometric mean (95%CI) plasma PfHRP2 was 1,611 (1,350-1,922) ng/mL in fatal cases (n = 381) versus 1,046 (991-1,104) ng/mL in survivors (n = 3,445, p<0.0001), without differences in parasitaemia as assessed by microscopy. There was a U-shaped association between log(10) plasma PfHRP2 and risk of death. Mortality increased 20% per log(10) increase in PfHRP2 above 174 ng/mL (adjusted odds ratio [AOR] 1.21, 95%CI 1.05-1.39, p = 0.009). A mechanistic model assuming a PfHRP2-independent risk of death in non-malaria illness closely fitted the observed data and showed malaria-attributable mortality less than 50% with plasma PfHRP2≤174 ng/mL. The odds ratio (OR) for death in artesunate versus quinine-treated patients was 0.61 (95%CI 0.44-0.83, p = 0.0018) in the highest PfHRP2 tertile, whereas there was no difference in the lowest tertile (OR 1.05; 95%CI 0.69-1.61; p = 0.82). A limitation of the study is that some conclusions are drawn from a mechanistic model, which is inherently dependent on certain assumptions. However, a sensitivity analysis of the model indicated that the results were robust to a plausible range of parameter estimates. Further studies are needed to validate our findings. CONCLUSIONS: Plasma PfHRP2 has prognostic significance in African children with severe falciparum malaria and provides a tool to stratify the risk of "true" severe malaria-attributable disease as opposed to other severe illnesses in parasitaemic African children.

Cited:

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Manske M, Miotto O, Campino S, Auburn S, Almagro-Garcia J, Maslen G, O'Brien J, Djimde A et al. 2012. Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing Nature, 487 (7407), pp. 375-379. | Show Abstract | Read more

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P.falciparum genome. © 2012 Macmillan Publishers Limited. All rights reserved.

Hendriksen IC, Ferro J, Montoya P, Chhaganlal KD, Seni A, Gomes E, Silamut K, Lee SJ et al. 2012. Diagnosis, clinical presentation, and in-hospital mortality of severe malaria in HIV-coinfected children and adults in Mozambique. Clin Infect Dis, 55 (8), pp. 1144-1153. | Show Abstract | Read more

BACKGROUND: Severe falciparum malaria with human immunodeficiency virus (HIV) coinfection is common in settings with a high prevalence of both diseases, but there is little information on whether HIV affects the clinical presentation and outcome of severe malaria. METHODS: HIV status was assessed prospectively in hospitalized parasitemic adults and children with severe malaria in Beira, Mozambique, as part of a clinical trial comparing parenteral artesunate versus quinine (ISRCTN50258054). Clinical signs, comorbidity, complications, and disease outcome were compared according to HIV status. RESULTS: HIV-1 seroprevalence was 11% (74/655) in children under 15 years and 72% (49/68) in adults with severe malaria. Children with HIV coinfection presented with more severe acidosis, anemia, and respiratory distress, and higher peripheral blood parasitemia and plasma Plasmodium falciparum histidine-rich protein-2 (PfHRP2). During hospitalization, deterioration in coma score, convulsions, respiratory distress, and pneumonia were more common in HIV-coinfected children, and mortality was 26% (19/74) versus 9% (53/581) in uninfected children (P < .001). In an age- and antimalarial treatment-adjusted logistic regression model, significant, independent predictors for death were renal impairment, acidosis, parasitemia, and plasma PfHRP2 concentration. CONCLUSIONS: Severe malaria in HIV-coinfected patients presents with higher parasite burden, more complications, and comorbidity, and carries a higher case fatality rate. Early identification of HIV coinfection is important for the clinical management of severe malaria.

Dondorp AM, Maude RJ, Hendriksen IC, Day NP, White NJ. 2012. Artesunate dosing in severe falciparum malaria. J Infect Dis, 206 (4), pp. 618-619. | Read more

Hanson J, Lam SW, Mahanta KC, Pattnaik R, Alam S, Mohanty S, Hasan MU, Hossain A et al. 2012. Relative contributions of macrovascular and microvascular dysfunction to disease severity in falciparum malaria. J Infect Dis, 206 (4), pp. 571-579. | Show Abstract | Read more

BACKGROUND: Sequestration of parasitized erythrocytes in the microcirculation is considered the central pathophysiological process in severe falciparum malaria. Hypovolemia with reduced oxygen delivery and microvascular obstruction have different implications for patient management; however, their relative contributions to disease severity are uncertain. METHODS: Adult patients (n = 28) with severe Plasmodium falciparum malaria were enrolled in a prospective hemodynamic study. Volume status and oxygen delivery were assessed using transpulmonary thermodilution. Microvascular sequestration was measured using orthogonal polarized spectroscopy. FINDINGS: Duration of therapy before study enrollment was correlated with the amount of directly visualized and quantitated microvascular sequestration (P = .03). The amount of sequestration correlated with plasma lactate (r(s )= 0.55; P = .003) and disease severity (r(s )= 0.41; P = .04). In patients who had received artesunate for <10 hours, sequestration was higher in fatal cases than in survivors: median (range) 45% (32-50) vs 15% (0-40); P = .03). Parasite biomass estimated from plasma P. falciparum histidine-rich protein 2 correlated positively with disease severity (r(s )= 0.48; P = .01) and was significantly higher in patients who died (P = .046). There was no relationship between oxygen delivery and disease severity (P = .64) or outcome (P = .74). INTERPRETATION: Vital organ dysfunction in severe malaria results primarily from sequestration of parasitized erythrocytes in the microvasculature rather than reduction in circulating blood volume and oxygen delivery.

Mayxay M, Khanthavong M, Chanthongthip O, Imwong M, Pongvongsa T, Hongvanthong B, Phompida S, Vanisaveth V, White NJ, Newton PN. 2012. Efficacy of artemether-lumefantrine, the nationally-recommended artemisinin combination for the treatment of uncomplicated falciparum malaria, in southern Laos. Malar J, 11 (1), pp. 184. | Show Abstract | Read more

BACKGROUND: The Lao Government changed the national policy for uncomplicated Plasmodium falciparum malaria from chloroquine to artemether-lumefantrine (AL) in 2005. Since then, no information on AL efficacy has been reported. With evidence of resistance to artemisinin derivatives in adjacent Cambodia, there has been a concern as to AL efficacy. Monitoring of AL efficacy would help the Lao Government to make decisions on appropriate malaria treatment. METHODS: The efficacy of a three-day, twice daily oral artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in Xepon District, Savannakhet Province, southern Laos was studied over 42 days follow-up. This was part of a trial of thiamin supplementation in falciparum malaria. RESULTS: Of 630 patients with P. falciparum enrolled in the trial of thiamin treatment, 549 (87%, 357 children ≤15 years and 192 adults) were included in this study. The per protocol 42-day cure rates were 97% (524/541) [96% (337/352) for children and 99% (187/189) for adults, p = 0.042]. By conventional intention-to-treat analysis, the 42-day cure rates adjusted for re-infection, were 97% (532/549) [96% (342/357) in children and 99% (190/192) in adults, p = 0.042]. The proportion of patients who remained parasitaemic at day 1 after treatment was significantly higher in children [33% (116/356)] compared to adults [15% (28/192)] (p < 0.001) and only one adult patient had detectable parasitaemia on day 2. There were no serious adverse events. Potential side effects after treatment were reported more commonly in adults (32%) compared to children (15%) (p < 0.001). Patients with recrudescent infections were significantly younger, had longer mean time to fever clearance, and had longer median time to parasite clearance compared to those who were cured. CONCLUSIONS: The current nationally-recommended anti-malarial treatment (artemether-lumefantrine) remains highly efficacious for the treatment of uncomplicated falciparum malaria five years after introduction in Laos. Regular monitoring is required in case artemisinin-resistant P. falciparum parasites should appear. TRIAL REGISTRATION: ISRCTN85411059.

Manske M, Miotto O, Campino S, Auburn S, Almagro-Garcia J, Maslen G, O'Brien J, Djimde A et al. 2012. Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing. Nature, 487 (7407), pp. 375-379. | Show Abstract | Read more

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

Deen J, von Seidlein L, Andersen F, Elle N, White NJ, Lubell Y. 2012. Community-acquired bacterial bloodstream infections in developing countries in south and southeast Asia: a systematic review. Lancet Infect Dis, 12 (6), pp. 480-487. | Show Abstract | Read more

Information about community-acquired bacteraemia in developing countries in south and southeast Asia is scarce. We aimed to establish the case fraction of bacteraemia in febrile patients admitted to hospital. We searched four databases and identified studies of south and southeast Asia published between 1990 and 2010 that prospectively assessed patients admitted to hospital and from whom a blood culture was taken. We reviewed 17 eligible studies describing 40,644 patients. Pathogenic organisms were isolated from 3506 patients (9%; range 1-51%); 1784 (12%) of 14,386 adults and 1722 (7%) of 26,258 children. Salmonella enterica serotype Typhi was the most common bacterial pathogen, accounting for 532 of 1798 (30%) isolates in adults and 432 of 1723 (25%) in children. Other commonly isolated organisms in adults were Staphylococcus aureus, Escherichia coli, and other gram-negative organisms, and in children were Streptococcus pneumoniae and Haemophilus influenzae. A substantial case fraction of bacteraemia occurs in patients admitted to hospital with fever in this region. Management could be improved if diagnostic microbiology facilities were more widely available. The prevailing organisms causing bacteraemia and their susceptibility patterns could inform empirical treatment regimens and prevention strategies.

Chotivanich K, Mungthin M, Ruengweerayuth R, Udomsangpetch R, Dondorp AM, Singhasivanon P, Pukrittayakamee S, White NJ. 2012. The effects of serum lipids on the in vitro activity of lumefantrine and atovaquone against Plasmodium falciparum. Malar J, 11 (1), pp. 177. | Show Abstract | Read more

BACKGROUND: Lumefantrine and atovaquone are highly lipophilic anti-malarial drugs. As a consequence absorption is increased when the drugs are taken together with a fatty meal, but the free fraction of active drug decreases in the presence of triglyceride-rich plasma lipoproteins. In this study, the consequences of lipidaemia on anti-malarial drug efficacy were assessed in vitro. METHODS: Serum was obtained from non-immune volunteers under fasting conditions and after ingestion of a high fat meal and used in standard Plasmodium falciparum in-vitro susceptibility assays. Anti-malarial drugs, including lumefantrine, atovaquone and chloroquine in five-fold dilutions (range 0.05 ng/ml-1 ug/mL) were diluted in culture medium supplemented with fasting or post-prandial 10% donor serum. The in-vitro drug susceptibility of parasite isolates was determined using the ³H-hypoxanthine uptake inhibition method and expressed as the concentration which gave 50% inhibition of hypoxanthine uptake (IC₅₀). RESULTS: Doubling plasma triglyceride concentrations (from 160 mg/dL to 320 mg/dL), resulted in an approximate doubling of the IC₅₀ for lumefantrine (191 ng/mL to 465 ng/mL, P < 0.01) and a 20-fold increase in the IC₅₀ for atovaquone (0.5 ng/mL to 12 ng/ml; P < 0.01). In contrast, susceptibility to the hydrophilic anti-malarial chloroquine did not change in relation to triglyceride content of the medium. CONCLUSIONS: Lipidaemia reduces the anti-malarial activity of lipophilic anti-malarial drugs. This is an important confounder in laboratory in vitro testing and it could have therapeutic relevance.

Jamsen KM, Duffull SB, Tarning J, Lindegardh N, White NJ, Simpson JA. 2012. Optimal designs for population pharmacokinetic studies of the partner drugs co-administered with artemisinin derivatives in patients with uncomplicated falciparum malaria. Malar J, 11 (1), pp. 143. | Show Abstract | Read more

BACKGROUND: Artemisinin-based combination therapy (ACT) is currently recommended as first-line treatment for uncomplicated malaria, but of concern, it has been observed that the effectiveness of the main artemisinin derivative, artesunate, has been diminished due to parasite resistance. This reduction in effect highlights the importance of the partner drugs in ACT and provides motivation to gain more knowledge of their pharmacokinetic (PK) properties via population PK studies. Optimal design methodology has been developed for population PK studies, which analytically determines a sampling schedule that is clinically feasible and yields precise estimation of model parameters. In this work, optimal design methodology was used to determine sampling designs for typical future population PK studies of the partner drugs (mefloquine, lumefantrine, piperaquine and amodiaquine) co-administered with artemisinin derivatives. METHODS: The optimal designs were determined using freely available software and were based on structural PK models from the literature and the key specifications of 100 patients with five samples per patient, with one sample taken on the seventh day of treatment. The derived optimal designs were then evaluated via a simulation-estimation procedure. RESULTS: For all partner drugs, designs consisting of two sampling schedules (50 patients per schedule) with five samples per patient resulted in acceptable precision of the model parameter estimates. CONCLUSIONS: The sampling schedules proposed in this paper should be considered in future population pharmacokinetic studies where intensive sampling over many days or weeks of follow-up is not possible due to either ethical, logistic or economical reasons.

Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, McGready R, ler Moo C, Al-Saai S et al. 2012. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet, 379 (9830), pp. 1960-1966. | Show Abstract | Read more

BACKGROUND: Artemisinin-resistant falciparum malaria has arisen in western Cambodia. A concerted international effort is underway to contain artemisinin-resistant Plasmodium falciparum, but containment strategies are dependent on whether resistance has emerged elsewhere. We aimed to establish whether artemisinin resistance has spread or emerged on the Thailand-Myanmar (Burma) border. METHODS: In malaria clinics located along the northwestern border of Thailand, we measured six hourly parasite counts in patients with uncomplicated hyperparasitaemic falciparum malaria (≥4% infected red blood cells) who had been given various oral artesunate-containing regimens since 2001. Parasite clearance half-lives were estimated and parasites were genotyped for 93 single nucleotide polymorphisms. FINDINGS: 3202 patients were studied between 2001 and 2010. Parasite clearance half-lives lengthened from a geometric mean of 2·6 h (95% CI 2·5-2·7) in 2001, to 3·7 h (3·6-3·8) in 2010, compared with a mean of 5·5 h (5·2-5·9) in 119 patients in western Cambodia measured between 2007 and 2010. The proportion of slow-clearing infections (half-life ≥6·2 h) increased from 0·6% in 2001, to 20% in 2010, compared with 42% in western Cambodia between 2007 and 2010. Of 1583 infections genotyped, 148 multilocus parasite genotypes were identified, each of which infected between two and 13 patients. The proportion of variation in parasite clearance attributable to parasite genetics increased from 30% between 2001 and 2004, to 66% between 2007 and 2010. INTERPRETATION: Genetically determined artemisinin resistance in P falciparum emerged along the Thailand-Myanmar border at least 8 years ago and has since increased substantially. At this rate of increase, resistance will reach rates reported in western Cambodia in 2-6 years. FUNDING: The Wellcome Trust and National Institutes of Health.

Chairat K, Tarning J, White NJ, Lindegardh N. 2012. Pharmacokinetic Properties of Anti-Influenza Neuraminidase Inhibitors. J Clin Pharmacol, 53 (2), pp. 119-139. | Show Abstract | Read more

Neuraminidase inhibitors are the mainstay of anti-influenza treatment. Oseltamivir is the most widely used drug but is currently available only as an oral formulation. Resistance spreads rapidly in seasonal H1N1 influenza A viruses, which were universally resistant in 2008, because of the H275Y mutation in the neuraminidase (NA) gene. Oseltamivir is a prodrug for the active carboxylate metabolite. Ex vivo conversion in blood samples may have confounded early pharmacokinetic studies. Oseltamivir shows dose linear kinetics, and oseltamivir carboxylate has an elimination half-life (t(&frac12;β)) after oral administration in healthy individuals of approximately 7.7 hours. Oseltamivir carboxylate is eliminated primarily by tubular secretion, and both clearance and tissue distribution are reduced by probenecid. The H275Y mutation in NA confers high-level oseltamivir resistance and intermediate peramivir resistance but does not alter zanamivir susceptibility. Zanamivir is available as a powder for inhalation, and a parenteral form is under development. Zanamivir distributes in an apparent volume of distribution approximating that of extracellular water and is rapidly eliminated (t(&frac12;β) of approximately 3.0 hours). Peramivir is slowly eliminated (t(&frac12;β) of 7.7-20.8 hours) and is prescribed as either a once-daily injection or as a single infusion. Laninamivir is a recently developed slowly eliminated compound for administration by inhalation.

von Seidlein L, Olaosebikan R, Hendriksen IC, Lee SJ, Adedoyin OT, Agbenyega T, Nguah SB, Bojang K et al. 2012. Predicting the clinical outcome of severe falciparum malaria in african children: findings from a large randomized trial. Clin Infect Dis, 54 (8), pp. 1080-1090. | Show Abstract | Read more

BACKGROUND: Data from the largest randomized, controlled trial for the treatment of children hospitalized with severe malaria were used to identify such predictors of a poor outcome from severe malaria. METHODS: African children (<15 years) with severe malaria participated in a randomized comparison of parenteral artesunate and parenteral quinine in 9 African countries. Detailed clinical assessment was performed on admission. Parasite densities were assessed in a reference laboratory. Predictors of death were examined using a multivariate logistic regression model. RESULTS: Twenty indicators of disease severity were assessed, out of which 5 (base deficit, impaired consciousness, convulsions, elevated blood urea, and underlying chronic illness) were associated independently with death. Tachypnea, respiratory distress, deep breathing, shock, prostration, low pH, hyperparasitemia, severe anemia, and jaundice were statistically significant indicators of death in the univariate analysis but not in the multivariate model. Age, glucose levels, axillary temperature, parasite density, heart rate, blood pressure, and blackwater fever were not related to death in univariate models. CONCLUSIONS: Acidosis, cerebral involvement, renal impairment, and chronic illness are key independent predictors for a poor outcome in African children with severe malaria. Mortality is markedly increased in cerebral malaria combined with acidosis. Clinical Trial Registration. ISRCTN50258054.

Mayxay M, Khanthavong M, Chanthongthip O, Imwong M, Lee SJ, Stepniewska K, Soonthornsata B, Pongvongsa T et al. 2012. No evidence for spread of Plasmodium falciparum artemisinin resistance to Savannakhet Province, Southern Laos. Am J Trop Med Hyg, 86 (3), pp. 403-408. | Show Abstract | Read more

We conducted an open-label, randomized clinical trial to assess parasite clearance times (PCT) and the efficacy of 4 mg/kg (group 1, n = 22) and 2 mg/kg (group 2, n = 22) of oral artesunate for three days followed by artemether-lumefantrine in patients with uncomplicated Plasmodium falciparum malaria at Xepon Interdistrict Hospital, Savannakhet Province in southern Laos. Slides were read in duplicate. The overall mean (95% confidence interval; range) PCT in hours was 23.2 (21.2-25.3; 12-46) and 22.4 (20.3-24.5; 12-46) for the first and second microscopists, respectively (P = 0.57). Ten (23%) patients remained parasitemic on day 1 after treatment (4 [18%] in group 1 and 6 [27%] in group 2; P = 0.47). No patient had patent asexual parasitemia on the second and third days of treatment. The 42-day polymerase chain reaction-corrected cure rates were 100% in both treatment groups. Serious adverse events did not develop during or after treatment in any patients. In conclusion, no evidence of P. falciparum in vivo resistance to artesunate was found in southern Laos.

Tarning J, Zongo I, Somé FA, Rouamba N, Parikh S, Rosenthal PJ, Hanpithakpong W, Jongrak N et al. 2012. Population pharmacokinetics and pharmacodynamics of piperaquine in children with uncomplicated falciparum malaria. Clin Pharmacol Ther, 91 (3), pp. 497-505. | Show Abstract | Read more

Dihydroartemisinin-piperaquine is being increasingly used as a first-line artemisinin combination treatment for malaria. The aim of this study was to describe the pharmacokinetic and pharmacodynamic properties of piperaquine in 236 children with uncomplicated falciparum malaria in Burkina Faso. They received a standard body weight-based oral 3-day fixed-dose dihydroartemisinin-piperaquine regimen. Capillary plasma concentration-time profiles were characterized using nonlinear mixed-effects modeling. The population pharmacokinetics of piperaquine were described accurately by a two-transit-compartment absorption model and a three-compartment distribution model. Body weight was a significant covariate affecting clearance and volume parameters. The individually predicted day 7 capillary plasma concentration of piperaquine was an important predictor (P < 0.0001) of recurrent malaria infection after treatment. Young children (2-5 years of age) received a significantly higher body weight-normalized dose than older children (P = 0.025) but had significantly lower day 7 piperaquine concentrations (P = 0.024) and total piperaquine exposures (P = 0.021), suggesting that an increased dose regimen for young children should be evaluated.

Tarning J, Rijken MJ, McGready R, Phyo AP, Hanpithakpong W, Day NP, White NJ, Nosten F, Lindegardh N. 2012. Population pharmacokinetics of dihydroartemisinin and piperaquine in pregnant and nonpregnant women with uncomplicated malaria. Antimicrob Agents Chemother, 56 (4), pp. 1997-2007. | Show Abstract | Read more

Pregnant women are particularly vulnerable to malaria. The pharmacokinetic properties of antimalarial drugs are often affected by pregnancy, resulting in lower drug concentrations and a consequently higher risk of treatment failure. The objective of this study was to evaluate the population pharmacokinetic properties of piperaquine and dihydroartemisinin in pregnant and nonpregnant women with uncomplicated malaria. Twenty-four pregnant and 24 matched nonpregnant women on the Thai-Myanmar boarder were treated with a standard fixed oral 3-day treatment, and venous plasma concentrations of both drugs were measured frequently for pharmacokinetic evaluation. Population pharmacokinetics were evaluated with nonlinear mixed-effects modeling. The main pharmacokinetic finding was an unaltered total exposure to piperaquine but reduced exposure to dihydroartemisinin in pregnant compared to nonpregnant women with uncomplicated malaria. Piperaquine was best described by a three-compartment disposition model with a 45% higher elimination clearance and a 47% increase in relative bioavailability in pregnant women compared with nonpregnant women. The resulting net effect of pregnancy was an unaltered total exposure to piperaquine but a shorter terminal elimination half-life. Dihydroartemisinin was best described by a one-compartment disposition model with a 38% lower relative bioavailability in pregnant women than nonpregnant women. The resulting net effect of pregnancy was a decreased total exposure to dihydroartemisinin. The shorter terminal elimination half-life of piperaquine and lower exposure to dihydroartemisinin will shorten the posttreatment prophylactic effect and might affect cure rates. The clinical impact of these pharmacokinetic findings in pregnant women with uncomplicated malaria needs to be evaluated in larger series.

Ponsford MJ, Medana IM, Prapansilp P, Hien TT, Lee SJ, Dondorp AM, Esiri MM, Day NP, White NJ, Turner GD. 2012. Sequestration and microvascular congestion are associated with coma in human cerebral malaria. J Infect Dis, 205 (4), pp. 663-671. | Show Abstract | Read more

The pathogenesis of coma in severe Plasmodium falciparum malaria remains poorly understood. Obstruction of the brain microvasculature because of sequestration of parasitized red blood cells (pRBCs) represents one mechanism that could contribute to coma in cerebral malaria. Quantitative postmortem microscopy of brain sections from Vietnamese adults dying of malaria confirmed that sequestration in the cerebral microvasculature was significantly higher in patients with cerebral malaria (CM; n = 21) than in patients with non-CM (n = 23). Sequestration of pRBCs and CM was also significantly associated with increased microvascular congestion by infected and uninfected erythrocytes. Clinicopathological correlation showed that sequestration and congestion were significantly associated with deeper levels of premortem coma and shorter time to death. Microvascular congestion and sequestration were highly correlated as microscopic findings but were independent predictors of a clinical diagnosis of CM. Increased microvascular congestion accompanies coma in CM, associated with parasite sequestration in the cerebral microvasculature.

Turner C, Turner P, Cararra V, Eh Lwe N, Watthanaworawit W, Day NP, White NJ, Goldblatt D, Nosten F. 2012. A high burden of respiratory syncytial virus associated pneumonia in children less than two years of age in a South East Asian refugee population. PLoS One, 7 (11), pp. e50100. | Show Abstract | Read more

BACKGROUND: Pneumonia is a major cause of childhood mortality and morbidity approximately 1.6 million deaths and 150 million episodes occur annually in children <5 years. Respiratory syncytial virus (RSV) may be responsible for up to 25% of cases and 12% of deaths making it an important potential vaccine target, although data from South East Asia is scarce. METHODS: We followed a birth cohort of Burmese refugee children, born over a one year period, for two years. Pneumonia episodes were diagnosed using WHO criteria. A chest radiograph, nasopharyngeal aspirate and non-specific markers of infection were taken during each episode. RESULTS: The incidence of RSV-associated pneumonia was 0.24 (95% CI 0.22-0.26) episodes per child year. All children with pneumonia received antibiotic treatment, following WHO guidelines. The highest incidence was in the 2-12 month age group. The commonest diagnosis in a child with RSV-associated pneumonia was non-severe pneumonia (239/362:66.0%), however the incidence of RSV-associated severe or very severe pneumonia was 0.08 (95% CI 0.01-0.10) episodes per child year. Birth in the wet season increased the risk of severe disease in children who had their first episode of RSV-associated pneumonia aged 2-11 months (OR 28.7, 95% CI 6.6-125.0, p<0.001). RSV episodes were highly seasonal being responsible for 80.0% of all the pneumonia episodes occurring each October and November over the study period. CONCLUSIONS: There was a high incidence of RSV associated pneumonia in this refugee population. Interventions to prevent RSV infection have the potential to reduce the incidence of clinically diagnosed pneumonia and hence unnecessary antibiotic usage in this population.

Turner P, Turner C, Jankhot A, Helen N, Lee SJ, Day NP, White NJ, Nosten F, Goldblatt D. 2012. A longitudinal study of Streptococcus pneumoniae carriage in a cohort of infants and their mothers on the Thailand-Myanmar border. PLoS One, 7 (5), pp. e38271. | Show Abstract | Read more

BACKGROUND: Pneumococcal disease is a major cause of childhood death. Almost a third of the world's children live in Southeast Asia, but there are few data from the region on pneumococcal colonization or disease. Our aim was to document the dynamics of pneumococcal carriage in a rural SE Asian birth cohort. METHODS: We studied 234 Karen mother-infant pairs in Northwestern Thailand. Infants were followed from birth and nasopharyngeal swabs were taken from mother and infant at monthly intervals until 24 months old. RESULTS: 8,386 swabs were cultured and 4,396 pneumococci characterized. Infants became colonized early (median 45.5 days; 95% confidence interval [CI] 44.5-46.0) and by 24 months had a median of seven (range 0-15) carriage episodes. Maternal smoking and young children in the house were associated with earlier colonization (hazard ratio [HR] 1.5 (95% CI 1.1-2.1) and 1.4 (95% CI 1.0-1.9)). For the four commonest serotypes and non-typeable pneumococci, previous exposure to homologous or heterologous serotypes resulted in an extended interval to reacquisition of the same serotype. Previous colonization by serotypes 14 and 19F was also associated with reduced carriage duration if subsequently reacquired (HR [first reacquisition] 4.1 (95% CI 1.4-12.6) and 2.6 (1.5-4.7)). Mothers acquired pneumococci less frequently, and carried them for shorter periods, than infants (acquisition rate 0.5 vs. 1.1 /100 person-days, p<0.001; median duration 31.0 vs. 60.5 days, p = 0.001). 55.8% of pneumococci from infants were vaccine serotypes (13-valent pneumococcal conjugate vaccine, PCV13), compared with 27.5% from mothers (p<0.001). Non-typeable pneumococcal carriage was common, being carried at least once by 55.1% of infants and 32.0% of mothers. CONCLUSIONS: Pneumococcal carriage frequency and duration are influenced by previous exposure to both homologous and heterologous serotypes. These data will inform vaccination strategies in this population.

Cheeseman IH, Miller BA, Nair S, Nkhoma S, Tan A, Tan JC, Al Saai S, Phyo AP et al. 2012. A major genome region underlying artemisinin resistance in malaria. Science, 336 (6077), pp. 79-82. | Show Abstract | Read more

Evolving resistance to artemisinin-based compounds threatens to derail attempts to control malaria. Resistance has been confirmed in western Cambodia and has recently emerged in western Thailand, but is absent from neighboring Laos. Artemisinin resistance results in reduced parasite clearance rates (CRs) after treatment. We used a two-phase strategy to identify genome region(s) underlying this ongoing selective event. Geographical differentiation and haplotype structure at 6969 polymorphic single-nucleotide polymorphisms (SNPs) in 91 parasites from Cambodia, Thailand, and Laos identified 33 genome regions under strong selection. We screened SNPs and microsatellites within these regions in 715 parasites from Thailand, identifying a selective sweep on chromosome 13 that shows strong association (P = 10(-6) to 10(-12)) with slow CRs, illustrating the efficacy of targeted association for identifying the genetic basis of adaptive traits.

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Amaratunga C, Sreng S, Suon S, Phelps ES, Stepniewska K, Lim P, Zhou C, Mao S et al. 2012. Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: A parasite clearance rate study The Lancet Infectious Diseases, 12 (11), pp. 851-858. | Show Abstract | Read more

Background: Artemisinin-resistant . Plasmodium falciparum has been reported in Pailin, western Cambodia, detected as a slow parasite clearance rate in vivo. Emergence of this phenotype in western Thailand and possibly elsewhere threatens to compromise the effectiveness of all artemisinin-based combination therapies. Parasite genetics is associated with parasite clearance rate but does not account for all variation. We investigated contributions of both parasite genetics and host factors to the artemisinin-resistance phenotype in Pursat, western Cambodia. Methods: Between June 19 and Nov 28, 2009, and June 26 and Dec 6, 2010, we enrolled patients aged 10 years or older with uncomplicated falciparum malaria, a density of asexual parasites of at least 10 000 per μL of whole blood, no symptoms or signs of severe malaria, no other cause of febrile illness, and no chronic illness. We gave participants 4 mg/kg artesunate at 0, 24, and 48 h, 15 mg/kg mefloquine at 72 h, and 10 mg/kg mefloquine at 96 h. We assessed parasite density on thick blood films every 6 h until undetectable. The parasite clearance half-life was calculated from the parasite clearance curve. We genotyped parasites with 18 microsatellite markers and patients for haemoglobin E, α-thalassaemia, and a mutation of . G6PD, which encodes glucose-6-phosphate dehydrogenase. To account for the possible effects of acquired immunity on half-life, we used three surrogates for increased likelihood of exposure to . P falciparum: age, sex, and place of residence. This study is registered with . ClinicalTrials.gov, number . NCT00341003. Findings: We assessed 3504 individuals from all six districts of Pursat province seeking treatment for malaria symptoms. We enrolled 168 patients with falciparum malaria who met inclusion criteria. The geometric mean half-life was 5·85 h (95% CI 5·54-6·18) in Pursat, similar to that reported in Pailin (p=0·109). We identified two genetically different parasite clone groups: parasite group 1 (PG1) and parasite group 2 (PG2). Non-significant increases in parasite clearance half-life were seen in patients with haemoglobin E (0·55 h; p=0·078), those of male sex (0·96 h; p=0·064), and in 2010 (0·68 h; p=0·068); PG1 was associated with a significant increase (0·79 h; p=0·033). The mean parasite heritability of half-life was 0·40 (SD 0·17). Interpretation: Heritable artemisinin resistance is established in a second Cambodian province. To accurately identify parasites that are intrinsically susceptible or resistant to artemisinins, future studies should explore the effect of erythrocyte polymorphisms and specific immune responses on half-life variation. Funding: Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health. © 2012 Elsevier Ltd.

Boyer C, Gaudin K, Kauss T, Gaubert A, Boudis A, Verschelden J, Franc M, Roussille J et al. 2012. Development of NIRS method for quality control of drug combination artesunate-azithromycin for the treatment of severe malaria. J Pharm Biomed Anal, 67-68 pp. 10-15. | Show Abstract | Read more

Near infrared spectroscopy (NIRS) methods were developed for the determination of analytical content of an antimalarial-antibiotic (artesunate and azithromycin) co-formulation in hard gelatin capsule (HGC). The NIRS consists of pre-processing treatment of spectra (raw spectra and first-derivation of two spectral zones), a unique principal component analysis model to ensure the specificity and then two partial least-squares regression models for the determination content of each active pharmaceutical ingredient. The NIRS methods were developed and validated with no reference method, since the manufacturing process of HGC is basically mixed excipients with active pharmaceutical ingredients. The accuracy profiles showed β-expectation tolerance limits within the acceptance limits (±5%). The analytical control approach performed by reversed phase (HPLC) required two different methods involving two different preparation and chromatographic methods. NIRS offers advantages in terms of lower costs of equipment and procedures, time saving, environmentally friendly.

McGready R, Boel M, Rijken MJ, Ashley EA, Cho T, Moo O, Paw MK, Pimanpanarak M et al. 2012. Effect of early detection and treatment on malaria related maternal mortality on the north-western border of Thailand 1986-2010. PLoS One, 7 (7), pp. e40244. | Show Abstract | Read more

INTRODUCTION: Maternal mortality is high in developing countries, but there are few data in high-risk groups such as migrants and refugees in malaria-endemic areas. Trends in maternal mortality were followed over 25 years in antenatal clinics prospectively established in an area with low seasonal transmission on the north-western border of Thailand. METHODS AND FINDINGS: All medical records from women who attended the Shoklo Malaria Research Unit antenatal clinics from 12(th) May 1986 to 31(st) December 2010 were reviewed, and maternal death records were analyzed for causality. There were 71 pregnancy-related deaths recorded amongst 50,981 women who attended antenatal care at least once. Three were suicide and excluded from the analysis as incidental deaths. The estimated maternal mortality ratio (MMR) overall was 184 (95%CI 150-230) per 100,000 live births. In camps for displaced persons there has been a six-fold decline in the MMR from 499 (95%CI 200-780) in 1986-90 to 79 (40-170) in 2006-10, p<0.05. In migrants from adjacent Myanmar the decline in MMR was less significant: 588 (100-3260) to 252 (150-430) from 1996-2000 to 2006-2010. Mortality from P. falciparum malaria in pregnancy dropped sharply with the introduction of systematic screening and treatment and continued to decline with the reduction in the incidence of malaria in the communities. P. vivax was not a cause of maternal death in this population. Infection (non-puerperal sepsis and P. falciparum malaria) accounted for 39.7 (27/68) % of all deaths. CONCLUSIONS: Frequent antenatal clinic screening allows early detection and treatment of falciparum malaria and substantially reduces maternal mortality from P. falciparum malaria. No significant decline has been observed in deaths from sepsis or other causes in refugee and migrant women on the Thai-Myanmar border.

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Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, McGready R, Moo CL, Al-Saai S et al. 2012. Emergence of artemisinin-resistant malaria on the western border of Thailand: A longitudinal study The Lancet, 379 (9830), pp. 1960-1966. | Show Abstract | Read more

Artemisinin-resistant falciparum malaria has arisen in western Cambodia. A concerted international eff ort is underway to contain artemisinin-resistant Plasmodium falciparum, but containment strategies are dependent on whether resistance has emerged elsewhere. We aimed to establish whether artemisinin resistance has spread or emerged on the Thailand-Myanmar (Burma) border. Methods In malaria clinics located along the northwestern border of Thailand, we measured six hourly parasite counts in patients with uncomplicated hyperparasitaemic falciparum malaria (≤4% infected red blood cells) who had been given various oral artesunate-containing regimens since 2001. Parasite clearance half-lives were estimated and parasites were genotyped for 93 single nucleotide polymorphisms. Findings 3202 patients were studied between 2001 and 2010. Parasite clearance half-lives lengthened from a geometric mean of 2·6 h (95% CI 2·5-2·7) in 2001, to 3·7 h (3·6-3·8) in 2010, compared with a mean of 5·5 h (5·2-5·9) in 119 patients in western Cambodia measured between 2007 and 2010. The proportion of slow-clearing infections (half-life ≤6·2 h) increased from 0·6% in 2001, to 20% in 2010, compared with 42% in western Cambodia between 2007 and 2010. Of 1583 infections genotyped, 148 multilocus parasite genotypes were identifi ed, each of which infected between two and 13 patients. The proportion of variation in parasite clearance attributable to parasite genetics increased from 30% between 2001 and 2004, to 66% between 2007 and 2010. Interpretation Genetically determined artemisinin resistance in P falciparum emerged along the Thailand-Myanmar border at least 8 years ago and has since increased substantially. At this rate of increase, resistance will reach rates reported in western Cambodia in 2-6 years.

Kim JR, Nandy A, Maji AK, Addy M, Dondorp AM, Day NP, Pukrittayakamee S, White NJ, Imwong M. 2012. Genotyping of Plasmodium vivax reveals both short and long latency relapse patterns in Kolkata. PLoS One, 7 (7), pp. e39645. | Show Abstract | Read more

BACKGROUND: The Plasmodium vivax that was once prevalent in temperate climatic zones typically had an interval between primary infection and first relapse of 7-10 months, whereas in tropical areas P.vivax infections relapse frequently at intervals of 3-6 weeks. Defining the epidemiology of these two phenotypes from temporal patterns of illness in endemic areas is difficult or impossible, particularly if they overlap. METHODS: A prospective open label comparison of chloroquine (CQ) alone versus CQ plus unobserved primaquine for either 5 days or 14 days was conducted in patients presenting with acute vivax malaria in Kolkata. Patients were followed for 15 months and primary and recurrent infections were genotyped using three polymorphic antigen and up to 8 microsatellite markers. RESULTS: 151 patients were enrolled of whom 47 (31%) had subsequent recurrent infections. Recurrence proportions were similar in the three treatment groups. Parasite genotyping revealed discrete temporal patterns of recurrence allowing differentiation of probable relapse from newly acquired infections. This suggested that 32 of the 47 recurrences were probable relapses of which 22 (69%) were genetically homologous. The majority (81%) of probable relapses occurred within three months (16 homologous, 10 heterologous) and six genetically homologous relapses (19%) were of the long latency (8-10 month interval) phenotype. CONCLUSIONS: With long follow-up to assess temporal patterns of vivax malaria recurrence, genotyping of P.vivax can be used to assess relapse rates. A 14 day unobserved course of primaquine did not prevent relapse. Genotyping indicates that long latency P.vivax is prevalent in West Bengal, and that the first relapses after long latent periods are genetically homologous. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN14027467.

Maude RJ, Socheat D, Nguon C, Saroth P, Dara P, Li G, Song J, Yeung S et al. 2012. Optimising strategies for Plasmodium falciparum malaria elimination in Cambodia: primaquine, mass drug administration and artemisinin resistance. PLoS One, 7 (5), pp. e37166. | Show Abstract | Read more

BACKGROUND: Malaria elimination requires a variety of approaches individually optimized for different transmission settings. A recent field study in an area of low seasonal transmission in South West Cambodia demonstrated dramatic reductions in malaria parasite prevalence following both mass drug administration (MDA) and high treatment coverage of symptomatic patients with artemisinin-piperaquine plus primaquine. This study employed multiple combined strategies and it was unclear what contribution each made to the reductions in malaria. METHOD AND FINDINGS: A mathematical model fitted to the trial results was used to assess the effects of the various components of these interventions, design optimal elimination strategies, and explore their interactions with artemisinin resistance, which has recently been discovered in Western Cambodia. The modelling indicated that most of the initial reduction of P. falciparum malaria resulted from MDA with artemisinin-piperaquine. The subsequent continued decline and near elimination resulted mainly from high coverage with artemisinin-piperaquine treatment. Both these strategies were more effective with the addition of primaquine. MDA with artemisinin combination therapy (ACT) increased the proportion of artemisinin resistant infections, although much less than treatment of symptomatic cases with ACT, and this increase was slowed by adding primaquine. Artemisinin resistance reduced the effectiveness of interventions using ACT when the prevalence of resistance was very high. The main results were robust to assumptions about primaquine action, and immunity. CONCLUSIONS: The key messages of these modelling results for policy makers were: high coverage with ACT treatment can produce a long-term reduction in malaria whereas the impact of MDA is generally only short-term; primaquine enhances the effect of ACT in eliminating malaria and reduces the increase in proportion of artemisinin resistant infections; parasite prevalence is a better surveillance measure for elimination programmes than numbers of symptomatic cases; combinations of interventions are most effective and sustained efforts are crucial for successful elimination.

Chotivanich K, Udomsangpetch R, Suwanarusk R, Pukrittayakamee S, Wilairatana P, Beeson JG, Day NP, White NJ. 2012. Plasmodium vivax adherence to placental glycosaminoglycans. PLoS One, 7 (4), pp. e34509. | Show Abstract | Read more

BACKGROUND: Plasmodium vivax infections seldom kill directly but do cause indirect mortality by reducing birth weight and causing abortion. Cytoadherence and sequestration in the microvasculature are central to the pathogenesis of severe Plasmodium falciparum malaria, but the contribution of cytoadherence to pathology in other human malarias is less clear. METHODOLOGY: The adherence properties of P. vivax infected red blood cells (PvIRBC) were evaluated under static and flow conditions. PRINCIPAL FINDINGS: P. vivax isolates from 33 patients were studied. None adhered to immobilized CD36, ICAM-1, or thrombospondin, putative ligands for P. falciparum vascular cytoadherence, or umbilical vein endothelial cells, but all adhered to immobilized chondroitin sulphate A (CSA) and hyaluronic acid (HA), the receptors for adhesion of P. falciparum in the placenta. PvIRBC also adhered to fresh placental cells (N = 5). Pre-incubation with chondroitinase prevented PvIRBC adherence to CSA, and reduced binding to HA, whereas preincubation with hyaluronidase prevented adherence to HA, but did not reduce binding to CSA significantly. Pre-incubation of PvIRBC with soluble CSA and HA reduced binding to the immobilized receptors and prevented placental binding. PvIRBC adhesion was prevented by pre-incubation with trypsin, inhibited by heparin, and reduced by EGTA. Under laminar flow conditions the mean (SD) shear stress reducing maximum attachment by 50% was 0.06 (0.02) Pa but, having adhered, the PvIRBC could then resist detachment by stresses up to 5 Pa. At 37 °C adherence began approximately 16 hours after red cell invasion with maximal adherence at 30 hours. At 39 °C adherence began earlier and peaked at 24 hours. SIGNIFICANCE: Adherence of P. vivax-infected erythrocytes to glycosaminoglycans may contribute to the pathogenesis of vivax malaria and lead to intrauterine growth retardation.

Klein K, Aarons L, Ter Kuile FO, Nosten F, White NJ, Edstein MD, Teja-Isavadharm P. 2012. Population pharmacokinetics of halofantrine in healthy volunteers and patients with symptomatic falciparum malaria. J Pharm Pharmacol, 64 (11), pp. 1603-1613. | Show Abstract | Read more

AIMS: To investigate the population pharmacokinetics of the antimalarial halofantrine (HF) in healthy volunteers and patients with symptomatic falciparum malaria. METHODS: Healthy volunteer data were obtained from six volunteers who received three different doses of HF (250, 500 and 1000 mg) after an overnight fast with a washout period of at least 6 weeks between doses. Patient data (n = 188) were obtained from randomised controlled trials conducted on the Thai-Burmese border in the early 1990s. They were either assigned to receive a total HF dose of 24 mg/kg (8 mg/kg every 6 h for 24 h) or 72 mg/kg (8 mg/kg every 6 to 10 h for 3 days). The population pharmacokinetics of HF were evaluated using non-linear mixed effects modelling with a two-compartment model with first-order absorption. KEY FINDINGS: The population estimates of apparent clearance (CL), volume of compartment one (V1), distributional clearance (CLD) and volume of compartment two (V2) of HF in healthy volunteers were 2453 l/day (102 l/h), 2386 l, 716 l/day (29.8 l/h) and 2641 l, respectively. The population estimates of the PK parameters in patients were 429 l/day (17.9 l/h), 729 l, 178 l/day (7.42 l/h) and 1351 l, respectively. All PK parameters were significantly related to body weight and some were related to sex, sampling method, pre-treatment parasite density and whether patients vomited or not. When the two datasets were analysed jointly using a maximum likelihood method, the population estimates in patients were 196 l/day (8.17 l/h), 161 l, 65 l/day (2.71 l/h) and 89 l, respectively, and the parameters were significantly related to body weight and sex. Bayesian analysis of the patient data, with a diffuse prior based on the healthy volunteer data analysis results, yielded the population estimates 354 l/day (14.8 l/h), 728 l, 162 l/day (6.75 l/h) and 1939 l, respectively. CONCLUSIONS: The pharmacokinetic properties of HF in patients with malaria are affected by several demographic variables as well as other relevant covariates. Apparent differences between the healthy volunteer and the patient data analysis results are not entirely due to differences in bioavailability. For the patient data analysis, the Bayesian method was preferred, as the fitting procedure was more stable, allowing random effects to be estimated for all four dispositional parameters.

Lwin KM, Phyo AP, Tarning J, Hanpithakpong W, Ashley EA, Lee SJ, Cheah P, Singhasivanon P, White NJ, Lindegårdh N, Nosten F. 2012. Randomized, double-blind, placebo-controlled trial of monthly versus bimonthly dihydroartemisinin-piperaquine chemoprevention in adults at high risk of malaria. Antimicrob Agents Chemother, 56 (3), pp. 1571-1577. | Show Abstract | Read more

Intermittent preventive treatment (IPT) is increasingly used to reduce malaria morbidity and mortality in children and pregnant women. The efficacy of IPT depends on the pharmacokinetic and pharmacodynamic properties of the antimalarial drugs used. Healthy adult male volunteers whose occupation put them at high risk of malaria on the Northwest border of Thailand were randomized to receive a 3-day-treatment dose of dihydroartemisinin-piperaquine monthly (DPm) or every 2 months (DPalt) or an identical placebo with or without fat (6.4 g/dose) over a 9-month period. All volunteers were monitored weekly. One thousand adults were recruited. Dihydroartemisinin-piperaquine was well tolerated. There were 114 episodes of malaria (49 Plasmodium falciparum, 63 P. vivax, and 2 P. ovale). The protective efficacy against all malaria at 36 weeks was 98% (95% confidence interval [CI], 96% to 99%) in the DPm group and 86% (95% CI, 81% to 90%) in the DPalt group (for both, P < 0.0001 compared to the placebo group). As a result, the placebo group also had lower hematocrits during the study (P < 0.0001). Trough plasma piperaquine concentrations were the main determinant of efficacy; no malaria occurred in participants with a trough concentration above 31 ng/ml. Neither plasma piperaquine concentration nor efficacy was influenced by the coadministration of fat. DPm is safe to use and is effective in the prevention of malaria in adult males living in an area where P. vivax and multidrug-resistant P. falciparum malaria are endemic.

White NJ, Qiao LG, Qi G, Luzzatto L. 2012. Rationale for recommending a lower dose of primaquine as a Plasmodium falciparum gametocytocide in populations where G6PD deficiency is common. Malar J, 11 (1), pp. 418. | Show Abstract | Read more

In areas of low malaria transmission, it is currently recommended that a single dose of primaquine (0.75 mg base/kg; 45 mg adult dose) be added to artemisinin combination treatment (ACT) in acute falciparum malaria to block malaria transmission. Review of studies of transmission-blocking activity based on the infectivity of patients or volunteers to anopheline mosquitoes, and of haemolytic toxicity in glucose 6-dehydrogenase (G6PD) deficient subjects, suggests that a lower primaquine dose (0.25 mg base/kg) would be safer and equally effective. This lower dose could be deployed together with ACTs without G6PD testing wherever use of a specific gametocytocide is indicated.

White NJ, Imwong M. 2012. Relapse. Adv Parasitol, 80 pp. 113-150. | Show Abstract | Read more

Plasmodium vivax is a major cause of febrile illness in endemic areas of Asia, Central and South America, and the horn of Africa. P. vivax infections are characterized by relapses of malaria arising from persistent liver stages of the parasite (hypnozoites), which can be prevented currently only by 8-aminoquinoline anti-malarials. Tropical P. vivax infections relapse at approximately 3-week intervals if rapidly eliminated anti-malarials are given for treatment, whereas in temperate regions and parts of the sub-tropics, P. vivax infections are characterized by either a long incubation or a long-latency period between illness and relapse - in both cases approximating 8-10 months. The epidemiology of the different relapse phenotypes has not been defined adequately despite obvious relevance to malaria therapeutic assessment, control, and elimination. The number of sporozoites inoculated by the anopheline mosquito is an important determinant of both the timing and the number of relapses. The intervals between P. vivax relapses display a remarkable periodicity which has not been explained. Evidence is presented that the proportion of patients who have successive relapses is relatively constant and that the factor which activates hypnozoites and leads to regular interval relapse in vivax malaria is the systemic febrile illness itself. It is proposed that in endemic areas, a large proportion of the population harbours latent hypnozoites which can be activated by a systemic illness such as vivax or falciparum malaria. This explains the high rates of vivax following falciparum malaria, the high proportion of heterologous genotypes in relapses, the higher rates of relapse in people living in endemic areas compared with artificial infection studies, and, by facilitating recombination between different genotypes, contributes to P. vivax genetic diversity particularly in low transmission settings. Long-latency P. vivax phenotypes may be more widespread and more prevalent than currently thought. These observations have important implications for the assessment of radical treatment efficacy and for malaria control and elimination.

Taylor WR, Hanson J, Turner GD, White NJ, Dondorp AM. 2012. Respiratory manifestations of malaria. Chest, 142 (2), pp. 492-505. | Show Abstract | Read more

Respiratory distress develops in up to 25% of adults and 40% of children with severe falciparum malaria. Its diverse causes include respiratory compensation of metabolic acidosis, noncardiogenic pulmonary edema, concomitant pneumonia, and severe anemia. Patients with severe falciparum, vivax, and knowlesi malaria may develop acute lung injury (ALI) and ARDS, often several days after antimalarial drug treatment. ARDS rates, best characterized for severe Plasmodium falciparum, are 5% to 25% in adults and up to 29% in pregnant women; ARDS is rare in young children. ARDS pathophysiology centers on inflammatory-mediated increased capillary permeability or endothelial damage leading to diffuse alveolar damage that can continue after parasite clearance. The role of parasite sequestration in the pulmonary microvasculature is unclear, because sequestration occurs intensely in P falciparum, less so in P knowlesi, and has not been shown convincingly in P vivax. Because early markers of ALI/ARDS are lacking, fluid resuscitation in severe malaria should follow the old adage to "keep them dry." Bacteremia and hospital-acquired pneumonia can complicate severe malaria and may contribute to ALI/ARDS. Mechanical ventilation can save life in ALI/ARDS. Basic critical care facilities are increasingly available in tropical countries. The use of lung-protective ventilation has helped to reduce mortality from malaria-induced ALI/ARDS, but permissive hypercapnia in unconscious patients is not recommended because increased intracranial pressure and cerebral swelling may occur in cerebral malaria. The best antimalarial treatment of severe malaria is IV artesunate.

Kauss T, Gaudin K, Gaubert A, Ba B, Tagliaferri S, Fawaz F, Fabre JL, Boiron JM et al. 2012. Screening paediatric rectal forms of azithromycin as an alternative to oral or injectable treatment. Int J Pharm, 436 (1-2), pp. 624-630. | Show Abstract | Read more

The aim of this study was to identify a candidate formulation for further development of a home or near-home administrable paediatric rectal form of a broad-spectrum antibiotic - specially intended for (emergency) use in tropical rural settings, in particular for children who cannot take medications orally and far from health facilities where injectable treatments can be given. Azithromycin, a broad-spectrum macrolide used orally or intravenously for the treatment of respiratory tract, skin and soft tissue infections, was selected because of its pharmacokinetic and therapeutic properties. Azithromycin in vitro solubility and stability in physiologically relevant conditions were studied. Various pharmaceutical forms, i.e. rectal suspension, two different rectal gels, polyethylene glycol (PEG) suppository and hard gelatin capsule (HGC) were assessed for in vitro dissolution and in vivo bioavailability in the rabbit. Azithromycin PEG suppository appears to be a promising candidate.

Arrow KJ, Danzon PM, Gelband H, Jamison D, Laxminarayan R, Mills A, Mwabu G, Panosian C, Peto R, White NJ. 2012. The Affordable Medicines Facility--malaria: killing it slowly. Lancet, 380 (9857), pp. 1889-1890. | Read more

Douglas NM, Anstey NM, Buffet PA, Poespoprodjo JR, Yeo TW, White NJ, Price RN. 2012. The anaemia of Plasmodium vivax malaria. Malar J, 11 (1), pp. 135. | Show Abstract | Read more

Plasmodium vivax threatens nearly half the world's population and is a significant impediment to achievement of the millennium development goals. It is an important, but incompletely understood, cause of anaemia. This review synthesizes current evidence on the epidemiology, pathogenesis, treatment and consequences of vivax-associated anaemia. Young children are at high risk of clinically significant and potentially severe vivax-associated anaemia, particularly in countries where transmission is intense and relapses are frequent. Despite reaching lower densities than Plasmodium falciparum, Plasmodium vivax causes similar absolute reduction in red blood cell mass because it results in proportionately greater removal of uninfected red blood cells. Severe vivax anaemia is associated with substantial indirect mortality and morbidity through impaired resilience to co-morbidities, obstetric complications and requirement for blood transfusion. Anaemia can be averted by early and effective anti-malarial treatment.

Imwong M, Boel ME, Pagornrat W, Pimanpanarak M, McGready R, Day NP, Nosten F, White NJ. 2012. The first Plasmodium vivax relapses of life are usually genetically homologous. J Infect Dis, 205 (4), pp. 680-683. | Show Abstract | Read more

In a prospective infant cohort, 21 infants developed Plasmodium vivax malaria during their first year. Twelve of their mothers also had vivax malaria in the corresponding pregnancies or postpartum period. The genotypes of the maternal and infant infections were all different. Eight of the 12 mothers and 9 of the 21 infants had recurrent infections. Relapse parasite genotypes were different to the initial infection in 13 of 20 (65%) mothers compared with 5 of 24 (21%) infants (P = .02). The first P. vivax relapses of life are usually genetically homologous, whereas relapse in adults may result from activation of heterologous latent hypnozoites acquired from previous inoculations.

McGready R, Lee SJ, Wiladphaingern J, Ashley EA, Rijken MJ, Boel M, Simpson JA, Paw MK et al. 2012. Adverse effects of falciparum and vivax malaria and the safety of antimalarial treatment in early pregnancy: a population-based study. Lancet Infect Dis, 12 (5), pp. 388-396. | Show Abstract | Read more

BACKGROUND: The effects of malaria and its treatment in the first trimester of pregnancy remain an area of concern. We aimed to assess the outcome of malaria-exposed and malaria-unexposed first-trimester pregnancies of women from the Thai-Burmese border and compare outcomes after chloroquine-based, quinine-based, or artemisinin-based treatments. METHODS: We analysed all antenatal records of women in the first trimester of pregnancy attending Shoklo Malaria Research Unit antenatal clinics from May 12, 1986, to Oct 31, 2010. Women without malaria in pregnancy were compared with those who had a single episode of malaria in the first trimester. The association between malaria and miscarriage was estimated using multivariable logistic regression. FINDINGS: Of 48,426 pregnant women, 17,613 (36%) met the inclusion criteria: 16,668 (95%) had no malaria during the pregnancy and 945 (5%) had a single episode in the first trimester. The odds of miscarriage increased in women with asymptomatic malaria (adjusted odds ratio 2·70, 95% CI 2·04-3·59) and symptomatic malaria (3·99, 3·10-5·13), and were similar for Plasmodium falciparum and Plasmodium vivax. Other risk factors for miscarriage included smoking, maternal age, previous miscarriage, and non-malaria febrile illness. In women with malaria, additional risk factors for miscarriage included severe or hyperparasitaemic malaria (adjusted odds ratio 3·63, 95% CI 1·15-11·46) and parasitaemia (1·49, 1·25-1·78 for each ten-fold increase in parasitaemia). Higher gestational age at the time of infection was protective (adjusted odds ratio 0·86, 95% CI 0·81-0·91). The risk of miscarriage was similar for women treated with chloroquine (92 [26%] of 354), quinine (95 [27%) of 355), or artesunate (20 [31%] of 64; p=0·71). Adverse effects related to antimalarial treatment were not observed. INTERPRETATION: A single episode of falciparum or vivax malaria in the first trimester of pregnancy can cause miscarriage. No additional toxic effects associated with artesunate treatment occurred in early pregnancy. Prospective studies should now be done to assess the safety and efficacy of artemisinin combination treatments in early pregnancy.

Newton PN, Amin AA, Bird C, Passmore P, Dukes G, Tomson G, Simons B, Bate R, Guerin PJ, White NJ. 2011. The primacy of public health considerations in defining poor quality medicines. PLoS Med, 8 (12), pp. e1001139. | Read more

Hanson J, Lam SW, Mohanty S, Alam S, Hasan MM, Lee SJ, Schultz MJ, Charunwatthana P et al. 2011. Central venous catheter use in severe malaria: time to reconsider the World Health Organization guidelines? Malar J, 10 (1), pp. 342. | Show Abstract | Read more

BACKGROUND: To optimize the fluid status of adult patients with severe malaria, World Health Organization (WHO) guidelines recommend the insertion of a central venous catheter (CVC) and a target central venous pressure (CVP) of 0-5 cmH2O. However there are few data from clinical trials to support this recommendation. METHODS: Twenty-eight adult Indian and Bangladeshi patients admitted to the intensive care unit with severe falciparum malaria were enrolled in the study. All patients had a CVC inserted and had regular CVP measurements recorded. The CVP measurements were compared with markers of disease severity, clinical endpoints and volumetric measures derived from transpulmonary thermodilution. RESULTS: There was no correlation between the admission CVP and patient outcome (p = 0.67) or disease severity (p = 0.33). There was no correlation between the baseline CVP and the concomitant extravascular lung water (p = 0.62), global end diastolic volume (p = 0.88) or cardiac index (p = 0.44). There was no correlation between the baseline CVP and the likelihood of a patient being fluid responsive (p = 0.37). On the occasions when the CVP was in the WHO target range patients were usually hypovolaemic and often had pulmonary oedema by volumetric measures. Seven of 28 patients suffered a complication of the CVC insertion, although none were fatal. CONCLUSION: The WHO recommendation for the routine insertion of a CVC, and the maintenance of a CVP of 0-5 cmH2O in adults with severe malaria, should be reconsidered.

Flegg JA, Guerin PJ, White NJ, Stepniewska K. 2011. Standardizing the measurement of parasite clearance in falciparum malaria: the parasite clearance estimator. Malar J, 10 (1), pp. 339. | Show Abstract | Read more

BACKGROUND: A significant reduction in parasite clearance rates following artesunate treatment of falciparum malaria, and increased failure rates following artemisinin combination treatments (ACT), signaled emergent artemisinin resistance in Western Cambodia. Accurate measurement of parasite clearance is therefore essential to assess the spread of artemisinin resistance in Plasmodium falciparum. The slope of the log-parasitaemia versus time relationship is considered to be the most robust measure of anti-malarial effect. However, an initial lag phase of numerical instability often precedes a steady exponential decline in the parasite count after the start of anti-malarial treatment. This lag complicates the clearance estimation, introduces observer subjectivity, and may influence the accuracy and consistency of reported results. METHODS: To address this problem, a new approach to modelling clearance of malaria parasites from parasitaemia-time profiles has been explored and validated. The methodology detects when a lag phase is present, selects the most appropriate model (linear, quadratic or cubic) to fit log-transformed parasite data, and calculates estimates of parasite clearance adjusted for this lag phase. Departing from previous approaches, parasite counts below the level of detection are accounted for and not excluded from the calculation. RESULTS: Data from large clinical studies with frequent parasite counts were examined. The effect of a lag phase on parasite clearance rate estimates is discussed, using individual patient data examples. As part of the World Wide Antimalarial Resistance Network's (WWARN) efforts to make innovative approaches available to the malaria community, an automated informatics tool: the parasite clearance estimator has been developed. CONCLUSIONS: The parasite clearance estimator provides a consistent, reliable and accurate method to estimate the lag phase and malaria parasite clearance rate. It could be used to detect early signs of emerging resistance to artemisinin derivatives and other compounds which affect ring-stage clearance.

Phyo AP, Lwin KM, Price RN, Ashley EA, Russell B, Sriprawat K, Lindegardh N, Singhasivanon P, White NJ, Nosten F. 2011. Dihydroartemisinin-piperaquine versus chloroquine in the treatment of Plasmodium vivax malaria in Thailand: a randomized controlled trial. Clin Infect Dis, 53 (10), pp. 977-984. | Show Abstract | Read more

BACKGROUND: Chloroquine (CQ) remains the treatment of choice for Plasmodium vivax malaria. Initially confined to parts of Indonesia and Papua, resistance of P. vivax to CQ seems to be spreading, and alternative treatments are required. METHODS: We conducted a randomized controlled study to compare the efficacy and the tolerability of CQ and dihydroartemisinin-piperaquine (DP) in 500 adults and children with acute vivax malaria on the Northwestern border of Thailand. RESULTS: Both drugs were well tolerated. Fever and parasite clearance times were slower in the CQ than in the DP group (P < .001). By day 28, recurrent infections had emerged in 18 of 207 CQ recipients compared with 5 of 230 treated with DP (relative risk, 4.0; 95% confidence interval [CI], 1.51-10.58; P = .0046). The cumulative risk of recurrence with P. vivax at 9 weeks was 79.1% (95% CI, 73.5%-84.8%) in patients treated with CQ compared with 54.9% (95% CI, 48.2%-61.6%) in those receiving DP (hazard ratio [HR], 2.27; 95% CI, 1.8-2.9; P < .001). Children <5 years old were at greater risk of recurrent P. vivax infection (74.4%; 95% CI, 63.2%-85.6%) than older patients (55.3% [95% CI, 50.2%-60.4%]; HR, 1.58 [95% CI, 1.1-2.2]; P = .005). In vitro susceptibility testing showed that 13% of the tested isolates had a CQ median inhibitory concentration >100 nmol/L, suggesting reduced susceptibility. CONCLUSIONS: The efficacy of CQ in the treatment of P. vivax infections is declining on the Thai-Myanmar border. DP is an effective alternative treatment.

White NJ. 2011. A vaccine for malaria. N Engl J Med, 365 (20), pp. 1926-1927. | Read more

Turner C, Aye Mya Thein N, Turner P, Nosten F, White NJ. 2012. Rectal pH in well and unwell infants. J Trop Pediatr, 58 (4), pp. 311-313. | Show Abstract | Read more

Prompt antibiotic treatment for infants with sepsis has the potential to save lives. A rectal formulation of an antibiotic could be used at a village level before referral to hospital. The development of such a preparation needs to take into account the rectal pH of infants that will affect drug partitioning and absorption. Rectal pH measurements were taken in 100 well and 45 unwell infants. We also measured rectal pH in 14 infants sequentially over the course of their illness. The mean rectal pH was 6.75 with no significant difference in well or unwell infants. The mean (95% CI) rectal pH of well neonates was significantly lower than that of older infants (>28 days): 6.47 (6.29-6.65) vs. 6.90 (6.68 to 7.12) p = 0.003.

McGready R, Phyo AP, Rijken MJ, Tarning J, Lindegardh N, Hanpithakpon W, Than HH, Hlaing N et al. 2012. Artesunate/dihydroartemisinin pharmacokinetics in acute falciparum malaria in pregnancy: absorption, bioavailability, disposition and disease effects. Br J Clin Pharmacol, 73 (3), pp. 467-477. | Show Abstract | Read more

AIM: To determine if reported lower plasma concentrations of artemisinin derivatives for malaria in pregnancy result from reduced oral bioavailability, expanded volume of distribution or increased clearance. METHODS: In a sequentially assigned crossover treatment study, pregnant women with uncomplicated falciparum malaria received i.v. artesunate (i.v. ARS) (4mgkg(-1) ) on the first day and oral ARS (4mgkg(-1) ) on the second, or, oral on the first and i.v. on the second, in both groups followed by oral ARS (4mgkg(-1) day(-1) ) for 5 days. Plasma concentrations of ARS and dihyroartemisinin (DHA) were measured by liquid chromatography-mass-spectrometry on days 0, 1, 2 and 6. Controls were the same women restudied when healthy (3 months post partum). RESULTS: I.v. ARS administration resulted in similar ARS and DHA pharmacokinetics in pregnant women with malaria (n= 20) and in controls (n= 14). Oral administration resulted in higher total drug exposure in pregnancy [AUC (95% CI) in (ngml(-1) h)/(mgkg(-1) )] of 55.1 (30.1, 100.0) vs. 26.5 (12.2, 54.3) for ARS, P= 0.002 and 673 (386, 1130) vs. 523 (351, 724) for DHA, P= 0.007. The corresponding median absolute oral bioavailability (F%) was 21.7 (12.6, 75.1) vs. 9.9 (6.0, 36.81) for ARS (P= 0.046) and 77.0 (42.2, 129) vs. 72.7 (42.0, 87.7) for DHA, P= 0.033. Total DHA exposure was lower at day 6 in pregnant women with malaria (P < 0.001) compared with day 0 or 1, but not in the controls (P= 0.084). CONCLUSIONS: This study demonstrates the effects of malaria on oral ARS drug disposition are greater than those of pregnancy. This probably results from a disease related reduction in first pass metabolism. The data are reassuring regarding current dosing recommendations.

Larsen RA, Bauer M, Pitisuttithum P, Sanchez A, Tansuphaswadikul S, Wuthiekanun V, Peacock SJ, Simpson AJ et al. 2011. Correlation of susceptibility of Cryptococcus neoformans to amphotericin B with clinical outcome. Antimicrob Agents Chemother, 55 (12), pp. 5624-5630. | Show Abstract | Read more

Testing of Cryptococcus neoformans for susceptibility to antifungal drugs by standard microtiter methods has not been shown to correlate with clinical outcomes. This report describes a modified quantitative broth macrodilution susceptibility method showing a correlation with both the patient's quantitative biological response in the cerebrospinal fluid (CSF) and the survival of 85 patients treated with amphotericin B (AMB). The Spearman rank correlation between the quantitative in vitro measure of susceptibility and the quantitative measure of the number of organisms in the patient's CSF was 0.37 (P < 0.01; 95% confidence interval [95% CI], 0.20, 0.60) for the first susceptibility test replicate and 0.46 (P < 0.001; 95% CI, 0.21, 0.62) for the second susceptibility test replicate. The median in vitro estimated response (defined as the fungal burden after AMB treatment) at 1.5 mg/liter AMB for patients alive at day 14 was 5 CFU (95% CI, 3, 8), compared to 57 CFU (95% CI, 4, 832) for those who died before day 14. These exploratory results suggest that patients whose isolates show a quantitative in vitro susceptibility response below 10 CFU/ml were more likely to survive beyond day 14.

Imwong M, Nakeesathit S, Day NP, White NJ. 2011. A review of mixed malaria species infections in anopheline mosquitoes. Malar J, 10 (1), pp. 253. | Show Abstract | Read more

BACKGROUND: In patients with malaria mixed species infections are common and under reported. In PCR studies conducted in Asia mixed infection rates often exceed 20%. In South-East Asia, approximately one third of patients treated for falciparum malaria experience a subsequent Plasmodium vivax infection with a time interval suggesting relapse. It is uncertain whether the two infections are acquired simultaneously or separately. To determine whether mixed species infections in humans are derived from mainly from simultaneous or separate mosquito inoculations the literature on malaria species infection in wild captured anopheline mosquitoes was reviewed. METHODS: The biomedical literature was searched for studies of malaria infection and species identification in trapped wild mosquitoes and artificially infected mosquitoes. The study location and year, collection methods, mosquito species, number of specimens, parasite stage examined (oocysts or sporozoites), and the methods of parasite detection and speciation were tabulated. The entomological results in South East Asia were compared with mixed infection rates documented in patients in clinical studies. RESULTS: In total 63 studies were identified. Individual anopheline mosquitoes were examined for different malaria species in 28 of these. There were 14 studies from Africa; four with species evaluations in individual captured mosquitoes (SEICM). One study, from Ghana, identified a single mixed infection. No mixed infections were identified in Central and South America (seven studies, two SEICM). 42 studies were conducted in Asia and Oceania (11 from Thailand; 27 SEICM). The proportion of anophelines infected with Plasmodium falciparum parasites only was 0.51% (95% CI: 0.44 to 0.57%), for P. vivax only was 0.26% (95% CI: 0.21 to 0.30%), and for mixed P. falciparum and P. vivax infections was 0.036% (95% CI: 0.016 to 0.056%). The proportion of mixed infections in mosquitoes was significantly higher than expected by chance (P < 0.001), but was one fifth of that sufficient to explain the high rates of clinical mixed infections by simultaneous inoculation. CONCLUSIONS: There are relatively few data on mixed infection rates in mosquitoes from Africa. Mixed species malaria infections may be acquired by simultaneous inoculation of sporozoites from multiply infected anopheline mosquitoes but this is relatively unusual. In South East Asia, where P. vivax infection follows P. falciparum malaria in one third of cases, the available entomological information suggests that the majority of these mixed species malaria infections are acquired from separate inoculations.

White LJ, Lee SJ, Stepniewska K, Simpson JA, Dwell SL, Arunjerdja R, Singhasivanon P, White NJ, Nosten F, McGready R. 2012. Estimation of gestational age from fundal height: a solution for resource-poor settings. J R Soc Interface, 9 (68), pp. 503-510. | Show Abstract | Read more

Many women in resource-poor settings lack access to reliable gestational age assessment because they do not know their last menstrual period; there is no ultrasound (US) and methods of newborn gestational age dating are not practised by birth attendants. A bespoke multiple-measures model was developed to predict the expected date of delivery determined by US. The results are compared with both a linear and a nonlinear model. Prospectively collected early US and serial symphysis-pubis fundal height (SFH) data were used in the models. The data were collected from Karen and Burmese women attending antenatal care on the Thai-Burmese border. The multiple-measures model performed best, resulting in a range of accuracy depending on the number of SFH measures recorded per mother (for example six SFH measurements resulted in a prediction accuracy of ±2 weeks). SFH remains the proxy for gestational age in much of the resource-poor world. While more accurate measures should be encouraged, we demonstrate that a formula that incorporates at least three SFH measures from an individual mother and the slopes between them provide a significant increase in the accuracy of prediction compared with the linear and nonlinear formulae also using multiple SFH measures.

Mok S, Imwong M, Mackinnon MJ, Sim J, Ramadoss R, Yi P, Mayxay M, Chotivanich K et al. 2011. Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription. BMC Genomics, 12 (1), pp. 391. | Show Abstract | Read more

BACKGROUND: Artemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates. RESULTS: In the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype. CONCLUSIONS: The decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes in vivo.

Hanson J, Hasan MM, Royakkers AA, Alam S, Charunwatthana P, Maude RJ, Douthwaite ST, Yunus EB et al. 2011. Laboratory prediction of the requirement for renal replacement in acute falciparum malaria. Malar J, 10 (1), pp. 217. | Show Abstract | Read more

BACKGROUND: Acute renal failure is a common complication of severe malaria in adults, and without renal replacement therapy (RRT), it carries a poor prognosis. Even when RRT is available, delaying its initiation may increase mortality. Earlier identification of patients who will need RRT may improve outcomes. METHOD: Prospectively collected data from two intervention studies in adults with severe malaria were analysed focusing on laboratory features on presentation and their association with a later requirement for RRT. In particular, laboratory indices of acute tubular necrosis (ATN) and acute kidney injury (AKI) that are used in other settings were examined. RESULTS: Data from 163 patients were available for analysis. Whether or not the patients should have received RRT (a retrospective assessment determined by three independent reviewers) was used as the reference. Forty-three (26.4%) patients met criteria for dialysis, but only 19 (44.2%) were able to receive this intervention due to the limited availability of RRT. Patients with impaired renal function on admission (creatinine clearance < 60 ml/min) (n = 84) had their laboratory indices of ATN/AKI analysed. The plasma creatinine level had the greatest area under the ROC curve (AUC): 0.83 (95% confidence interval 0.74-0.92), significantly better than the AUCs for, urinary sodium level, the urea to creatinine ratio (UCR), the fractional excretion of urea (FeUN) and the urinary neutrophil gelatinase-associated lipocalcin (NGAL) level. The AUC for plasma creatinine was also greater than the AUC for blood urea nitrogen level, the fractional excretion of sodium (FeNa), the renal failure index (RFI), the urinary osmolality, the urine to plasma creatinine ratio (UPCR) and the creatinine clearance, although the difference for these variables did not reach statistical significance. CONCLUSIONS: In adult patients with severe malaria and impaired renal function on admission, none of the evaluated laboratory indices was superior to the plasma creatinine level when used to predict a later requirement for renal replacement therapy.

Maude RJ, Saralamba S, Lewis A, Sherwood D, White NJ, Day NP, Dondorp AM, White LJ. 2011. Modelling malaria elimination on the internet. Malar J, 10 (1), pp. 191. | Show Abstract | Read more

BACKGROUND: Unprecedented efforts are underway to eliminate malaria. Mathematical modelling can help to determine the optimal strategies for malaria elimination in different epidemiological settings. This is necessary as there is limited scope for expensive and time-consuming field studies and failure of planned elimination strategies is likely to discourage ongoing investment by funders. However, there has been very little modelling of malaria elimination and little direct involvement of policymakers in its development. There is thus an urgent need for user-friendly and accessible models purpose-designed in collaboration with policymakers to answer pertinent questions arising from the field. RESULTS: An internet site is presented with a simple mathematical modelling platform for population level models of malaria elimination. It is freely accessible to all and designed to be flexible so both the platform and models can be developed through interaction with users. The site is an accessible introduction to modelling for a non-mathematical audience, and lessons learned from the project will help inform future development of mathematical models and improve communication of modelling results. Currently it hosts a simple model of strategies for malaria elimination and this will be developed, and more models added, over time. The iterative process of feedback and development will result in an educational and planning tool for non-modellers to assist with malaria elimination efforts worldwide. CONCLUSIONS: By collaboration with end users, iterative development of mathematical models of malaria elimination through this internet platform will maximize its potential as an educational and public health policy planning tool. It will also assist with preliminary optimisation of local malaria elimination strategies before commitment of valuable resources.

Lubell Y, Turner P, Ashley EA, White NJ. 2011. Susceptibility of bacterial isolates from community-acquired infections in sub-Saharan Africa and Asia to macrolide antibiotics. Trop Med Int Health, 16 (10), pp. 1192-1205. | Show Abstract | Read more

OBJECTIVE: To review the literature on the susceptibility of common community pathogens in sub-Saharan Africa and Asia to the macrolide antibiotics. METHODS: Inclusion criteria required that isolates were collected since 2004 to ensure results were of contemporary relevance. The data were aggregated by region, age group and sterility of site of culture sample. RESULTS: A total of 51 studies were identified, which reported the macrolide antimicrobial susceptibilities of common bacterial pathogens isolated since 2004. In general, there was less macrolide resistance in African than in Asian isolates. Most African studies reported high levels of macrolide susceptibility in Streptococcus pneumoniae, whereas most Chinese studies reported high levels of resistance. There was very little information available for Gram-negative organisms. CONCLUSIONS: Susceptibility of the pneumococcus to macrolides in SSA remains high in many areas, and good activity of azithromycin has been shown against Salmonellae spp. in Asia. In urban areas where high antibiotic consumption is prevalent, there was evidence of increased resistance to macrolides. However, there is no information on susceptibility from large areas in both continents.

Lubell Y, Riewpaiboon A, Dondorp AM, von Seidlein L, Mokuolu OA, Nansumba M, Gesase S, Kent A et al. 2011. Cost-effectiveness of parenteral artesunate for treating children with severe malaria in sub-Saharan Africa. Bull World Health Organ, 89 (7), pp. 504-512. | Show Abstract | Read more

OBJECTIVE: To explore the cost-effectiveness of parenteral artesunate for the treatment of severe malaria in children and its potential impact on hospital budgets. METHODS: The costs of inpatient care of children with severe malaria were assessed in four of the 11 sites included in the African Quinine Artesunate Malaria Treatment trial, conducted with over 5400 children. The drugs, laboratory tests and intravenous fluids provided to 2300 patients from admission to discharge were recorded, as was the length of inpatient stay, to calculate the cost of inpatient care. The data were matched with pooled clinical outcomes and entered into a decision model to calculate the cost per disability-adjusted life year (DALY) averted and the cost per death averted. FINDINGS: The mean cost of treating severe malaria patients was similar in the two study groups: 63.5 United States dollars (US$) (95% confidence interval, CI: 61.7-65.2) in the quinine arm and US$ 66.5 (95% CI: 63.7-69.2) in the artesunate arm. Children treated with artesunate had 22.5% lower mortality than those treated with quinine and the same rate of neurological sequelae: (artesunate arm: 2.3 DALYs per patient; quinine arm: 3.0 DALYs per patient). Compared with quinine as a baseline, artesunate showed an incremental cost per DALY averted and an incremental cost per death averted of US$ 3.8 and US$ 123, respectively. CONCLUSION: Artesunate is a highly cost-effective and affordable alternative to quinine for treating children with severe malaria. The budgetary implications of adopting artesunate for routine use in hospital-based care are negligible.

Jamsen KM, Duffull SB, Tarning J, Lindegardh N, White NJ, Simpson JA. 2011. Optimal designs for population pharmacokinetic studies of oral artesunate in patients with uncomplicated falciparum malaria. Malar J, 10 (1), pp. 181. | Show Abstract | Read more

BACKGROUND: Currently, population pharmacokinetic (PK) studies of anti-malarial drugs are designed primarily by the logistical and ethical constraints of taking blood samples from patients, and the statistical models that are fitted to the data are not formally considered. This could lead to imprecise estimates of the target PK parameters, and/or designs insufficient to estimate all of the parameters. Optimal design methodology has been developed to determine blood sampling schedules that will yield precise parameter estimates within the practical constraints of sampling the study populations. In this work optimal design methods were used to determine sampling designs for typical future population PK studies of dihydroartemisinin, the principal biologically active metabolite of oral artesunate. METHODS: Optimal designs were derived using freely available software and were based on appropriate structural PK models from an analysis of data or the literature and key sampling constraints identified in a questionnaire sent to active malaria researchers (3-4 samples per patient, at least 15 minutes between samples). The derived optimal designs were then evaluated via simulation-estimation. RESULTS: The derived optimal sampling windows were 17 to 29 minutes, 30 to 57 minutes, 2.5 to 3.7 hours and 5.8 to 6.6 hours for non-pregnant adults; 16 to 29 minutes, 31 minutes to 1 hour, 2.0 to 3.4 hours and 5.5 to 6.6 hours for designs with non-pregnant adults and children and 35 to 59 minutes, 1.2 to 3.4 hours, 3.4 to 4.9 hours and 6.0 to 8.0 hours for pregnant women. The optimal designs resulted in acceptable precision of the PK parameters. CONCLUSIONS: The proposed sampling designs in this paper are robust and efficient and should be considered in future PK studies of oral artesunate where only three or four blood samples can be collected.

Lindegardh N, Hanpithakpong W, Kamanikom B, Pattayaso J, Singhasivanon P, White NJ, Day NP. 2011. Quantification of dihydroartemisinin, artesunate and artemisinin in human blood: overcoming the technical challenge of protecting the peroxide bridge. Bioanalysis, 3 (14), pp. 1613-1624. | Show Abstract | Read more

BACKGROUND: Quantification of artemisinin (ARN) and its derivatives in whole blood has hitherto been thought impossible. RESULTS: A LC-MS/MS method for the analysis of artesunate (ARS), its metabolite dihydroartemisinin (DHA) and artemisinin in human whole blood has been developed and successfully validated. The method includes stabilization of the blood matrix at the time of collection and at the time of analysis. Addition of potassium dichromate to the blood samples deactivated the Fe(2+) core in hemoglobin, while deferoxamine chelated Fe(3+) and prevented back conversion into Fe(2+). A pilot study showed that the blood:plasma ratio for ARS and DHA is approximately 0.75, indicating a significantly lower uptake in red blood cells than had previously been estimated using radiolabeled drug methodology. CONCLUSIONS: The developed LC-MS/MS assay is the first method available for quantification of ARN and its derivatives in blood and opens up new possibilities of studying these drugs inside infected red blood cells.

Rijken MJ, McGready R, Jullien V, Tarning J, Lindegardh N, Phyo AP, Win AK, Hsi P et al. 2011. Pharmacokinetics of amodiaquine and desethylamodiaquine in pregnant and postpartum women with Plasmodium vivax malaria. Antimicrob Agents Chemother, 55 (9), pp. 4338-4342. | Show Abstract | Read more

In order to study the pharmacokinetic properties of amodiaquine and desethylamodiaquine during pregnancy, 24 pregnant women in the second and third trimesters of pregnancy and with Plasmodium vivax malaria were treated with amodiaquine (10 mg/kg of body weight/day) for 3 days. The same women were studied again at 3 months postpartum. Plasma was analyzed for amodiaquine and desethylamodiaquine by use of a liquid chromatography-tandem mass spectrometry method. Individual concentration-time data were evaluated using noncompartmental analysis. There were no clinically relevant differences in the pharmacokinetics of amodiaquine and desethylamodiaquine between pregnant (n = 24) and postpartum (n = 18) women. The results suggest that the current amodiaquine dosing regimen is adequate for the treatment of P. vivax infections during pregnancy.

Ashley EA, Lubell Y, White NJ, Turner P. 2011. Antimicrobial susceptibility of bacterial isolates from community acquired infections in Sub-Saharan Africa and Asian low and middle income countries. Trop Med Int Health, 16 (9), pp. 1167-1179. | Show Abstract | Read more

OBJECTIVE: Antimicrobial resistance has arisen across the globe in both nosocomial and community settings as a consequence of widespread antibiotic consumption. Poor availability of laboratory diagnosis means that resistance frequently goes unrecognised and may only be detected as clinical treatment failure. In this review, we provide an overview of the reported susceptibility of common community acquired bacterial pathogens in Sub-Saharan Africa and Asia to the antibiotics that are most widely used in these areas. METHODS: We reviewed the literature for reports of the susceptibility of prevalent pathogens in the community in SSA and Asia to a range of commonly prescribed antibiotics. Inclusion criteria required that isolates were collected since 2004 and that they were obtained from either normally sterile sites or urine. The data were aggregated by region and by age group. RESULTS: Eighty-three studies were identified since 2004 which reported the antimicrobial susceptibilities of common bacterial pathogens. Different methods were used to assess in-vitro susceptibility in the different studies. The quality of testing (evidenced by resistance profiles) also varied considerably. For Streptococcus pneumoniae and Neisseria meningitidis most drugs maintained relatively high efficacy, apart from co-trimoxazole to which there were high levels of resistance in most of the pathogens surveyed. CONCLUSIONS: Compared with the enormous infectious disease burden and widespread use of antibiotics there are relatively few reliable data on antimicrobial susceptibility from tropical Asia and Africa upon which to draw firm conclusions, although it is evident that many commonly used antibiotics face considerable resistance in prevalent bacterial pathogens. This is likely to exacerbate morbidity and mortality. Investment in improved antimicrobial susceptibility testing and surveillance systems is likely to be a highly cost-effective strategy and should be complemented by centralized and readily accessible information resources.

Pukrittayakamee S, Jittamala P, Stepniewska K, Lindegardh N, Chueasuwanchai S, Leowattana W, Phakdeeraj A, Permpunpanich S et al. 2011. An open-label crossover study to evaluate potential pharmacokinetic interactions between oral oseltamivir and intravenous zanamivir in healthy Thai adults. Antimicrob Agents Chemother, 55 (9), pp. 4050-4057. | Show Abstract | Read more

There is no parenteral formulation of the neuraminidase inhibitor oseltamivir, the most widely used anti-influenza virus drug. Oseltamivir resistance is an increasing problem. Zanamivir is effective against the most prevalent oseltamivir-resistant influenza viruses. A parenteral formulation of zanamivir is in development for the treatment of severe influenza. It is not known if there is any pharmacokinetic interaction between the two drugs. Sixteen healthy Thai adult volunteers were studied in an open-label, four-period, randomized two-sequence crossover pharmacokinetic study in which zanamivir was given by constant-rate infusion or slow intravenous injection either alone or together with oral oseltamivir. Plasma concentration profiles of oseltamivir, the active metabolite oseltamivir carboxylate, and zanamivir were measured by liquid chromatography-mass spectrometry-mass spectrometry. Both drugs were well tolerated alone and in combination. The maximum plasma concentrations and the areas under the plasma concentration-time curves (AUC) of oseltamivir and oseltamivir carboxylate were not significantly different when oseltamivir was given separately or together with zanamivir. Maximum plasma concentrations of zanamivir were 10% (95% confidence interval, 7 to 12%) higher when zanamivir was infused concurrently with oral oseltamivir than with infusions before or after oral oseltamivir. The plasma zanamivir total AUC was positively correlated with the total oseltamivir carboxylate AUC (Pearson's correlation coefficient [r(P)] = 0.720, P = 0.002, n = 16) but not with the oseltamivir AUC (r(p) = 0.121, n = 16). There is no clinically significant pharmacokinetic interaction between oseltamivir and zanamivir.

Ford NP, de Smet M, Kolappa K, White NJ. 2011. Responding to the evidence for the management of severe malaria. Trop Med Int Health, 16 (9), pp. 1085-1086. | Read more

Lacaze C, Kauss T, Kiechel JR, Caminiti A, Fawaz F, Terrassin L, Cuart S, Grislain L et al. 2011. The initial pharmaceutical development of an artesunate/amodiaquine oral formulation for the treatment of malaria: a public-private partnership. Malar J, 10 (1), pp. 142. | Show Abstract | Read more

BACKGROUND: Artemisinin-based combination therapy is currently recommended worldwide for the treatment of uncomplicated malaria. Fixed-dose combinations are preferred as they favour compliance. This paper reports on the initial phases of the pharmaceutical development of an artesunate-amodiaquine (ASAQ) bilayer co-formulation tablet, undertaken following pre-formulation studies by a network of scientists and industrials from institutions of both industrialized and low income countries. METHODS: Pharmaceutical development was performed by a research laboratory at the University Bordeaux Segalen, School of Pharmacy, for feasibility and early stability studies of various drug formulations, further transferred to a company specialized in pharmaceutical development, and then provided to another company for clinical batch manufacturing. The work was conducted by a regional public-private not-for-profit network (TropiVal) within a larger Public Private partnership (the FACT project), set up by WHO/TDR, Médecins Sans Frontières and the Drugs for Neglected Disease initiative (DNDi). RESULTS: The main pharmaceutical goal was to combine in a solid oral form two incompatible active principles while preventing artesunate degradation under tropical conditions. Several options were attempted and failed to provide satisfactory stability results: incorporating artesunate in the external phase of the tablets, adding a pH regulator, alcoholic wet granulation, dry granulation, addition of an hydrophobic agent, tablet manufacturing in controlled conditions. However, long-term stability could be achieved, in experimental batches under GMP conditions, by physical separation of artesunate and amodiaquine in a bilayer co-formulation tablet in alu-alu blisters. Conduction of the workplan was monitored by DNDi. CONCLUSIONS: Collaborations between research and industrial groups greatly accelerated the process of development of the bi-layered ASAQ tablet. Lack of public funding was the main obstacle hampering the development process, and no intellectual property right was claimed. This approach resulted in a rapid technology transfer to the drug company Sanofi-Aventis, finalizing the process of development, registration and WHO pre-qualification of the fixed-dose co-formulation together with DNDi. The bi-layered tablet is made available under the names of Coarsucam® and Artesunate amodiaquine Winthrop®, Sanofi-Aventis. The issue related to the difficulty of public institutions to valorise their participation in such initiative by lack of priority and funding of applied research is discussed.

Hendriksen IC, Mtove G, Pedro AJ, Gomes E, Silamut K, Lee SJ, Mwambuli A, Gesase S et al. 2011. Evaluation of a PfHRP2 and a pLDH-based rapid diagnostic test for the diagnosis of severe malaria in 2 populations of African children. Clin Infect Dis, 52 (9), pp. 1100-1107. | Show Abstract | Read more

BACKGROUND: Rapid diagnostic tests (RDTs) now play an important role in the diagnosis of falciparum malaria in many countries where the disease is endemic. Although these tests have been extensively evaluated in uncomplicated falciparum malaria, reliable data on their performance for diagnosing potentially lethal severe malaria is lacking. METHODS: We compared a Plasmodium falciparum histidine-rich-protein2 (PfHRP₂)-based RDT and a Plasmodium lactate dehydrogenase (pLDH)-based RDT with routine microscopy of a peripheral blood slide and expert microscopy as a reference standard for the diagnosis of severe malaria in 1898 children who presented with severe febrile illness at 2 centers in Mozambique and Tanzania. RESULTS: The overall sensitivity, specificity, positive predictive value, and negative predictive values of the PfHRP₂-based test were 94.0%, 70.9%, 85.4%, and 86.8%, respectively, and for the pLDH-based test, the values were 88.0%, 88.3%, 93.2%, and 80.3%, respectively. At parasite counts < 1000 parasites/μL (n = 173), sensitivity of the pLDH-based test was low (45.7%), compared with that of the PfHRP₂-based test (69.9%). Both RDTs performed better than did the routine slide reading in a clinical laboratory as assessed in 1 of the centers. CONCLUSION: The evaluated PfHRP2-based RDT is an acceptable alternative to routine microscopy for diagnosing severe malaria in African children and performed better than did the evaluated pLDH-based RDT.

Dondorp AM, Fanello CI, von Seidlein L, Day NPJ, White NJ. 2011. Artesunate for severe malaria in African children Reply LANCET, 377 (9772), pp. 1154-1154.

Koh GC, Maude RR, Schreiber MF, Limmathurotsakul D, Wiersinga WJ, Wuthiekanun V, Lee SJ, Mahavanakul W et al. 2011. Glyburide is anti-inflammatory and associated with reduced mortality in melioidosis. Clin Infect Dis, 52 (6), pp. 717-725. | Show Abstract | Read more

BACKGROUND: Patients with diabetes mellitus are more prone to bacterial sepsis, but there are conflicting data on whether outcomes are worse in diabetics after presentation with sepsis. Glyburide is an oral hypoglycemic agent used to treat diabetes mellitus. This K(ATP)-channel blocker and broad-spectrum ATP-binding cassette (ABC) transporter inhibitor has broad-ranging effects on the immune system, including inhibition of inflammasome assembly and would be predicted to influence the host response to infection. METHODS: We studied a cohort of 1160 patients with gram-negative sepsis caused by a single pathogen (Burkholderia pseudomallei), 410 (35%) of whom were known to have diabetes. We subsequently studied prospectively diabetics with B. pseudomallei infection (n = 20) to compare the gene expression profile of peripheral whole blood leukocytes in patients who were taking glyburide against those not taking any sulfonylurea. RESULTS: Survival was greater in diabetics than in nondiabetics (38% vs 45%, respectively, P = .04), but the survival benefit was confined to the patient group taking glyburide (adjusted odds ratio .47, 95% confidence interval .28-.74, P = .005). We identified differential expression of 63 immune-related genes (P = .001) in patients taking glyburide, the sum effect of which we predict to be antiinflammatory in the glyburide group. CONCLUSIONS: We present observational evidence for a glyburide-associated benefit during human melioidosis and correlate this with an anti-inflammatory effect of glyburide on the immune system.

Nantakomol D, Dondorp AM, Krudsood S, Udomsangpetch R, Pattanapanyasat K, Combes V, Grau GE, White NJ, Viriyavejakul P, Day NP, Chotivanich K. 2011. Circulating red cell-derived microparticles in human malaria. J Infect Dis, 203 (5), pp. 700-706. | Show Abstract | Read more

In patients with falciparum malaria, plasma concentrations of cell-derived microparticles correlate with disease severity. Using flow cytometry, we quantified red blood cell-derived microparticles (RMPs) in patients with malaria and identified the source and the factors associated with production. RMP concentrations were increased in patients with Plasmodium falciparum (n = 29; median, 457 RMPs/μL [range, 13-4,342 RMPs/μL]), Plasmodium vivax (n = 5; median, 409 RMPs/μL [range, 281-503/μL]), and Plasmodium malariae (n = 2; median, 163 RMPs/μL [range, 127-200 RMPs/μL]) compared with those in healthy subjects (n = 11; median, 8 RMPs/μL [range, 3-166 RMPs/μL]; P = .01). RMP concentrations were highest in patients with severe falciparum malaria (P = .01). Parasitized red cells produced >10 times more RMPs than did unparasitized cells, but the overall majority of RMPs still derived from uninfected red blood cells (URBCs). In cultures, RMP production increased as the parasites matured. Hemin and parasite products induced RMP production in URBCs, which was inhibited by N-acetylcysteine, suggesting heme-mediated oxidative stress as a pathway for the generation of RMPs.

Douglas NM, Nosten F, Ashley EA, Phaiphun L, van Vugt M, Singhasivanon P, White NJ, Price RN. 2011. Plasmodium vivax recurrence following falciparum and mixed species malaria: risk factors and effect of antimalarial kinetics. Clin Infect Dis, 52 (5), pp. 612-620. | Show Abstract | Read more

BACKGROUND: Plasmodium vivax malaria commonly follows treatment of falciparum malaria in regions of co-endemicity. This is an important cause of preventable morbidity. METHODS: We examined the factors contributing to the risk of recurrence of P. vivax infection after treatment of acute falciparum malaria in a series of clinical trials conducted on the Thai-Myanmar border from 1991 through 2005. RESULTS: Overall, 10,549 patients (4960 children aged <15 years and 5589 adults) were treated for falciparum malaria; of these patients, 9385 (89.0%) had Plasmodium falciparum monoinfection and 1164 (11.0%) had mixed P. falciparum/P. vivax infections according to microscopic examinations performed at screening. The cumulative proportion of patients with P. falciparum infection recurrence by day 63 was 21.5% (95% confidence interval [CI], 20.3%-22.8%), and the cumulative proportion with P. vivax infection recurrence was 31.5% (95% CI, 30.1%-33.0%). Significant risk factors for P. vivax infection recurrence were mixed infection at enrollment, male sex, younger age, lower hematocrit, higher asexual P. falciparum parasite density (P < .001 for all factors), and P. falciparum gametocytemia at enrollment (P = .001). By day 63, the cumulative risk of vivax malaria after P. falciparum monoinfection was 51.1% (95% CI, 46.1%-56.2%) after treatment with rapidly eliminated drugs (t(1/2) <1 day), 35.3% (95% CI, 31.8%-39.0%) after treatment with intermediate half-life drugs (t(1/2) 1-7 days), and 19.6% (95% CI, 18.1%-21.3%) after treatment with slowly eliminated drugs (t(1/2) > 7 days) (P < .001, by test for trend). Artemisinin-based combinations containing mefloquine or piperaquine, compared with the artemether-lumefantrine and artesunate-atovaquone-proguanil combinations, were associated with a 3.6-fold to 4.2-fold lower adjusted hazard ratio for P. vivax infection recurrence within 63 days after pure or mixed P. falciparum infections (P < .001, for comparisons with artesunate-mefloquine). CONCLUSIONS: On the Thai-Myanmar border, P. vivax is the most common cause of parasitological failure after treatment for falciparum malaria. Slowly eliminated antimalarials reduce the risk of early P. vivax infection recurrence.

Lubell Y, Staedke SG, Greenwood BM, Kamya MR, Molyneux M, Newton PN, Reyburn H, Snow RW et al. 2011. Likely health outcomes for untreated acute febrile illness in the tropics in decision and economic models; a Delphi survey. PLoS One, 6 (2), pp. e17439. | Show Abstract | Read more

BACKGROUND: Modelling is widely used to inform decisions about management of malaria and acute febrile illnesses. Most models depend on estimates of the probability that untreated patients with malaria or bacterial illnesses will progress to severe disease or death. However, data on these key parameters are lacking and assumptions are frequently made based on expert opinion. Widely diverse opinions can lead to conflicting outcomes in models they inform. METHODS AND FINDINGS: A Delphi survey was conducted with malaria experts aiming to reach consensus on key parameters for public health and economic models, relating to the outcome of untreated febrile illnesses. Survey questions were stratified by malaria transmission intensity, patient age, and HIV prevalence. The impact of the variability in opinion on decision models is illustrated with a model previously used to assess the cost-effectiveness of malaria rapid diagnostic tests. Some consensus was reached around the probability that patients from higher transmission settings with untreated malaria would progress to severe disease (median 3%, inter-quartile range (IQR) 1-5%), and the probability that a non-malaria illness required antibiotics in areas of low HIV prevalence (median 20%). Children living in low transmission areas were considered to be at higher risk of progressing to severe malaria (median 30%, IQR 10-58%) than those from higher transmission areas (median 13%, IQR 7-30%). Estimates of the probability of dying from severe malaria were high in all settings (medians 60-73%). However, opinions varied widely for most parameters, and did not converge on resurveying. CONCLUSIONS: This study highlights the uncertainty around potential consequences of untreated malaria and bacterial illnesses. The lack of consensus on most parameters, the wide range of estimates, and the impact of variability in estimates on model outputs, demonstrate the importance of sensitivity analysis for decision models employing expert opinion. Results of such models should be interpreted cautiously. The diversity of expert opinion should be recognised when policy options are debated.

Maude RJ, White NJ, White LJ. 2011. Feasibility of malaria elimination. Lancet, 377 (9766), pp. 638. | Read more

Lang T, Cheah PY, White NJ. 2011. Clinical research: time for sensible global guidelines. Lancet, 377 (9777), pp. 1553-1555. | Read more

Khounnorath S, Chamberlain K, Taylor AM, Soukaloun D, Mayxay M, Lee SJ, Phengdy B, Luangxay K et al. 2011. Clinically unapparent infantile thiamin deficiency in Vientiane, Laos. PLoS Negl Trop Dis, 5 (2), pp. e969. | Show Abstract | Read more

BACKGROUND: Beriberi occurs in Vientiane, Lao PDR, among breastfed infants. Clinical disease may be the tip of an iceberg with subclinical thiamin deficiency contributing to other illnesses. Thiamin treatment could improve outcome. METHODOLOGY/PRINCIPAL FINDINGS: A cohort of 778 sick infants admitted during one year without clinical evidence of beriberi were studied prospectively and erythrocyte transketolase assays (ETK) performed. Biochemical thiamin deficiency was defined both in terms of the activation coefficient (α>31%) and basal ETK activity <0.59 micromoles/min/gHb. Of the 778 infants, median (range) age was 5 (0-12) months, 79.2% were breastfed, 5.1% had α>31% and 13.4 % basal ETK<0.59 micromoles/min/gHb. Infants≥2 months old had a higher frequency of biochemical markers of thiamin deficiency. Mortality was 5.5% but, among infants ≥2 months old, mortality was higher in those with basal ETK<0.59 micromoles/min/gHb (3/47, 6.4%) than in those with basal ETK≥0.59 micromoles/min/gHb (1/146, 0.7%) (P=0.045, relative risk=9.32 (95%CI 0.99 to 87.5)). Multivariate regression analysis indicated that infant age≥2 months and fewer maternal years of schooling were independently associated with infant basal ETK<0.59 micromoles/min/gHb. CONCLUSIONS/SIGNIFICANCE: Clinically unapparent thiamin deficiency is common among sick infants (≥2 months old) admitted to hospital in Vientiane. This may contribute to mortality and a low clinical threshold for providing thiamin to sick infants may be needed.

Annerberg A, Lwin KM, Lindegardh N, Khrutsawadchai S, Ashley E, Day NP, Singhasivanon P, Tarning J, White NJ, Nosten F. 2011. A small amount of fat does not affect piperaquine exposure in patients with malaria. Antimicrob Agents Chemother, 55 (9), pp. 3971-3976. | Show Abstract | Read more

Dihydroartemisinin-piperaquine is a new, highly effective, and well-tolerated combination treatment for uncomplicated falciparum malaria. The lipophilic characteristic of piperaquine suggests that administration together with fat will increase the oral bioavailability of the drug, and this has been reported for healthy volunteers. This pharmacokinetic study monitored 30 adult patients with uncomplicated falciparum malaria for 4.5 months to evaluate the effects of the concomitant intake of fat on the total piperaquine exposure. The fixed-drug combination of dihydroartemisinin-piperaquine was given with water to fasting patients (n = 15) or was coadministered with 200 ml milk containing 6.4 g fat (n = 15). The drug combination was generally well tolerated, and there were no severe adverse effects reported for either group during the study. Total piperaquine exposure (area under the concentration-time curve from zero to infinity [AUC(0-∞)]; results are given as medians [ranges]) were not statistically different between fed (29.5 h · μg/ml [20.6 to 58.7 h · μg/ml]) and fasting (23.9 h · μg/ml [11.9 to 72.9 h · μg/ml]) patients, but the interindividual variation was reduced in the fed group. Overall, none of the pharmacokinetic parameters differed statistically between the groups. Total piperaquine exposure correlated well with the day 7 concentrations in the fasted group, but the fed group showed a poor correlation. In conclusion, the coadministration of 6.4 g fat did not have any significant effect on piperaquine pharmacokinetics in the treatment of uncomplicated malaria.

Pukrittayakamee S, Jittamala P, Stepniewska K, Lindegardh N, Chueasuwanchai S, Leowattana W, Phakdeeraj A, Permpunpanich S et al. 2011. An open-label crossover study to evaluate potential pharmacokinetic interactions between oral oseltamivir and intravenous zanamivir in healthy thai adults Antimicrobial Agents and Chemotherapy, 55 (9), pp. 4050-4057. | Show Abstract | Read more

There is no parenteral formulation of the neuraminidase inhibitor oseltamivir, the most widely used anti-influenza virus drug. Oseltamivir resistance is an increasing problem. Zanamivir is effective against the most prevalent oseltamivir-resistant influenza viruses. A parenteral formulation of zanamivir is in development for the treatment of severe influenza. It is not known if there is any pharmacokinetic interaction between the two drugs. Sixteen healthy Thai adult volunteers were studied in an open-label, four-period, randomized two-sequence crossover pharmacokinetic study in which zanamivir was given by constant-rate infusion or slow intravenous injection either alone or together with oral oseltamivir. Plasma concentration profiles of oseltamivir, the active metabolite oseltamivir carboxylate, and zanamivir were measured by liquid chromatography-mass spectrometry-mass spectrometry. Both drugs were well tolerated alone and in combination. The maximum plasma concentrations and the areas under the plasma concentration-time curves (AUC) of oseltamivir and oseltamivir carboxylate were not significantly different when oseltamivir was given separately or together with zanamivir. Maximum plasma concentrations of zanamivir were 10% (95% confidence interval, 7 to 12%) higher when zanamivir was infused concurrently with oral oseltamivir than with infusions before or after oral oseltamivir. The plasma zanamivir total AUC was positively correlated with the total oseltamivir carboxylate AUC (Pearson's correlation coefficient [rP] = 0.720, P = 0.002, n = 16) but not with the oseltamivir AUC (rp =0.121, n = 16). There is no clinically significant pharmacokinetic interaction between oseltamivir and zanamivir. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

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Ashley EA, Lubell Y, White NJ, Turner P. 2011. Antimicrobial susceptibility of bacterial isolates from community acquired infections in Sub-Saharan Africa and Asian low and middle income countries Tropical Medicine and International Health, 16 (9), pp. 1167-1179. | Show Abstract | Read more

Objective Antimicrobial resistance has arisen across the globe in both nosocomial and community settings as a consequence of widespread antibiotic consumption. Poor availability of laboratory diagnosis means that resistance frequently goes unrecognised and may only be detected as clinical treatment failure. In this review, we provide an overview of the reported susceptibility of common community acquired bacterial pathogens in Sub-Saharan Africa and Asia to the antibiotics that are most widely used in these areas. Methods We reviewed the literature for reports of the susceptibility of prevalent pathogens in the community in SSA and Asia to a range of commonly prescribed antibiotics. Inclusion criteria required that isolates were collected since 2004 and that they were obtained from either normally sterile sites or urine. The data were aggregated by region and by age group. Results Eighty-three studies were identified since 2004 which reported the antimicrobial susceptibilities of common bacterial pathogens. Different methods were used to assess in-vitro susceptibility in the different studies. The quality of testing (evidenced by resistance profiles) also varied considerably. For Streptococcus pneumoniae and Neisseria meningitidis most drugs maintained relatively high efficacy, apart from co-trimoxazole to which there were high levels of resistance in most of the pathogens surveyed. Conclusions Compared with the enormous infectious disease burden and widespread use of antibiotics there are relatively few reliable data on antimicrobial susceptibility from tropical Asia and Africa upon which to draw firm conclusions, although it is evident that many commonly used antibiotics face considerable resistance in prevalent bacterial pathogens. This is likely to exacerbate morbidity and mortality. Investment in improved antimicrobial susceptibility testing and surveillance systems is likely to be a highly cost-effective strategy and should be complemented by centralized and readily accessible information resources. © 2011 Blackwell Publishing Ltd.

Dondorp AM, Fanello CI, von Seidlein L, Day NPJ, White NJ. 2011. Artesunate for severe malaria in African children – Authors' reply The Lancet, 377 (9772), pp. 1154-1154. | Read more

Cited:

35

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Nantakomol D, Dondorp AM, Krudsood S, Udomsangpetch R, Pattanapanyasat K, Combes V, Grau GE, White NJ, Viriyavejakul P, Day NPJ, Chotivanich K. 2011. Circulating red cell-derived microparticles in human malaria Journal of Infectious Diseases, 203 (5), pp. 700-706. | Show Abstract | Read more

In patients with falciparum malaria, plasma concentrations of cell-derived microparticles correlate with disease severity. Using flow cytometry, we quantified red blood cell-derived microparticles (RMPs) in patients with malaria and identified the source and the factors associated with production. RMP concentrations were increased in patients with Plasmodium falciparum (n = 29; median, 457 RMPs/μL [range, 13-4,342 RMPs/μL]), Plasmodium vivax (n = 5; median, 409 RMPs/μL [range, 281-503/μL]), and Plasmodium malariae (n = 2; median, 163 RMPs/μL [range, 127-200 RMPs/μL]) compared with those in healthy subjects (n = 11; median, 8 RMPs/μL [range, 3-166 RMPs/μL]; P = .01). RMP concentrations were highest in patients with severe falciparum malaria (P=.01). Parasitized red cells produced >10 times more RMPs than did unparasitized cells, but the overall majority of RMPs still derived from uninfected red blood cells (URBCs). In cultures, RMP production increased as the parasites matured. Hemin and parasite products induced RMP production in URBCs, which was inhibited by N-acetylcysteine, suggesting heme-mediated oxidative stress as a pathway for the generation of RMPs. © The Author 2011.

Lang T, Cheah PY, White NJ. 2011. Clinical research: Time for sensible global guidelines The Lancet, 377 (9777), pp. 1153-1555. | Read more

Medana IM, Day NP, Sachanonta N, Mai NT, Dondorp AM, Pongponratn E, Hien TT, White NJ, Turner GD. 2011. Coma in fatal adult human malaria is not caused by cerebral oedema. Malar J, 10 (1), pp. 267. | Show Abstract | Read more

BACKGROUND: The role of brain oedema in the pathophysiology of cerebral malaria is controversial. Coma associated with severe Plasmodium falciparum malaria is multifactorial, but associated with histological evidence of parasitized erythrocyte sequestration and resultant microvascular congestion in cerebral vessels. To determine whether these changes cause breakdown of the blood-brain barrier and resultant perivascular or parenchymal cerebral oedema, histology, immunohistochemistry and image analysis were used to define the prevalence of histological patterns of oedema and the expression of specific molecular pathways involved in water balance in the brain in adults with fatal falciparum malaria. METHODS: The brains of 20 adult Vietnamese patients who died of severe malaria were examined for evidence of disrupted vascular integrity. Immunohistochemistry and image analysis was performed on brainstem sections for activation of the vascular endothelial growth factor (VEGF) receptor 2 and expression of the aquaporin 4 (AQP4) water channel protein. Fibrinogen immunostaining was assessed as evidence of blood-brain barrier leakage and perivascular oedema formation. Correlations were performed with clinical, biochemical and neuropathological parameters of severe malaria infection. RESULTS: The presence of oedema, plasma protein leakage and evidence of VEGF signalling were heterogeneous in fatal falciparum malaria and did not correlate with pre-mortem coma. Differences in vascular integrity were observed between brain regions with the greatest prevalence of disruption in the brainstem, compared to the cortex or midbrain. There was a statistically non-significant trend towards higher AQP4 staining in the brainstem of cases that presented with coma (P = .02). CONCLUSIONS: Histological evidence of cerebral oedema or immunohistochemical evidence of localised loss of vascular integrity did not correlate with the occurrence of pre-mortem coma in adults with fatal falciparum malaria. Enhanced expression of AQP4 water channels in the brainstem may, therefore, reflect a mix of both neuropathological or attempted neuroprotective responses to oedema formation.

White NJ. 2011. Determinants of relapse periodicity in Plasmodium vivax malaria. Malar J, 10 (1), pp. 297. | Show Abstract | Read more

Plasmodium vivax is a major cause of febrile illness in endemic areas of Asia, Central and South America, and the horn of Africa. Plasmodium vivax infections are characterized by relapses of malaria arising from persistent liver stages of the parasite (hypnozoites) which can be prevented only by 8-aminoquinoline anti-malarials. Tropical P. vivax relapses at three week intervals if rapidly eliminated anti-malarials are given for treatment, whereas in temperate regions and parts of the sub-tropics P. vivax infections are characterized either by a long incubation or a long-latency period between illness and relapse - in both cases approximating 8-10 months. The epidemiology of the different relapse phenotypes has not been defined adequately despite obvious relevance to malaria control and elimination. The number of sporozoites inoculated by the anopheline mosquito is an important determinant of both the timing and the number of relapses. The intervals between relapses display a remarkable periodicity which has not been explained. Evidence is presented that the proportion of patients who have successive relapses is relatively constant and that the factor which activates hypnozoites and leads to regular interval relapse in vivax malaria is the systemic febrile illness itself. It is proposed that in endemic areas a large proportion of the population harbours latent hypnozoites which can be activated by a systemic illness such as vivax or falciparum malaria. This explains the high rates of vivax following falciparum malaria, the high proportion of heterologous genotypes in relapses, the higher rates of relapse in people living in endemic areas compared with artificial infection studies, and, by facilitating recombination between different genotypes, contributes to P. vivax genetic diversity particularly in low transmission settings. Long-latency P. vivax phenotypes may be more widespread and more prevalent than currently thought. These observations have important implications for the assessment of radical treatment efficacy and for malaria control and elimination.

Soukaloun D, Lee SJ, Chamberlain K, Taylor AM, Mayxay M, Sisouk K, Soumphonphakdy B, Latsavong K et al. 2011. Erythrocyte transketolase activity, markers of cardiac dysfunction and the diagnosis of infantile beriberi. PLoS Negl Trop Dis, 5 (2), pp. e971. | Show Abstract | Read more

BACKGROUND: Infantile beriberi is a potentially lethal manifestation of thiamin deficiency, associated with traditional post-partum maternal food avoidance, which persists in the Lao PDR (Laos). There are few data on biochemical markers of infantile thiamin deficiency or indices of cardiac dysfunction as potential surrogate markers. METHODOLOGY/PRINCIPAL FINDINGS: A case control study of 47 infants with beriberi and age-matched afebrile and febrile controls was conducted in Vientiane, Laos. Basal and activated erythrocyte transketolase activities (ETK) and activation (α) coefficients were assayed along with plasma brain natriuretic peptide, N-terminal pro-brain natriuretic peptide and troponin T. Basal ETK (and to a lesser extent activated ETK) and plasma troponin T were the only infant biochemical markers that predicted infantile beriberi. A basal ETK ≤ 0.59 micromoles/min/gHb gave a sensitivity (95%CI) of 75.0 (47.6 to 92.7)% and specificity (95%CI) of 85.2 (66.3 to 95.8)% for predicting infantile beriberi (OR (95%CI) 15.9 (2.03-124.2); p = 0.008) (area under ROC curve = 0.80). In contrast, the α coefficient did not discriminate between cases and controls. Maternal basal ETK was linearly correlated with infant basal ETK (Pearson's r = 0.66, p < 0.001). The odds of beriberi in infants with detectable plasma troponin T was 3.4 times higher in comparison to infants without detectable troponin T (OR 3.4, 95%CI 1.22-9.73, p = 0.019). Detectable troponin T had a sensitivity (95%CI) of 78.6 (59.0 to 91.7) % and specificity (95%CI) of 56.1 (39.7 to 71.5) % for predicting infantile beriberi. CONCLUSIONS/SIGNIFICANCE: Basal ETK is a more accurate biochemical marker of infantile beriberi than the activation coefficient. Raised plasma troponin T may be a useful indicator of infantile beriberi in infants at risk and in the absence of other evident causes.

McGready R, White NJ, Nosten F. 2011. Parasitological efficacy of antimalarials in the treatment and prevention of falciparum malaria in pregnancy 1998 to 2009: a systematic review. BJOG, 118 (2), pp. 123-135. | Show Abstract | Read more

BACKGROUND: Pregnant women are at increased risk from malaria. Resistance to all classes of antimalarials has affected the treatment and prevention of malaria in pregnancy. OBJECTIVES: To review the therapeutic efficacy of antimalarials used for treatment and intermittent preventive treatment (IPT) in pregnancy. SEARCH STRATEGY: We searched MEDLINE and the Cochrane Library between January 1998 and December 2009 for publications using the medical subject headings: efficacy, antimalarials, malaria, pregnancy, pharmacokinetics, treatment, IPT and placenta positive. In May 2010 we searched the register of clinical trials (http://clinicaltrials.gov/) and of WHO (http://apps.who.int/trialsearch/) using 'malaria', and 'pregnancy' and 'treatment'. SELECTION CRITERIA: We identified 233 abstracts, reviewed 83 full text articles and included 60 studies. DATA COLLECTION AND ANALYSIS: Two authors entered extracted data to an excel spreadsheet. MAIN RESULTS: Parasitological failure rates, placenta positivity rates (assessed by microscopy) or both were reported in 44% (21/48), 46% (22/48) and 10% (5/48) of articles, respectively. Most pharmacokinetic studies (9/12) suggested dose optimisation. In 23 treatment studies 17 different antimalarial drugs were delivered in 53 study arms; 43.4% (23/53) reported a failure rate of < 5%; 83.3% of sulphadoxine-pyrimethamine (SP) arms and 9% of artemisinin combination therapy (ACT) arms had failure rates ≥ 10%. Placenta-positive rates (mostly reported in the context of IPT in pregnancy) were > 10% in 68% (23/34) of SP trial arms and > 15% in all seven chloroquine arms. The ACT provided lower parasitological failure and gametocyte carriage rates. AUTHOR'S CONCLUSIONS: Drugs used in pregnancy should aim for 95% efficacy but many currently deployed regimens are associated with much lower cure rates.

Campino S, Auburn S, Kivinen K, Zongo I, Ouedraogo JB, Mangano V, Djimde A, Doumbo OK et al. 2011. Population genetic analysis of Plasmodium falciparum parasites using a customized Illumina GoldenGate genotyping assay. PLoS One, 6 (6), pp. e20251. | Show Abstract | Read more

The diversity in the Plasmodium falciparum genome can be used to explore parasite population dynamics, with practical applications to malaria control. The ability to identify the geographic origin and trace the migratory patterns of parasites with clinically important phenotypes such as drug resistance is particularly relevant. With increasing single-nucleotide polymorphism (SNP) discovery from ongoing Plasmodium genome sequencing projects, a demand for high SNP and sample throughput genotyping platforms for large-scale population genetic studies is required. Low parasitaemias and multiple clone infections present a number of challenges to genotyping P. falciparum. We addressed some of these issues using a custom 384-SNP Illumina GoldenGate assay on P. falciparum DNA from laboratory clones (long-term cultured adapted parasite clones), short-term cultured parasite isolates and clinical (non-cultured isolates) samples from East and West Africa, Southeast Asia and Oceania. Eighty percent of the SNPs (n = 306) produced reliable genotype calls on samples containing as little as 2 ng of total genomic DNA and on whole genome amplified DNA. Analysis of artificial mixtures of laboratory clones demonstrated high genotype calling specificity and moderate sensitivity to call minor frequency alleles. Clear resolution of geographically distinct populations was demonstrated using Principal Components Analysis (PCA), and global patterns of population genetic diversity were consistent with previous reports. These results validate the utility of the platform in performing population genetic studies of P. falciparum.

Gardner KB, Sinha I, Bustamante LY, Day NPJ, White NJ, Woodrow CJ. 2011. Protein-based signatures of functional evolution in Plasmodium falciparum BMC Evolutionary Biology, 11 (1), | Show Abstract | Read more

Abstract. Background: It has been known for over a decade that Plasmodium falciparum proteins are enriched in non-globular domains of unknown function. The potential for these regions of protein sequence to undergo high levels of genetic drift provides a fundamental challenge to attempts to identify the molecular basis of adaptive change in malaria parasites. Results: Evolutionary comparisons were undertaken using a set of forty P. falciparum metabolic enzyme genes, both within the hominid malaria clade (P. reichenowi) and across the genus (P. chabaudi). All genes contained coding elements highly conserved across the genus, but there were also a large number of regions of weakly or non-aligning coding sequence. These displayed remarkable levels of non-synonymous fixed differences within the hominid malaria clade indicating near complete release from purifying selection (dN/dS ratio at residues non-aligning across genus: 0.64, dN/dS ratio at residues identical across genus: 0.03). Regions of low conservation also possessed high levels of hydrophilicity, a marker of non-globularity. The propensity for such regions to act as potent sources of non-synonymous genetic drift within extant P. falciparum isolates was confirmed at chromosomal regions containing genes known to mediate drug resistance in field isolates, where 150 of 153 amino acid variants were located in poorly conserved regions. In contrast, all 22 amino acid variants associated with drug resistance were restricted to highly conserved regions. Additional mutations associated with laboratory-selected drug resistance, such as those in PfATPase4 selected by spiroindolone, were similarly restricted while mutations in another calcium ATPase (PfSERCA, a gene proposed to mediate artemisinin resistance) that reach significant frequencies in field isolates were located exclusively in poorly conserved regions consistent with genetic drift. Conclusion: Coding sequences of malaria parasites contain prospectively definable domains subject to neutral or nearly neutral evolution on a scale that appears unrivalled in biology. This distinct evolutionary landscape has potential to confound analytical methods developed for other genera. Against this tide of genetic drift, polymorphisms mediating functional change stand out to such an extent that evolutionary context provides a useful signal for identifying the molecular basis of drug resistance in malaria parasites, a finding that is of relevance to both genome-wide and candidate gene studies in this genus. © 2011 Gardner et al; licensee BioMed Central Ltd.

Lindegardh N, Hanpithakpong W, Kamanikom B, Farrar J, Hien TT, Singhasivanon P, White NJ, Day NP. 2011. Quantification of the anti-influenza drug zanamivir in plasma using high-throughput HILIC-MS/MS. Bioanalysis, 3 (2), pp. 157-165. | Show Abstract | Read more

BACKGROUND: parenteral zanamivir is a promising drug for the treatment of severe influenza. However, quantification of this polar drug in biological matrices has traditionally been difficult and the methods developed have been relatively insensitive. RESULTS: a high-throughput bioanalytical method for the analysis of zanamivir in human plasma using SPE in the 96-well plate format and LC coupled to positive MS/MS has been developed and validated according to US FDA guidelines. The method uses 50 microl of plasma and covers a large working range from 1-50, 000 ng/ml with a LOD of 0.50 ng/ml. CONCLUSION: this new LC-MS/MS assay is more sensitive than previous methods despite using a small plasma volume sample. It is particularly suitable for clinical studies on both parenteral and inhaled zanamivir.

Ford NP, de Smet M, Kolappa K, White NJ. 2011. Responding to the evidence for the management of severe malaria Tropical Medicine and International Health, 16 (9), pp. 1085-1086. | Read more

Gaudin K, Kauss T, Gaubert A, Viaud V, Dubost J-P, Olliaro P, White NJ, Millet P. 2011. SIMULTANEOUS DETERMINATION OF ARTEMETHER AND AZITHROMYCIN IN SUPPOSITORIES BY REVERSED PHASE HPLC ANALYTICAL LETTERS, 44 (16), pp. 2732-2743. | Show Abstract | Read more

Chromatographic parameter assessments for RP-HPLC-UV method development for the simultaneous analysis of artemether and azithromycin for the pharmaceutical analysis of a rectal coformulation currently under development for the treatment of malaria infected children. Using methanol based mobile phase for the analysis of both artemether and azithromycin provided a more robust method in terms of resolution and peak symmetry. The method validated for suppository used 80% methanol and 20% phosphate buffer 15 mM at pH 9. The UV detection was at 210 nm. The accuracy profiles indicated a method validation between 80-120% for both active pharmaceutical ingredients. The preparation process of the suppository was validated based on theoretical values of artemether and azithromycin present in the formulation; active pharmaceutical ingredients were homogenously distributed within the suppository. © 2011 Copyright Taylor and Francis Group, LLC.

Wangdi K, Kaewkungwal J, Singhasivanon P, Silawan T, Lawpoolsri S, White NJ. 2011. Spatio-temporal patterns of malaria infection in Bhutan: a country embarking on malaria elimination. Malar J, 10 (1), pp. 89. | Show Abstract | Read more

BACKGROUND: At the verge of elimination of malaria in Bhutan, this study was carried out to analyse the trend of malaria in the endemic districts of Bhutan and to identify malaria clusters at the sub-districts. The findings would aid in implementing the control activities. Poisson regression was performed to study the trend of malaria incidences at district level from 1994 to 2008. Spatial Empirical Bayesian smoothing was deployed to identify clusters of malaria at the sub-district level from 2004 to 2008. RESULTS: Trend of the overall districts and most of the endemic districts have decreased except Pemagatshel, which has an increase in the trend. Spatial cluster-outlier analysis showed that malaria clusters were mostly concentrated in the central and eastern Bhutan in three districts of Dagana, Samdrup Jongkhar and Sarpang. The disease clusters were reported throughout the year. Clusters extended to the non-transmission areas in the eastern Bhutan. CONCLUSIONS: There is significant decrease in the trend of malaria with the elimination at the sight. The decrease in the trend can be attributed to the success of the control and preventive measures. In order to realize the target of elimination of malaria, the control measure needs to be prioritized in these high-risk clusters of malaria.

Lubell Y, Turner P, Ashley EA, White NJ. 2011. Susceptibility of bacterial isolates from community-acquired infections in sub-Saharan Africa and Asia to macrolide antibiotics Tropical Medicine and International Health, 16 (10), pp. 1192-1205. | Show Abstract | Read more

Objective To review the literature on the susceptibility of common community pathogens in sub-Saharan Africa and Asia to the macrolide antibiotics. Methods Inclusion criteria required that isolates were collected since 2004 to ensure results were of contemporary relevance. The data were aggregated by region, age group and sterility of site of culture sample. Results A total of 51 studies were identified, which reported the macrolide antimicrobial susceptibilities of common bacterial pathogens isolated since 2004. In general, there was less macrolide resistance in African than in Asian isolates. Most African studies reported high levels of macrolide susceptibility in Streptococcus pneumoniae, whereas most Chinese studies reported high levels of resistance. There was very little information available for Gram-negative organisms. Conclusions Susceptibility of the pneumococcus to macrolides in SSA remains high in many areas, and good activity of azithromycin has been shown against Salmonellae spp. in Asia. In urban areas where high antibiotic consumption is prevalent, there was evidence of increased resistance to macrolides. However, there is no information on susceptibility from large areas in both continents. © 2011 Blackwell Publishing Ltd.

White NJ. 2011. The parasite clearance curve. Malar J, 10 (1), pp. 278. | Show Abstract | Read more

Parasite clearance rates are important measures of anti-malarial drug efficacy. They are particularly important in the assessment of artemisinin resistance. The slope of the log-linear segment in the middle of the parasite clearance curve has the least inter-individual variance and is the focus of therapeutic assessment. The factors affecting parasite clearance are reviewed. Methods of presentation and the approaches to analysis are discussed.

Maude RJ, Hoque G, Hasan MU, Sayeed A, Akter S, Samad R, Alam B, Yunus EB et al. 2011. Timing of enteral feeding in cerebral malaria in resource-poor settings: a randomized trial. PLoS One, 6 (11), pp. e27273. | Show Abstract | Read more

BACKGROUND: Early start of enteral feeding is an established treatment strategy in intubated patients in intensive care since it reduces invasive bacterial infections and length of hospital stay. There is equipoise whether early enteral feeding is also beneficial in non-intubated patients with cerebral malaria in resource poor settings. We hypothesized that the risk of aspiration pneumonia might outweigh the potential benefits of earlier recovery and prevention of hypoglycaemia. METHOD AND FINDINGS: A randomized trial of early (day of admission) versus late (after 60 hours in adults or 36 hours in children) start of enteral feeding was undertaken in patients with cerebral malaria in Chittagong, Bangladesh from May 2008 to August 2009. The primary outcome measures were incidence of aspiration pneumonia, hypoglycaemia and coma recovery time. The trial was terminated after inclusion of 56 patients because of a high incidence of aspiration pneumonia in the early feeding group (9/27 (33%)), compared to the late feeding group (0/29 (0%)), p = 0.001). One patient in the late feeding group, and none in the early group, had hypoglycaemia during admission. There was no significant difference in overall mortality (9/27 (33%) vs 6/29 (21%), p = 0.370), but mortality was 5/9 (56%) in patients with aspiration pneumonia. CONCLUSIONS: In conclusion, early start of enteral feeding is detrimental in non-intubated patients with cerebral malaria in many resource-poor settings. Evidence gathered in resource rich settings is not necessarily transferable to resource-poor settings. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN57488577.

Lourens C, Watkins WM, Barnes KI, Sibley CH, Guerin PJ, White NJ, Lindegardh N. 2010. Implementation of a reference standard and proficiency testing programme by the World Wide Antimalarial Resistance Network (WWARN). Malar J, 9 (1), pp. 375. | Show Abstract | Read more

BACKGROUND: The Worldwide Antimalarial Resistance Network (WWARN) is a global collaboration to support the objective that anyone affected by malaria receives effective and safe drug treatment. The Pharmacology module aims to inform optimal anti-malarial drug selection. There is an urgent need to define the drug exposure - effect relationship for most anti-malarial drugs. Few anti-malarials have had their therapeutic blood concentration levels defined. One of the main challenges in assessing safety and efficacy data in relation to drug concentrations is the comparability of data generated from different laboratories. To explain differences in anti-malarial pharmacokinetics in studies with different measurement laboratories it is necessary to confirm the accuracy of the assay methods. This requires the establishment of an external quality assurance process to assure results that can be compared. This paper describes this process. METHODS: The pharmacology module of WWARN has established a quality assurance/quality control (QA/QC) programme consisting of two separate components:1. A proficiency testing programme where blank human plasma spiked with certified reference material (CRM) in different concentrations is sent out to participating bioanalytical laboratories.2. A certified reference standard programme where accurately weighed amounts of certified anti-malarial reference standards, metabolites, and internal standards are sent to participating bioanalytical and in vitro laboratories. CONCLUSION: The proficiency testing programme is designed as a cooperative effort to help participating laboratories assess their ability to carry out drug analysis, resolve any potential problem areas and to improve their results - and, in so doing, to improve the quality of anti-malarial pharmacokinetic data published and shared with WWARN.By utilizing the same source of standards for all laboratories, it is possible to minimize bias arising from poor quality reference standards. By providing anti-malarial drug standards from a central point, it is possible to lower the cost of these standards.

Saralamba S, Pan-Ngum W, Maude RJ, Lee SJ, Tarning J, Lindegårdh N, Chotivanich K, Nosten F et al. 2011. Intrahost modeling of artemisinin resistance in Plasmodium falciparum. Proc Natl Acad Sci U S A, 108 (1), pp. 397-402. | Show Abstract | Read more

Artemisinin-resistant Plasmodium falciparum malaria has emerged in western Cambodia. Resistance is characterized by prolonged in vivo parasite clearance times (PCTs) following artesunate treatment. The biological basis is unclear. The hypothesis that delayed parasite clearance results from a stage-specific reduction in artemisinin sensitivity of the circulating young asexual parasite ring stages was examined. A mathematical model was developed, describing the intrahost parasite stage-specific pharmacokinetic-pharmacodynamic relationships. Model parameters were estimated using detailed pharmacokinetic and parasite clearance data from 39 patients with uncomplicated falciparum malaria treated with artesunate from Pailin (western Cambodia) where artemisinin resistance was evident and 40 patients from Wang Pha (northwestern Thailand) where efficacy was preserved. The mathematical model reproduced the observed parasite clearance for each patient with an accurate goodness of fit (rmsd: 0.03-0.67 in log(10) scale). The parameter sets that provided the best fits with the observed in vivo data consist of a highly conserved concentration-effect relationship for the trophozoite and schizont parasite stages, but a variable relationship for the ring stages. The model-derived assessment suggests that the efficacy of artesunate on ring stage parasites is reduced significantly in Pailin. This result supports the hypothesis that artemisinin resistance mainly reflects reduced ring-stage susceptibility and predicts that doubling the frequency of dosing will accelerate clearance of artemisinin-resistant parasites.

White NJ. 2010. Artemisinin resistance--the clock is ticking. Lancet, 376 (9758), pp. 2051-2052. | Read more

Mayxay M, Keomany S, Khanthavong M, Souvannasing P, Stepniewska K, Khomthilath T, Keola S, Pongvongsa T et al. 2010. A phase III, randomized, non-inferiority trial to assess the efficacy and safety of dihydroartemisinin-piperaquine in comparison with artesunate-mefloquine in patients with uncomplicated Plasmodium falciparum malaria in southern Laos. Am J Trop Med Hyg, 83 (6), pp. 1221-1229. | Show Abstract | Read more

We conducted an open, randomized clinical trial of oral dihydroartemisinin-piperaquine (DP) versus artesunate-mefloquine (AM) in 300 patients in Laos with uncomplicated Plasmodium falciparum malaria as part of a multicentre study in Asia. Survival analysis and adjustment for re-infection showed that the 63-day cure rates (95% confidence interval [CI]) were 100% for AM and 99.5% (96.4-99.8%) for DP. The 63-day cure rates per protocol were 99% (97 of 98) for AM and 99.5% (196 of 197) for DP (P = 0.55). The difference (AM minus DP) in cure rates (95% CI) was -0.5% (-5.1 to 2.0%), which is within the 5% non-inferiority margin. The median fever and parasite clearance times were also similar for AM and DP. The proportion of patients with at least one recorded potential adverse event was significantly higher in the AM group (38 of 87, 44%) than in the DP group (57 of 182, 31%) (relative risk = 0.6, 95% CI = 0.4-0.9; P = 0.04). Dihydroartemisinin-piperaquine is not inferior to AM in the treatment of uncomplicated P. falciparum malaria in Laos and is associated with fewer adverse effects. The results of this study were similar to those of the larger multicentre study.

McGready R, Wuthiekanun V, Ashley EA, Tan SO, Pimanpanarak M, Viladpai-Nguen SJ, Jesadapanpong W, Blacksell SD et al. 2010. Diagnostic and treatment difficulties of pyelonephritis in pregnancy in resource-limited settings. Am J Trop Med Hyg, 83 (6), pp. 1322-1329. | Show Abstract | Read more

Limited microbiology services impede adequate diagnosis and treatment of common infections such as pyelonephritis in resource-limited settings. Febrile pregnant women attending antenatal clinics at Shoklo Malaria Research Unit were offered urine dipstick, sediment microscopy, urine culture, and a 5-mL blood culture. The incidence of pyelonephritis was 11/1,000 deliveries (N = 53 in 4,819 pregnancies) between January 7, 2004 and May 17, 2006. Pyelonephritis accounted for 20.2% (41/203) of fever cases in pregnancy. Escherichia coli was the most commonly isolated pathogen: 87.5% (28/32) of organisms cultured. Susceptibility of E. coli to ampicillin (14%), cotrimoxazole (21%), and amoxicillin-clavulanic acid (48%) was very low. E. coli was susceptible to ceftriaxone and ciprofloxacin. The rate of extended spectrum β-lactamase (4.2%; 95% confidence interval = 0.7-19.5) was low. The rate and causes of pyelonephritis in pregnant refugee and migrant women were comparable with those described in developed countries. Diagnostic innovation in microbiology that permits affordable access is a high priority for resource-poor settings.

Lubell Y, Ashley EA, Turner C, Turner P, White NJ. 2011. Susceptibility of community-acquired pathogens to antibiotics in Africa and Asia in neonates--an alarmingly short review. Trop Med Int Health, 16 (2), pp. 145-151. | Show Abstract | Read more

OBJECTIVE: To assess the susceptibility of community-acquired pathogens in neonatal sepsis to commonly prescribed antibiotics in sub-Saharan Africa and Asia since 2002. METHODS: Literature review in PubMed and Embase. Susceptibility was estimated for pathogens individually and stratified by region. Isolates were also classified into Gram positive and Gram negative pathogens to estimate their pooled susceptibility. RESULTS AND CONCLUSIONS: Only nine studies met the inclusion criteria. The available data indicated poor susceptibility to almost all commonly used antibiotics in pathogens such as Staphylococcus aureus and Klebsiella spp. Only Streptococcus pneumoniae exhibited good susceptibility to all drugs other than cotrimoxazole. The extreme scarcity of data prevents drawing any firm conclusions beyond the urgent need for more studies to identify the best treatments for neonatal sepsis in the developing world.

Dondorp AM, Fanello CI, Hendriksen IC, Gomes E, Seni A, Chhaganlal KD, Bojang K, Olaosebikan R et al. 2010. Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, randomised trial. Lancet, 376 (9753), pp. 1647-1657. | Show Abstract | Read more

BACKGROUND: Severe malaria is a major cause of childhood death and often the main reason for paediatric hospital admission in sub-Saharan Africa. Quinine is still the established treatment of choice, although evidence from Asia suggests that artesunate is associated with a lower mortality. We compared parenteral treatment with either artesunate or quinine in African children with severe malaria. METHODS: This open-label, randomised trial was undertaken in 11 centres in nine African countries. Children (<15 years) with severe falciparum malaria were randomly assigned to parenteral artesunate or parenteral quinine. Randomisation was in blocks of 20, with study numbers corresponding to treatment allocations kept inside opaque sealed paper envelopes. The trial was open label at each site, and none of the investigators or trialists, apart from for the trial statistician, had access to the summaries of treatment allocations. The primary outcome measure was in-hospital mortality, analysed by intention to treat. This trial is registered, number ISRCTN50258054. FINDINGS: 5425 children were enrolled; 2712 were assigned to artesunate and 2713 to quinine. All patients were analysed for the primary outcome. 230 (8·5%) patients assigned to artesunate treatment died compared with 297 (10·9%) assigned to quinine treatment (odds ratio [OR] stratified for study site 0·75, 95% CI 0·63-0·90; relative reduction 22·5%, 95% CI 8·1-36·9; p=0·0022). Incidence of neurological sequelae did not differ significantly between groups, but the development of coma (65/1832 [3·5%] with artesunate vs 91/1768 [5·1%] with quinine; OR 0·69 95% CI 0·49-0·95; p=0·0231), convulsions (224/2712 [8·3%] vs 273/2713 [10·1%]; OR 0·80, 0·66-0·97; p=0·0199), and deterioration of the coma score (166/2712 [6·1%] vs 208/2713 [7·7%]; OR 0·78, 0·64-0·97; p=0·0245) were all significantly less frequent in artesunate recipients than in quinine recipients. Post-treatment hypoglycaemia was also less frequent in patients assigned to artesunate than in those assigned to quinine (48/2712 [1·8%] vs 75/2713 [2·8%]; OR 0·63, 0·43-0·91; p=0·0134). Artesunate was well tolerated, with no serious drug-related adverse effects. INTERPRETATION: Artesunate substantially reduces mortality in African children with severe malaria. These data, together with a meta-analysis of all trials comparing artesunate and quinine, strongly suggest that parenteral artesunate should replace quinine as the treatment of choice for severe falciparum malaria worldwide. FUNDING: The Wellcome Trust.

Smithuis F, Kyaw MK, Phe O, Win T, Aung PP, Oo AP, Naing AL, Nyo MY et al. 2010. Effectiveness of five artemisinin combination regimens with or without primaquine in uncomplicated falciparum malaria: an open-label randomised trial. Lancet Infect Dis, 10 (10), pp. 673-681. | Show Abstract | Read more

BACKGROUND: Artemisinin-combination therapy (ACT) is recommended as first-line treatment of falciparum malaria throughout the world, and fixed-dose combinations are preferred by WHO; whether a single gametocytocidal dose of primaquine should be added is unknown. We aimed to compare effectiveness of four fixed-dose ACTs and a loose tablet combination of artesunate and mefloquine, and assess the addition of a single gametocytocidal dose of primaquine. METHODS: In an open-label randomised trial in clinics in Rakhine state, Kachin state, and Shan state in Myanmar (Burma) between Dec 30, 2008, and March 20, 2009, we compared the effectiveness of all four WHO-recommended fixed-dose ACTs (artesunate-mefloquine, artesunate-amodiaquine, dihydroartemisinin-piperaquine, artemether-lumefantrine) and loose artesunate-mefloquine in Burmese adults and children. Eligible patients were those who presented to the clinics with acute uncomplicated Plasmodium falciparum malaria or mixed infection, who were older than 6 months, and who weighed more than 5 kg. Treatments were randomised in equal numbers within blocks of 50 and allocation was in sealed envelopes. All patients were also randomly assigned to receive either a single dose of primaquine 0·75 mg base/kg or not. Patients were followed up for 63 days. Treatment groups were compared by analysis of variance and multiple logistic regression. The primary outcome was the 63 day recrudescence rate. This study is registered with clinicaltrials.gov, number NCT00902811. FINDINGS: 155 patients received artesunate-amodiaquine, 162 artemether-lumefantrine, 169 artesunate-mefloquine, 161 loose artesunate-mefloquine, and 161 dihydroartemisinin-piperaquine. By day 63 of follow-up, 14 patients (9·4%; 95% CI 5·7-15·3%) on artesunate-amodiaquine had recrudescent P falciparum infections, a rate significantly higher than for artemether-lumefantrine (two patients; 1·4%; 0·3-5·3; p=0·0013), fixed-dose artesunate-mefloquine (0 patients; 0-2·3; p<0·0001), loose artesunate-mefloquine (two patients; 1·3%; 0·3-5·3; p=0·0018), and dihydroartemisinin-piperaquine (two patients 1·3%; 0·3-5·2%; p=0·0012). Hazard ratios for re-infection (95% CI) after artesunate-amodiaquine were 3·2 (1·3-8·0) compared with the two artesunate-mefloquine groups (p=0·01), 2·6 (1·0-6-0) compared with artemether-lumefantrine (p=0·04), and 2·3 (0·9-6·0) compared with dihydroartemisinin-piperaquine (p=0·08). Mixed falciparum and vivax infections were common: 129 (16%) had a mixed infection at presentation and 330 (41%) patients had one or more episodes of Plasmodium vivax infection during follow-up. The addition of a single dose of primaquine (0·75 mg/kg) reduced P falciparum gametocyte carriage substantially: rate ratio 11·9 (95% CI 7·4-20·5). All regimens were well tolerated. Adverse events were reported by 599 patients, most commonly vomiting and dizziness. Other side-effects were less common and were not related to a specific treatment. INTERPRETATION: Artesunate-amodiaquine should not be used in Myanmar, because the other ACTs are substantially more effective. Artesunate-mefloquine provided the greatest post-treatment suppression of malaria. Adding a single dose of primaquine would substantially reduce transmission potential. Vivax malaria, not recurrent falciparum malaria, is the main complication after treatment of P falciparum infections in this region. FUNDING: Médecins sans Frontières (Holland) and the Wellcome Trust Mahidol University Oxford Tropical Medicine Research Programme.

Wangdi K, Singhasivanon P, Silawan T, Lawpoolsri S, White NJ, Kaewkungwal J. 2010. Development of temporal modelling for forecasting and prediction of malaria infections using time-series and ARIMAX analyses: a case study in endemic districts of Bhutan. Malar J, 9 (1), pp. 251. | Show Abstract | Read more

BACKGROUND: Malaria still remains a public health problem in some districts of Bhutan despite marked reduction of cases in last few years. To strengthen the country's prevention and control measures, this study was carried out to develop forecasting and prediction models of malaria incidence in the endemic districts of Bhutan using time series and ARIMAX. METHODS: This study was carried out retrospectively using the monthly reported malaria cases from the health centres to Vector-borne Disease Control Programme (VDCP) and the meteorological data from Meteorological Unit, Department of Energy, Ministry of Economic Affairs. Time series analysis was performed on monthly malaria cases, from 1994 to 2008, in seven malaria endemic districts. The time series models derived from a multiplicative seasonal autoregressive integrated moving average (ARIMA) was deployed to identify the best model using data from 1994 to 2006. The best-fit model was selected for each individual district and for the overall endemic area was developed and the monthly cases from January to December 2009 and 2010 were forecasted. In developing the prediction model, the monthly reported malaria cases and the meteorological factors from 1996 to 2008 of the seven districts were analysed. The method of ARIMAX modelling was employed to determine predictors of malaria of the subsequent month. RESULTS: It was found that the ARIMA (p, d, q) (P, D, Q)s model (p and P representing the auto regressive and seasonal autoregressive; d and D representing the non-seasonal differences and seasonal differencing; and q and Q the moving average parameters and seasonal moving average parameters, respectively and s representing the length of the seasonal period) for the overall endemic districts was (2,1,1)(0,1,1)12; the modelling data from each district revealed two most common ARIMA models including (2,1,1)(0,1,1)12 and (1,1,1)(0,1,1)12. The forecasted monthly malaria cases from January to December 2009 and 2010 varied from 15 to 82 cases in 2009 and 67 to 149 cases in 2010, where population in 2009 was 285,375 and the expected population of 2010 to be 289,085. The ARIMAX model of monthly cases and climatic factors showed considerable variations among the different districts. In general, the mean maximum temperature lagged at one month was a strong positive predictor of an increased malaria cases for four districts. The monthly number of cases of the previous month was also a significant predictor in one district, whereas no variable could predict malaria cases for two districts. CONCLUSIONS: The ARIMA models of time-series analysis were useful in forecasting the number of cases in the endemic areas of Bhutan. There was no consistency in the predictors of malaria cases when using ARIMAX model with selected lag times and climatic predictors. The ARIMA forecasting models could be employed for planning and managing malaria prevention and control programme in Bhutan.

Tanomsing N, Imwong M, Theppabutr S, Pukrittayakamee S, Day NP, White NJ, Snounou G. 2010. Accurate and sensitive detection of Plasmodium species in humans by use of the dihydrofolate reductase-thymidylate synthase linker region. J Clin Microbiol, 48 (10), pp. 3735-3737. | Show Abstract | Read more

A nested-PCR protocol based on the linker region of the Plasmodium dihydrofolate reductase-thymidylate synthase gene (dhfr-ts) was developed. This provides highly sensitive specific detection and identification of the five parasite species that infect humans.

Medana IM, Day NP, Roberts R, Sachanonta N, Turley H, Pongponratn E, Hien TT, White NJ, Turner GD. 2010. Induction of the vascular endothelial growth factor pathway in the brain of adults with fatal falciparum malaria is a non-specific response to severe disease. Histopathology, 57 (2), pp. 282-294. | Show Abstract | Read more

AIMS: Pathological or neuroprotective mechanisms in the brain in severe malaria may arise from microvascular obstruction with malaria-parasitized erythrocytes. This study aimed to investigate the role of hypoxia and induction of the vascular endothelial growth factor (VEGF) pathway in the neuropathophysiology of severe malaria. METHODS AND RESULTS: Immunohistochemistry was performed on post mortem brain tissue sections from 20 cases of severe malaria and examined for the expression of transcriptional regulators of VEGF [hypoxia-inducible factor-1 alpha (HIF-1alpha), HIF-2alpha], DEC-1, VEGF, VEGF receptors 1 and 2, and the activated, phosphorylated VEGF receptor 2 (pKDR). HIFs showed limited protein expression and/or translocation to cell nuclei in severe malaria, but DEC-1, which is more stable and regulated by HIF-1alpha, was observed. There was heterogeneous expression of VEGF and its receptors in severe malaria and non-malarial disease controls. pKDR expression on vessels was greater in malaria cases than in controls but did not correlate with parasite sequestration. VEGF uptake by malaria parasites was observed. CONCLUSIONS: VEGF and its receptor expression levels in severe malaria reflect a non-specific response to severe systemic disease. Potential manipulation of events at the vasculature by the parasite requires further investigation.

Preechapornkul P, Chotivanich K, Imwong M, Dondorp AM, Lee SJ, Day NP, White NJ, Pukrittayakamee S. 2010. Optimizing the culture of Plasmodium falciparum in hollow fiber bioreactors. Southeast Asian J Trop Med Public Health, 41 (4), pp. 761-769. | Show Abstract

The hollow fiber bioreactor (HFBR) is a cell culturing system allowing continuous perfusion of medium. It was designed to grow microorganisms in a dynamically altering medium mimicking change in the in vivo intravascular and extravascular compartments. The cell compartment (extra capillary space) and medium compartment (intra capillary space) are connected through pores of semipermeable fiber membranes. These membranes allow exchange of gas and nutrients. We have adapted this system for the ex vivo culture of Plasmodiumfalciparum at high parasite densities. A Thai P. falciparum isolate (TM036) cultured in RPMI, supplemented with 0.5% Albumax II, could be maintained continuously in the system by daily changes of a small volumes of medium. Under optimized conditions the HFBR cultures attained 8% parasitemia in 40% hematocrit, thereby providing a total parasite biomass of 6.0 x 10(9) parasitized erythrocytes. The main problem encountered was clogging of micropores in the hollow fiber system by cellular debris over time. Although 'reverse flushing' partly prevented this, a larger pore size might be needed to overcome this problem. The system opens new possibilities for the study of in vitro drug sensitivity under conditions mimicking in vivo pharmacokinetics, and the selection of anti-malarial drug resistance and associated parasite biological and genomic changes.

Sutherland CJ, Tanomsing N, Nolder D, Oguike M, Jennison C, Pukrittayakamee S, Dolecek C, Hien TT et al. 2010. Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally. J Infect Dis, 201 (10), pp. 1544-1550. | Show Abstract | Read more

BACKGROUND: Malaria in humans is caused by apicomplexan parasites belonging to 5 species of the genus Plasmodium. Infections with Plasmodium ovale are widely distributed but rarely investigated, and the resulting burden of disease is not known. Dimorphism in defined genes has led to P. ovale parasites being divided into classic and variant types. We hypothesized that these dimorphs represent distinct parasite species. METHODS: Multilocus sequence analysis of 6 genetic characters was carried out among 55 isolates from 12 African and 3 Asia-Pacific countries. RESULTS: Each genetic character displayed complete dimorphism and segregated perfectly between the 2 types. Both types were identified in samples from Ghana, Nigeria, São Tomé, Sierra Leone, and Uganda and have been described previously in Myanmar. Splitting of the 2 lineages is estimated to have occurred between 1.0 and 3.5 million years ago in hominid hosts. CONCLUSIONS: We propose that P. ovale comprises 2 nonrecombining species that are sympatric in Africa and Asia. We speculate on possible scenarios that could have led to this speciation. Furthermore, the relatively high frequency of imported cases of symptomatic P. ovale infection in the United Kingdom suggests that the morbidity caused by ovale malaria has been underestimated.

Anderson TJ, Nair S, Nkhoma S, Williams JT, Imwong M, Yi P, Socheat D, Das D et al. 2010. High heritability of malaria parasite clearance rate indicates a genetic basis for artemisinin resistance in western Cambodia. J Infect Dis, 201 (9), pp. 1326-1330. | Show Abstract | Read more

In western Cambodia, malaria parasites clear slowly from the blood after treatment with artemisinin derivatives, but it is unclear whether this results from parasite, host, or other factors specific to this population. We measured heritability of clearance rate by evaluating patients infected with identical or nonidentical parasite genotypes, using methods analogous to human twin studies. A substantial proportion (56%-58%) of the variation in clearance rate is explained by parasite genetics. This has 2 important implications: (1) selection with artemisinin derivatives will tend to drive resistance spread and (2) because heritability is high, the genes underlying parasite clearance rate may be identified by genome-wide association.

Imwong M, Dondorp AM, Nosten F, Yi P, Mungthin M, Hanchana S, Das D, Phyo AP et al. 2010. Exploring the contribution of candidate genes to artemisinin resistance in Plasmodium falciparum. Antimicrob Agents Chemother, 54 (7), pp. 2886-2892. | Show Abstract | Read more

The reduced in vivo sensitivity of Plasmodium falciparum has recently been confirmed in western Cambodia. Identifying molecular markers for artemisinin resistance is essential for monitoring the spread of the resistant phenotype and identifying the mechanisms of resistance. Four candidate genes, including the P. falciparum mdr1 (pfmdr1) gene, the P. falciparum ATPase6 (pfATPase6) gene, the 6-kb mitochondrial genome, and ubp-1, encoding a deubiquitinating enzyme, of artemisinin-resistant P. falciparum strains from western Cambodia were examined and compared to those of sensitive strains from northwestern Thailand, where the artemisinins are still very effective. The artemisinin-resistant phenotype did not correlate with pfmdr1 amplification or mutations (full-length sequencing), mutations in pfATPase6 (full-length sequencing) or the 6-kb mitochondrial genome (full-length sequencing), or ubp-1 mutations at positions 739 and 770. The P. falciparum CRT K76T mutation was present in all isolates from both study sites. The pfmdr1 copy numbers in western Cambodia were significantly lower in parasite samples obtained in 2007 than in those obtained in 2005, coinciding with a local change in drug policy replacing artesunate-mefloquine with dihydroartemisinin-piperaquine. Artemisinin resistance in western Cambodia is not linked to candidate genes, as was suggested by earlier studies.

Awab GR, Pukrittayakamee S, Imwong M, Dondorp AM, Woodrow CJ, Lee SJ, Day NP, Singhasivanon P, White NJ, Kaker F. 2010. Dihydroartemisinin-piperaquine versus chloroquine to treat vivax malaria in Afghanistan: an open randomized, non-inferiority, trial. Malar J, 9 (1), pp. 105. | Show Abstract | Read more

BACKGROUND: Afghanistan's national guidelines recommend chloroquine for the treatment of Plasmodium vivax infection, the parasite responsible for the majority of its malaria burden. Chloroquine resistance in P. vivax is emerging in Asia. Therapeutic responses across Afghanistan have not been evaluated in detail. METHODS: Between July 2007 and February 2009, an open-label, randomized controlled trial of chloroquine and dihydroartemisinin-piperaquine in patients aged three months and over with slide-confirmed P. vivax mono-infections was conducted. Consistent with current national guidelines, primaquine was not administered. Subjects were followed up daily during the acute phase of illness (days 0-3) and weekly until day 56. The primary endpoint was the overall cumulative parasitological failure rate at day 56 after the start of treatment, with the hypothesis being that dihydroartemisinin-piperaquine was non-inferior compared to chloroquine (Delta = 5% difference in proportion of failures). RESULTS: Of 2,182 individuals with positive blood films for P. vivax, 536 were enrolled in the trial. The day 28 cure rate was 100% in both treatment groups. Parasite clearance was more rapid with dihydroartemisinin-piperaquine than chloroquine. At day 56, there were more recurrent infections in the chloroquine arm (8.9%, 95% CI 6.0-13.1%) than the dihydroartemisinin-piperaquine arm (2.8%, 95% CI 1.4-5.8%), a difference in cumulative recurrence rate of 6.1% (2-sided 90%CI +2.6 to +9.7%). The log-rank test comparing the survival curves confirmed the superiority of dihydroartemisinin-piperaquine over chloroquine (p = 0.003). Multivariate analysis showed that a lower initial haemoglobin concentration was also independently associated with recurrence. Both regimens were well tolerated and no serious adverse events were reported. CONCLUSIONS: Chloroquine remains an efficacious treatment for the treatment of vivax malaria in Afghanistan. In a setting where radical therapy cannot be administered, dihydroartemisinin-piperaquine provides additional benefit in terms of post-treatment prophylaxis, reducing the incidence of recurrence from 4-8 weeks after treatment.

Phu NH, Tuan PQ, Day N, Mai NT, Chau TT, Chuong LV, Sinh DX, White NJ, Farrar J, Hien TT. 2010. Randomized controlled trial of artesunate or artemether in Vietnamese adults with severe falciparum malaria. Malar J, 9 (1), pp. 97. | Show Abstract | Read more

BACKGROUND: Both artemether and artesunate have been shown to be superior to quinine for the treatment of severe falciparum malaria in Southeast Asian adults, although the magnitude of the superiority has been greater for artesunate than artemether. These two artemisinin derivatives had not been compared in a randomized trial. METHODS: A randomized double blind trial in 370 adults with severe falciparum malaria; 186 received intramuscular artesunate (2.4 mg/kg immediately followed by 1.2 mg/kg at 12 hours then 24 hours then daily) and 184 received intramuscular artemether (3.6 mg per kilogram immediately followed by 1.8 mg per kilogram daily) was conducted in Viet Nam. Both drugs were given for a minimum of 72 hours. RESULTS: There were 13 deaths in the artesunate group (7 percent) and 24 in the artemether group (13 percent); P = 0.052; relative risk of death in the patients given artesunate, 0.54; (95 percent confidence interval 0.28-1.02). Parasitaemia declined more rapidly in the artesunate group. Both drugs were very well tolerated. CONCLUSIONS: Intramuscular artesunate may be superior to intramuscular artemether for the treatment of severe malaria in adults.

Syhavong B, Rasachack B, Smythe L, Rolain JM, Roque-Afonso AM, Jenjaroen K, Soukkhaserm V, Phongmany S et al. 2010. The infective causes of hepatitis and jaundice amongst hospitalised patients in Vientiane, Laos. Trans R Soc Trop Med Hyg, 104 (7), pp. 475-483. | Show Abstract | Read more

There is little information on the diverse infectious causes of jaundice and hepatitis in the Asiatic tropics. Serology (hepatitis A, B, C and E, leptospirosis, dengue, rickettsia), antigen tests (dengue), PCR assays (hepatitis A, C and E) and blood cultures (septicaemia) were performed on samples from 392 patients admitted with jaundice or raised transaminases (> or =x3) to Mahosot Hospital, Vientiane, Laos over 3 years. Conservative definitions suggested diagnoses of dengue (8.4%), rickettsioses (7.3%), leptospirosis (6.8%), hepatitis B (4.9%), hepatitis C (4.9%), community-acquired septicaemia (3.3%) and hepatitis E (1.6%). Although anti-hepatitis A virus (HAV) IgM antibody results suggested that 35.8% of patients had acute HAV infections, anti-HAV IgG antibody avidity and HAV PCR suggested that 82% had polyclonal activation and not acute HAV infections. Scrub typhus, murine typhus or leptospirosis were present in 12.8% of patients and were associated with meningism and relatively low AST and ALT elevation. These patients would be expected to respond to empirical doxycycline therapy which, in the absence of virological diagnosis and treatment, may be an appropriate cost-effective intervention in Lao patients with jaundice/hepatitis.

Pukrittayakamee S, Imwong M, Chotivanich K, Singhasivanon P, Day NP, White NJ. 2010. A comparison of two short-course primaquine regimens for the treatment and radical cure of Plasmodium vivax malaria in Thailand. Am J Trop Med Hyg, 82 (4), pp. 542-547. | Show Abstract | Read more

Thai adult males (N = 85) with acute Plasmodium vivax malaria and normal glucose-6-phosphate dehydrogenase screening were randomized to receive 30 mg or 60 mg primaquine daily for 7 days (N = 43 and 42, respectively). The regimens were well tolerated and all patients recovered fully. Median fever clearance (47 hours; range 4 to 130 hours), mean + or - SD parasite clearance times (87.7 + or - 25.3 hours), gametocyte clearance, and adverse effects were similar in the 2 groups. Two patients, 1 from each group, had a 30% reduction in hematocrit. The cumulative 28 day relapse rate (95% confidence interval) by Kaplan Meier survival analysis was 29% (16-49%) in the 30 mg group compared with 7% (2-24%) in the 60 mg group; P = 0.027. Comparison with previous data obtained at this same site suggests that the recurrences comprised approximately 17% recrudescences and 12% relapses in the 30 mg/day group compared with 3% recrudescences and 4% relapses in the 60 mg/day group. These data suggest that the dose-response relationships for primaquine's asexual stage and hypnozoitocidal activities in-vivo are different. A 1 week course of primaquine 60 mg daily is an effective treatment of vivax malaria in this region.

Brouwer AE, Teparrukkul P, Rajanuwong A, Chierakul W, Mahavanakul W, Chantratita W, White NJ, Harrison TS. 2010. Cerebrospinal fluid HIV-1 viral load during treatment of cryptococcal Meningitis. J Acquir Immune Defic Syndr, 53 (5), pp. 668-669. | Read more

Choowongkomon K, Theppabutr S, Songtawee N, Day NP, White NJ, Woodrow CJ, Imwong M. 2010. Computational analysis of binding between malarial dihydrofolate reductases and anti-folates. Malar J, 9 (1), pp. 65. | Show Abstract | Read more

BACKGROUND: Plasmodium falciparum readily develops resistance to the anti-folates pyrimethamine and proguanil via a characteristic set of mutations in the dihydrofolate reductase (PfDHFR) gene that leads to reduced competitive drug binding at the enzyme's active site. Analogous mutations can be found in the DHFR gene in isolates of Plasmodium vivax (PvDHFR) although anti-folates have not been widely used for the treatment of this infection. Here the interactions between DHFR inhibitors and modelled structures of the DHFR enzymes of Plasmodium malariae (PmDHFR) and Plasmodium ovale (PoDHFR) are described, along with an investigation of the effect of recently reported mutations within PmDHFR. METHODS: DHFR models for PmDHFR and PoDHFR were constructed using the solved PfDHFR-TS and PvDHFR structures respectively as templates. The modelled structures were docked with three DHFR inhibitors as ligands and more detailed interactions were explored via simulation of molecular dynamics. RESULTS: Highly accurate models were obtained containing sets of residues that mediate ligand binding which are highly comparable to those mediating binding in known crystal structures. Within this set, there were differences in the relative contribution of individual residues to inhibitor binding. Modelling of PmDHFR mutant sequences revealed that PmDHFR I170M was associated with a significant reduction in binding energy to all DHFR inhibitors studied, while the other predicted resistance mutations had lesser or no effects on ligand binding. CONCLUSIONS: Binding of DHFR inhibitors to the active sites of all four Plasmodium enzymes is broadly similar, being determined by an analogous set of seven residues. PmDHFR mutations found in field isolates influenced inhibitor interactions to a varying extent. In the case of the isolated I170M mutation, the loss of interaction with pyrimethamine suggests that DHFR-inhibitor interactions in P. malariae are different to those seen for DHFRs from P. falciparum and P. vivax.

Mu J, Myers RA, Jiang H, Liu S, Ricklefs S, Waisberg M, Chotivanich K, Wilairatana P et al. 2010. Plasmodium falciparum genome-wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs. Nat Genet, 42 (3), pp. 268-271. | Show Abstract | Read more

Antimalarial drugs impose strong selective pressure on Plasmodium falciparum parasites and leave signatures of selection in the parasite genome; screening for genes under selection may suggest potential drug or immune targets. Genome-wide association studies (GWAS) of parasite traits have been hampered by the lack of high-throughput genotyping methods, inadequate knowledge of parasite population history and time-consuming adaptations of parasites to in vitro culture. Here we report the first Plasmodium GWAS, which included 189 culture-adapted P. falciparum parasites genotyped using a custom-built Affymetrix molecular inversion probe 3K malaria panel array with a coverage of approximately 1 SNP per 7 kb. Population structure, variation in recombination rate and loci under recent positive selection were detected. Parasite half-maximum inhibitory concentrations for seven antimalarial drugs were obtained and used in GWAS to identify genes associated with drug responses. This study provides valuable tools and insight into the P. falciparum genome.

Stepniewska K, Ashley E, Lee SJ, Anstey N, Barnes KI, Binh TQ, D'Alessandro U, Day NP et al. 2010. In vivo parasitological measures of artemisinin susceptibility. J Infect Dis, 201 (4), pp. 570-579. | Show Abstract | Read more

Parasite clearance data from 18,699 patients with falciparum malaria treated with an artemisinin derivative in areas of low (n=14,539), moderate (n=2077), and high (n=2083) levels of malaria transmission across the world were analyzed to determine the factors that affect clearance rates and identify a simple in vivo screening measure for artemisinin resistance. The main factor affecting parasite clearance time was parasite density on admission. Clearance rates were faster in high-transmission settings and with more effective partner drugs in artemisinin-based combination treatments (ACTs). The result of the malaria blood smear on day 3 (72 h) was a good predictor of subsequent treatment failure and provides a simple screening measure for artemisinin resistance. Artemisinin resistance is highly unlikely if the proportion of patients with parasite densities of <100,000 parasites/microL given the currently recommended 3-day ACT who have a positive smear result on day 3 is <3%; that is, for n patients the observed number with a positive smear result on day 3 does not exceed (n + 60)/24.

Hanson J, Lee SJ, Mohanty S, Faiz MA, Anstey NM, Charunwatthana P, Yunus EB, Mishra SK et al. 2010. A simple score to predict the outcome of severe malaria in adults. Clin Infect Dis, 50 (5), pp. 679-685. | Show Abstract | Read more

BACKGROUND: World Health Organization treatment guidelines recommend that adults with severe malaria be admitted to an intensive care unit (ICU). However, ICU facilities are limited in the resource-poor settings where most malaria occurs. Identification of patients at greater risk of complications may facilitate their triage and resource allocation. METHODS: With use of data from a trial conducted in Southeast Asia (n=868), a logistic regression model was built to identify independent predictors of mortality among adults with severe malaria. A scoring system based on this model was tested in the original dataset and then validated in 2 series from Bangladesh (n=188) and Vietnam (n=292). RESULTS: Acidosis (base deficit) and cerebral malaria (measured as Glasgow Coma Score) were the main independent predictors of outcome. The 5-point Coma Acidosis Malaria (CAM) score was simply derived from these 2 variables. Mortality increased steadily with increasing score. A CAM score <2 predicted survival with a positive predictive value (PPV) of 95.8% (95% confidence interval [CI], 93%- 97.7%). Of the 14 of 331 patients who died with a CAM score <2, 11 (79%) had renal failure and death occurred late after hospital admission (median, 108 h; range, 40-360 h). Substitution of plasma bicarbonate as the measure of acidosis only slightly reduced the prognostic value of the model. Use of respiratory rate was inferior, but a score <2 still predicted survival with a PPV of 92.2% (95% CI, 89.1%-94.7%). CONCLUSIONS: Patients with a CAM score <2 at hospital admission may be safely treated in a general ward, provided that renal function can be monitored.

Lee N, Wong CK, Chan PK, Lindegardh N, White NJ, Hayden FG, Wong EH, Wong KS, Cockram CS, Sung JJ, Hui DS. 2010. Acute encephalopathy associated with influenza A infection in adults. Emerg Infect Dis, 16 (1), pp. 139-142. | Show Abstract | Read more

We report acute encephalopathy associated with influenza A infection in 3 adults. We detected high cerebrospinal fluid (CSF) and plasma concentrations of CXCL8/IL-8 and CCL2/MCP-1 (CSF/plasma ratios > or =3), and interleukin-6, CXCL10/IP-10, but no evidence of viral neuroinvasion. Patients recovered without sequelae. Hyperactivated cytokine response may play a role in pathogenesis.

McGready R, Ashley EA, Wuthiekanun V, Tan SO, Pimanpanarak M, Viladpai-Nguen SJ, Jesadapanpong W, Blacksell SD et al. 2010. Arthropod borne disease: the leading cause of fever in pregnancy on the Thai-Burmese border. PLoS Negl Trop Dis, 4 (11), pp. e888. | Show Abstract | Read more

BACKGROUND: Fever in pregnancy is dangerous for both mother and foetus. In the 1980's malaria was the leading cause of death in pregnant women in refugee camps on the Thai-Burmese border. Artemisinin combination therapy has significantly reduced the incidence of malaria in the population. The remaining causes of fever in pregnancy are not well documented. METHODOLOGY: Pregnant women attending antenatal care, where weekly screening for malaria is routine, were invited to have a comprehensive clinical and laboratory screen if they had fever. Women were admitted to hospital, treated and followed up weekly until delivery. A convalescent serum was collected on day 21. Delivery outcomes were recorded. PRINCIPAL FINDINGS: Febrile episodes (n = 438) occurred in 5.0% (409/8,117) of pregnant women attending antenatal clinics from 7-Jan-2004 to 17-May-2006. The main cause was malaria in 55.5% (227/409). A cohort of 203 (49.6% of 409) women had detailed fever investigations and follow up. Arthropod-borne (malaria, rickettsial infections, and dengue) and zoonotic disease (leptospirosis) accounted for nearly half of all febrile illnesses, 47.3% (96/203). Coinfection was observed in 3.9% (8/203) of women, mostly malaria and rickettsia. Pyelonephritis, 19.7% (40/203), was also a common cause of fever. Once malaria, pyelonephritis and acute respiratory illness are excluded by microscopy and/or clinical findings, one-third of the remaining febrile infections will be caused by rickettsia or leptospirosis. Scrub and murine typhus were associated with poor pregnancy outcomes including stillbirth and low birth weight. One woman died (no positive laboratory tests). CONCLUSION/SIGNIFICANCE: Malaria remains the leading cause of fever in pregnancy on the Thai-Burmese border. Scrub and murine typhus were also important causes of fever associated with poor pregnancy outcomes. Febrile pregnant women on the Thai-Burmese border who do not have malaria, pyelonephritis or respiratory tract infection should be treated with azithromycin, effective for typhus and leptospirosis.

Lang TA, White NJ, Tran HT, Farrar JJ, Day NP, Fitzpatrick R, Angus BJ, Denis E et al. 2010. Clinical research in resource-limited settings: enhancing research capacity and working together to make trials less complicated. PLoS Negl Trop Dis, 4 (6), pp. e619. | Read more

Cheah PY, Lwin KM, Phaiphun L, Maelankiri L, Parker M, Day NP, White NJ, Nosten F. 2010. Community engagement on the Thai-Burmese border: rationale, experience and lessons learnt. Int Health, 2 (2), pp. 123-129. | Show Abstract | Read more

Community engagement is increasingly promoted in developing countries, especially in international health research, but there is little published experience. The Shoklo Malaria Research Unit (SMRU) conducts research with refugees, migrant workers, displaced people, and day migrants on the Thai-Burmese border, and has recently facilitated the set up of the Tak Province Border Community Ethics Advisory Board (T-CAB). Valuable lessons have been learnt from consultation with the T-CAB especially in the area of participant recruitment and the informed consent process. A lot of new research questions have emerged from consultation with the T-CAB. This paper describes our experience, lessons learnt and the unique challenges faced working with the T-CAB from its initial conception to date. We conclude that consultation with the T-CAB has made improvements in our research in particular operational and ethical aspects of our studies.

Boel M, Carrara VI, Rijken M, Proux S, Nacher M, Pimanpanarak M, Paw MK, Moo O et al. 2010. Complex Interactions between soil-transmitted helminths and malaria in pregnant women on the Thai-Burmese border. PLoS Negl Trop Dis, 4 (11), pp. e887. | Show Abstract | Read more

BACKGROUND: Deworming is recommended by the WHO in girls and pregnant and lactating women to reduce anaemia in areas where hookworm and anaemia are common. There is conflicting evidence on the harm and the benefits of intestinal geohelminth infections on the incidence and severity of malaria, and consequently on the risks and benefits of deworming in malaria affected populations. We examined the association between geohelminths and malaria in pregnancy on the Thai-Burmese border. METHODOLOGY: Routine antenatal care (ANC) included active detection of malaria (weekly blood smear) and anaemia (second weekly haematocrit) and systematic reporting of birth outcomes. In 1996 stool samples were collected in cross sectional surveys from women attending the ANCs. This was repeated in 2007 when malaria incidence had reduced considerably. The relationship between geohelminth infection and the progress and outcome of pregnancy was assessed. PRINCIPAL FINDINGS: Stool sample examination (339 in 1996, 490 in 2007) detected a high prevalence of geohelminths 70% (578/829), including hookworm (42.8% (355)), A. lumbricoides (34.4% (285)) and T.trichuria (31.4% (250)) alone or in combination. A lower proportion of women (829) had mild (21.8% (181)) or severe (0.2% (2)) anaemia, or malaria 22.4% (186) (P.vivax monoinfection 53.3% (101/186)). A. lumbricoides infection was associated with a significantly decreased risk of malaria (any species) (AOR: 0.43, 95% CI: 0.23-0.84) and P.vivax malaria (AOR: 0.29, 95% CI: 0.11-0.79) whereas hookworm infection was associated with an increased risk of malaria (any species) (AOR: 1.66, 95% CI: 1.06-2.60) and anaemia (AOR: 2.41, 95% CI: 1.18-4.93). Hookworm was also associated with low birth weight (AOR: 1.81, 95% CI: 1.02-3.23). CONCLUSION/SIGNIFICANCE: A. lumbricoides and hookworm appear to have contrary associations with malaria in pregnancy.

Ashley EA, Stepniewska K, Lindegardh N, Annerberg A, Tarning J, McGready R, Phaiphun L, Singhasivanon P, White NJ, Nosten F. 2010. Comparison of plasma, venous and capillary blood levels of piperaquine in patients with uncomplicated falciparum malaria. Eur J Clin Pharmacol, 66 (7), pp. 705-712. | Show Abstract | Read more

PURPOSE: Dihydroartemisinin-piperaquine (DP) is a fixed-dose artemisinin-based combination treatment. Field pharmacokinetic studies would be simplified and facilitated by being able to use small volume capillary assays rather than venous blood. The aim of this study was to describe the relationship between piperaquine concentrations measured in capillary blood, venous blood and venous plasma. METHODS: Samples of plasma, whole blood obtained by venesection and capillary blood were taken simultaneously from patients with uncomplicated Plasmodium falciparum malaria treated with DP between 0 and 9 weeks after treatment. Piperaquine concentrations in venous and capillary samples were measured using solid phase extraction and analysis by liquid chromatography with ultraviolet detection. RESULTS: A total of 161 sets of the three measures were obtained from 54 patients. Piperaquine concentrations in the venous blood samples were approximately twofold higher and those in the capillary blood samples were threefold higher than the corresponding venous plasma concentrations. Capillary blood piperaquine concentrations were approximately 1.7-fold higher than venous blood concentrations, and this difference also increased with time. CONCLUSION: Differences in whole blood and plasma levels of piperaquine suggest compartmentalisation of the drug within blood cells, as also occurs with the structurally related quinoline chloroquine. The relationship between piperaquine concentrations in the venous plasma, venous blood and capillary blood is variable and unpredictable at low concentrations. However, within the range of concentrations usually present in patients between 3 and 21 days after treatment with currently recommended doses, the relationship between capillary and venous whole blood is predictable; consequently, capillary blood sampling can be used in field assessments.

Gaudin K, Millet P, Fawaz F, Olliaro P, White NJ, Cassus-Coussère C, Agbahoungha U, Dubost JP. 2010. Investigation of porous graphitic carbon at high-temperature liquid chromatography with evaporative light scattering detection for the analysis of the drug combination artesunate--azithromycin for the treatment of severe malaria. J Chromatogr A, 1217 (1), pp. 75-81. | Show Abstract | Read more

Artesunate combined therapies represent the best option for the treatment of malaria and require the development of new methods of analysis. Retention, selectivity and detection with high-temperature liquid chromatography-porous graphitic carbon-evaporative light scattering detection was studied for artesunate and azithromycin separation. Organic solvent, concentration of organic modifiers, temperature and flow rate were all relevant parameters to optimize this separation. The behaviour of artesunate in the tested conditions appeared close to a neutral compound. In CH(3)OH, only azithromycin retention was dramatically altered depending on the [triethylamine]/[formic acid] ratio and on the temperature, whereas in CH(3)CN, azithromycin, artesunate, artemisinin and dihydroartemisinin retentions decreased with the temperature increase whatever the organic modifier ratio. The best efficiency was obtained with CH(3)CN. 25% variation of the concentration values of the organic modifiers did not significantly influenced the retention. The sensitivity of ELSD increased with the flow rate decrease. Peak area and S/N ratio dramatically decreased with the flow rate increase by 10- and 5-fold for artesunate and azithromycin, respectively. Non-linear calibration curves were obtained for both artesunate and azithromycin.

Monatrakul P, Mungthin M, Dondorp AM, Krudsood S, Udomsangpetch R, Wilairatana P, White NJ, Chotivanich K. 2010. Modulating effects of plasma containing anti-malarial antibodies on in vitro anti-malarial drug susceptibility in Plasmodium falciparum. Malar J, 9 (1), pp. 326. | Show Abstract | Read more

BACKGROUND: The efficacy of anti-malarial drugs is determined by the level of parasite susceptibility, anti-malarial drug bioavailability and pharmacokinetics, and host factors including immunity. Host immunity improves the in vivo therapeutic efficacy of anti-malarial drugs, but the mechanism and magnitude of this effect has not been characterized. This study characterized the effects of 'immune' plasma to Plasmodium falciparumon the in vitro susceptibility of P. falciparum to anti-malarial drugs. METHODS: Titres of antibodies against blood stage antigens (mainly the ring-infected erythrocyte surface antigen [RESA]) were measured in plasma samples obtained from Thai patients with acute falciparum malaria. 'Immune' plasma was selected and its effects on in vitro parasite growth and multiplication of the Thai P. falciparum laboratory strain TM267 were assessed by light microscopy. The in vitro susceptibility to quinine and artesunate was then determined in the presence and absence of 'immune' plasma using the 3H-hypoxanthine uptake inhibition method. Drug susceptibility was expressed as the concentrations causing 50% and 90% inhibition (IC50 and IC90), of 3H-hypoxanthine uptake. RESULTS: Incubation with 'immune' plasma reduced parasite maturation and decreased parasite multiplication in a dose dependent manner. 3H-hypoxanthine incorporation after incubation with 'immune' plasma was decreased significantly compared to controls (median [range]; 181.5 [0 to 3,269] cpm versus 1,222.5 [388 to 5,932] cpm) (p= 0.001). As a result 'immune' plasma reduced apparent susceptibility to quinine substantially; median (range) IC50 6.4 (0.5 to 23.8) ng/ml versus 221.5 (174.4 to 250.4) ng/ml (p = 0.02), and also had a borderline effect on artesunate susceptibility; IC50 0.2 (0.02 to 0.3) ng/ml versus 0.8 (0.2 to 2.3) ng/ml (p = 0.08). Effects were greatest at low concentrations, changing the shape of the concentration-effect relationship. IC90 values were not significantly affected; median (range) IC90 448.0 (65 to > 500) ng/ml versus 368.8 (261 to 501) ng/ml for quinine (p > 0.05) and 17.0 (0.1 to 29.5) ng/ml versus 7.6 (2.3 to 19.5) ng/ml for artesunate (p = 0.4). CONCLUSIONS: 'Immune' plasma containing anti-malarial antibodies inhibits parasite development and multiplication and increases apparent in vitro anti-malarial drug susceptibility of P. falciparum. The IC90 was much less affected than the IC50 measurement.

Karema C, Imwong M, Fanello CI, Stepniewska K, Uwimana A, Nakeesathit S, Dondorp A, Day NP, White NJ. 2010. Molecular correlates of high-level antifolate resistance in Rwandan children with Plasmodium falciparum malaria. Antimicrob Agents Chemother, 54 (1), pp. 477-483. | Show Abstract | Read more

Antifolate drugs have an important role in the treatment of malaria. Polymorphisms in the genes encoding the dihydrofolate reductase and dihydropteroate synthetase enzymes cause resistance to the antifol and sulfa drugs, respectively. Rwanda has the highest levels of antimalarial drug resistance in Africa. We correlated the efficacy of chlorproguanil-dapsone plus artesunate (CPG-DDS+A) and amodiaquine plus sulfadoxine-pyrimethamine (AQ+SP) in children with uncomplicated malaria caused by Plasmodium falciparum parasites with pfdhfr and pfdhps mutations, which are known to confer reduced drug susceptibility, in two areas of Rwanda. In the eastern province, where the cure rates were low, over 75% of isolates had three or more pfdhfr mutations and two or three pfdhps mutations and 11% had the pfdhfr 164-Leu polymorphism. In the western province, where the cure rates were significantly higher (P < 0.001), the prevalence of multiple resistance mutations was lower and the pfdhfr I164L polymorphism was not found. The risk of treatment failure following the administration of AQ+SP more than doubled for each additional pfdhfr resistance mutation (odds ratio [OR] = 2.4; 95% confidence interval [CI] = 1.01 to 5.55; P = 0.048) and each pfdhps mutation (OR = 2.1; 95% CI = 1.21 to 3.54; P = 0.008). The risk of failure following CPG-DDS+A treatment was 2.2 times higher (95% CI = 1.34 to 3.7) for each additional pfdhfr mutation, whereas there was no association with mutations in the pfdhps gene (P = 0.13). The pfdhfr 164-Leu polymorphism is prevalent in eastern Rwanda. Antimalarial treatments with currently available antifol-sulfa combinations are no longer effective in Rwanda because of high-level resistance.

Maude RJ, Lubell Y, Socheat D, Yeung S, Saralamba S, Pongtavornpinyo W, Cooper BS, Dondorp AM, White NJ, White LJ. 2010. The role of mathematical modelling in guiding the science and economics of malaria elimination. Int Health, 2 (4), pp. 239-246. | Show Abstract | Read more

Unprecedented efforts are now underway to eliminate malaria from many regions. Despite the enormous financial resources committed, if malaria elimination is perceived as failing it is likely that this funding will not be sustained. It is imperative that methods are developed to use the limited data available to design site-specific, cost-effective elimination programmes. Mathematical modelling is a way of including mechanistic understanding to use available data to make predictions. Different strategies can be evaluated much more rapidly than is possible through trial and error in the field. Mathematical modelling has great potential as a tool to guide and inform current elimination efforts. Economic modelling weighs costs against characterised effects or predicted benefits in order to determine the most cost-efficient strategy but has traditionally used static models of disease not suitable for elimination. Dynamic mathematical modelling and economic modelling techniques need to be combined to contribute most effectively to ongoing policy discussions. We review the role of modelling in previous malaria control efforts as well as the unique nature of elimination and the consequent need for its explicit modelling, and emphasise the importance of good disease surveillance. The difficulties and complexities of economic evaluation of malaria control, particularly the end stages of elimination, are discussed.

Cited:

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Scopus

Imwong M, Tanomsing N, Pukrittayakamee S, Day NPJ, White NJ, Snounou G. 2009. Spurious Amplification of a Plasmodium vivax Small-Subunit RNA Gene by Use of Primers Currently Used To Detect P. knowlesi JOURNAL OF CLINICAL MICROBIOLOGY, 47 (12), pp. 4173-4175. | Show Abstract | Read more

The PCR primers commonly used to detect Plasmodium knowlesi infections in humans were found to cross-react stochastically with P. vivax genomic DNA. A nested primer set that targets one of the P. knowlesi small-subunit rRNA genes was validated for specificity and for sensitivity of detection of <10 parasite genomes. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

White NJ, Turner GD, Medana IM, Dondorp AM, Day NP. 2010. The murine cerebral malaria phenomenon. Trends Parasitol, 26 (1), pp. 11-15. | Show Abstract | Read more

P.berghei ANKA infection in CBA or CB57BL/6 mice is used widely as a murine 'model' of human cerebral malaria (HCM), despite markedly different histopathological features. The pathology of the murine model is characterised by marked inflammation with little or no intracerebral sequestration of parasitised erythrocytes, whereas HCM is associated with intense intracerebral sequestration, often with little inflammatory response. There are now more than ten times as many studies each year of the murine model than on HCM. Of 48 adjunctive interventions evaluated in the murine model, 44 (92%) were successful, compared with only 1 (6%) of 17 evaluated in HCM during the same period. The value of the mouse model in identifying pathological processes or therapeutic interventions in human cerebral malaria is questionable.

Dondorp AM, Nosten F, White NJ. 2009. Artemisinin Resistance in Plasmodium falciparum Malaria REPLY NEW ENGLAND JOURNAL OF MEDICINE, 361 (18), pp. 1808-1808.

Cheng AC, McBryde ES, Wuthiekanun V, Chierakul W, Amornchai P, Day NP, White NJ, Peacock SJ. 2009. Dosing regimens of cotrimoxazole (trimethoprim-sulfamethoxazole) for melioidosis. Antimicrob Agents Chemother, 53 (10), pp. 4193-4199. | Show Abstract | Read more

Melioidosis is an infectious disease with a propensity for relapse, despite prolonged antibiotic eradication therapy for 12 to 20 weeks. A pharmacokinetic (PK) simulation study was performed to determine the optimal dosing of cotrimoxazole (trimethoprim-sulfamethoxazole [TMP-SMX]) used in current eradication regimens in Thailand and Australia. Data for bioavailability, protein binding, and coefficients of absorption and elimination were taken from published literature. Apparent volumes of distribution were correlated with body mass and were estimated separately for Thai and Australian populations. In vitro experiments demonstrated concentration-dependent killing. In Australia, the currently used eradication regimen (320 [TMP]/1,600 [SMX] mg every 12 h [q12h]) was predicted to achieve the PK-pharmacodynamic (PD) target (an area under the concentration-time curve from 0 to 24 h/MIC ratio of >25 for both TMP and SMX) for strains with the MIC90 of Australian strains (< or = 1/19 mg/liter). In Thailand, the former regimen of 160/800 mg q12h would not be expected to attain the target for strains with an MIC of > or = 1/19 mg/liter, but the recently implemented weight-based regimen (<40 kg [body weight], 160/800 mg q12h; 40 to 60 kg, 240/1,200 mg q12h; >60 kg, 320/1,600 mg q12h) would be expected to achieve adequate concentrations for strains with an MIC of < or = 1/19 mg/liter. The results were sensitive to the variance of the PK parameters. Prospective PK-PD studies of Asian populations are needed to optimize TMP-SMX dosing in melioidosis.

Rijken MJ, Lee SJ, Boel ME, Papageorghiou AT, Visser GH, Dwell SL, Kennedy SH, Singhasivanon P, White NJ, Nosten F, McGready R. 2009. Obstetric ultrasound scanning by local health workers in a refugee camp on the Thai-Burmese border. Ultrasound Obstet Gynecol, 34 (4), pp. 395-403. | Show Abstract | Read more

OBJECTIVES: Ultrasound examination of the fetus is a powerful tool for assessing gestational age and detecting obstetric problems but is rarely available in developing countries. The aim of this study was to assess the intraobserver and interobserver agreement of fetal biometry by locally trained health workers in a refugee camp on the Thai-Burmese border. METHODS: One expatriate doctor and four local health workers participated in the study, which included examinations performed on every fifth pregnant woman with a singleton pregnancy between 16 and 40 weeks' gestation, and who had undergone an early dating ultrasound scan, attending the antenatal clinic in Maela refugee camp. At each examination, two examiners independently measured biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC) and femur length (FL), with one of the examiners obtaining duplicate measurements of each parameter. Intraobserver measurement error was assessed using the intraclass correlation coefficient (ICC) and interobserver error was assessed by the Bland and Altman 95% limits of agreement method. RESULTS: A total of 4188 ultrasound measurements (12 per woman) were obtained in 349 pregnancies at a median gestational age of 27 (range, 16-40) weeks in 2008. The ICC for BPD, HC, AC and FL was greater than 0.99 for all four trainees and the doctor (range, 0.996-0.998). For gestational ages between 18 and 24 weeks, interobserver 95% limits of agreement corresponding to differences in estimated gestational age of less than +/- 1 week were calculated for BPD, HC, AC and FL. Measurements by local health workers showed high levels of agreement with those of the expatriate doctor. CONCLUSIONS: Locally trained health workers working in a well organized unit with ongoing quality control can obtain accurate fetal biometry measurements for gestational age estimation. This experience suggests that training of local health workers in developing countries is possible and could allow effective use of obstetric ultrasound imaging.

Nontprasert A, Cheeramakara C, Pukrittayakamee S, Dance DAB, Pitt TL, Smith MD, Vanijanonta S, White NJ. 2009. SDS-PAGE analysis of whole cell protein and outer membrane protein patterns of clinical isolates of Burkholderia pseudomallei ASIAN PACIFIC JOURNAL OF TROPICAL MEDICINE, 2 (5), pp. 14-19.

Bicanic T, Muzoora C, Brouwer AE, Meintjes G, Longley N, Taseera K, Rebe K, Loyse A et al. 2009. Independent association between rate of clearance of infection and clinical outcome of HIV-associated cryptococcal meningitis: analysis of a combined cohort of 262 patients. Clin Infect Dis, 49 (5), pp. 702-709. | Show Abstract | Read more

BACKGROUND: Progress in therapy for cryptococcal meningitis has been slow because of the lack of a suitable marker of treatment response. Previously, we demonstrated the statistical power of a novel endpoint, the rate of clearance of infection, based on serial quantitative cultures of cerebrospinal fluid, to differentiate the fungicidal activity of alternative antifungal drug regimens. We hypothesized that the rate of clearance of infection should also be a clinically meaningful endpoint. METHODS: We combined data from cohorts of patients with human immunodeficiency virus-associated cryptococcal meningitis from Thailand, South Africa, and Uganda, for whom the rate of clearance of infection was determined, and clinical and laboratory data prospectively collected, and explored the association between the rate of clearance of infection and mortality by Cox survival analyses. RESULTS: The combined cohort comprised 262 subjects. Altered mental status at presentation, a high baseline organism load, and a slow rate of clearance of infection were independently associated with increased mortality at 2 and 10 weeks. Rate of clearance of infection was associated with antifungal drug regimen and baseline cerebrospinal fluid interferon-gamma levels. CONCLUSIONS: The results support the use of the rate of clearance of infection or early fungicidal activity as a means to explore antifungal drug dosages and combinations in phase II studies. An increased understanding of how the factors determining outcome interrelate may help clarify opportunities for intervention.

Tarning J, McGready R, Lindegardh N, Ashley EA, Pimanpanarak M, Kamanikom B, Annerberg A, Day NP et al. 2009. Population pharmacokinetics of lumefantrine in pregnant women treated with artemether-lumefantrine for uncomplicated Plasmodium falciparum malaria. Antimicrob Agents Chemother, 53 (9), pp. 3837-3846. | Show Abstract | Read more

Artemether-lumefantrine has become one of the most widely used antimalarial drugs in the world. The objective of this study was to determine the population pharmacokinetic properties of lumefantrine in pregnant women with uncomplicated multidrug-resistant Plasmodium falciparum malaria on the northwestern border of Thailand. Burmese and Karen women (n = 103) with P. falciparum malaria and in the second and third trimesters of pregnancy were treated with artemether-lumefantrine (80/480 mg) twice daily for 3 days. All patients provided five capillary plasma samples for drug quantification, and the collection times were randomly distributed over 14 days. The concentration-time profiles of lumefantrine were assessed by nonlinear mixed-effects modeling. The treatment failure rate (PCR-confirmed recrudescent infections at delivery) was high; 16.5% (95% confidence interval, 9.9 to 25.1). The population pharmacokinetics of lumefantrine were described well by a two-compartment open model with first-order absorption and elimination. The final model included interindividual variability in all pharmacokinetic parameters and a linear covariate relationship between the estimated gestational age and the central volume of distribution. A high proportion of all women (40%, 41/103) had day 7 capillary plasma concentrations of <355 ng/ml (which corresponds to approximately <280 ng/ml in venous plasma), a threshold previously associated with an increased risk of therapeutic failure in nonpregnant patients in this area. Predictive modeling suggests that a twice-daily regimen given for 5 days would be preferable in later pregnancy. In conclusion, altered pharmacokinetic properties of lumefantrine contribute to the high rates of failure of artemether-lumefantrine treatment in later pregnancy. Dose optimization is urgently needed.

McGready R, Stepniewska K, Lindegardh N, Ashley EA, La Y, Singhasivanon P, White NJ, Nosten F. 2009. The pharmacokinetics of artemether and lumefantrine in pregnant women with uncomplicated falciparum malaria. Eur J Clin Pharmacol, 65 (8), pp. 847-847. | Read more

Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, Ariey F et al. 2009. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med, 361 (5), pp. 455-467. | Show Abstract | Read more

BACKGROUND: Artemisinin-based combination therapies are the recommended first-line treatments of falciparum malaria in all countries with endemic disease. There are recent concerns that the efficacy of such therapies has declined on the Thai-Cambodian border, historically a site of emerging antimalarial-drug resistance. METHODS: In two open-label, randomized trials, we compared the efficacies of two treatments for uncomplicated falciparum malaria in Pailin, western Cambodia, and Wang Pha, northwestern Thailand: oral artesunate given at a dose of 2 mg per kilogram of body weight per day, for 7 days, and artesunate given at a dose of 4 mg per kilogram per day, for 3 days, followed by mefloquine at two doses totaling 25 mg per kilogram. We assessed in vitro and in vivo Plasmodium falciparum susceptibility, artesunate pharmacokinetics, and molecular markers of resistance. RESULTS: We studied 40 patients in each of the two locations. The overall median parasite clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours (interquartile range, 36 to 66) in Wang Pha (P<0.001). Recrudescence confirmed by means of polymerase-chain-reaction assay occurred in 6 of 20 patients (30%) receiving artesunate monotherapy and 1 of 20 (5%) receiving artesunate-mefloquine therapy in Pailin, as compared with 2 of 20 (10%) and 1 of 20 (5%), respectively, in Wang Pha (P=0.31). These markedly different parasitologic responses were not explained by differences in age, artesunate or dihydroartemisinin pharmacokinetics, results of isotopic in vitro sensitivity tests, or putative molecular correlates of P. falciparum drug resistance (mutations or amplifications of the gene encoding a multidrug resistance protein [PfMDR1] or mutations in the gene encoding sarco-endoplasmic reticulum calcium ATPase6 [PfSERCA]). Adverse events were mild and did not differ significantly between the two treatment groups. CONCLUSIONS: P. falciparum has reduced in vivo susceptibility to artesunate in western Cambodia as compared with northwestern Thailand. Resistance is characterized by slow parasite clearance in vivo without corresponding reductions on conventional in vitro susceptibility testing. Containment measures are urgently needed. (ClinicalTrials.gov number, NCT00493363, and Current Controlled Trials number, ISRCTN64835265.)

Maude RJ, Dondorp AM, Abu Sayeed A, Day NP, White NJ, Beare NA. 2009. The eye in cerebral malaria: what can it teach us? Trans R Soc Trop Med Hyg, 103 (7), pp. 661-664. | Show Abstract | Read more

The pathophysiology of coma in cerebral malaria (CM) is not well understood. Obstruction of microcirculatory flow is thought to play a central role, but other hypotheses include roles for parasite- and host-derived factors such as immune mediators, and for increased blood-brain barrier permeability leading to raised intracranial pressure. The retinal vasculature is a direct extension of the cerebral vasculature. It is the only vascular bed easily accessible for visualisation and provides a unique opportunity to observe vascular pathology and its effect on neurological tissue. A specific retinopathy has been well described in African children with CM and its severity correlates with outcome. This retinopathy has been less well described in adults. The central mechanism causing malarial retinopathy appears to be microvascular obstruction, which has been demonstrated in affected retinas by fluorescein angiography. The presence in a central nervous system tissue of microvascular obstruction strongly supports the hypothesis that the sequestration of erythrocytes in small blood vessels and consequent obstruction of microcirculatory flow is an important mechanism causing coma and death in CM. Despite advances in the antimalarial treatment of severe malaria, its mortality remains approximately 15-20%. Adjunctive treatment targeting sequestration is a promising strategy to further lower mortality.

Maude RJ, Beare NA, Abu Sayeed A, Chang CC, Charunwatthana P, Faiz MA, Hossain A, Yunus EB et al. 2009. The spectrum of retinopathy in adults with Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg, 103 (7), pp. 665-671. | Show Abstract | Read more

A specific retinopathy has been described in African children with cerebral malaria, but in adults this has not been extensively studied. Since the structure and function of the retinal vasculature greatly resembles the cerebral vasculature, study of retinal changes can reveal insights into the pathophysiology of cerebral malaria. A detailed observational study of malarial retinopathy in Bangladeshi adults was performed using high-definition portable retinal photography. Retinopathy was present in 17/27 adults (63%) with severe malaria and 14/20 adults (70%) with cerebral malaria. Moderate or severe retinopathy was more frequent in cerebral malaria (11/20, 55%) than in uncomplicated malaria (3/15, 20%; P=0.039), bacterial sepsis (0/5, 0%; P=0.038) or healthy controls (0/18, 0%; P<0.001). The spectrum of malarial retinopathy was similar to that previously described in African children, but no vessel discolouration was observed. The severity of retinal whitening correlated with admission venous plasma lactate (P=0.046), suggesting that retinal ischaemia represents systemic ischaemia. In conclusion, retinal changes related to microvascular obstruction were common in adults with severe falciparum malaria and correlated with disease severity and coma, suggesting that a compromised microcirculation has important pathophysiological significance in severe and cerebral malaria. Portable retinal photography has potential as a valuable tool to study malarial retinopathy.

White NJ, Webster RG, Govorkova EA, Uyeki TM. 2009. What is the optimal therapy for patients with H5N1 influenza? PLoS Med, 6 (6), pp. e1000091. | Show Abstract | Read more

BACKGROUND TO THE DEBATE: In a 2007 article in PLoS Medicine[10], Holger J. Schünemann and colleagues described a new process used by the World Health Organization for rapidly developing clinical management guidelines in emergency situations. These situations include outbreaks of emerging infectious diseases. The authors discussed how they developed such a "rapid advice" guideline for the pharmacological management of avian influenza A (H5N1) virus infection. The guideline recommends giving the antiviral drug oseltamivir at a dose of 75 mg twice daily for five days. In this Debate, Nicholas White argues that such dosing is inadequate, Robert Webster and Elena Govorkova say that combination antiviral therapy should be used, and Tim Uyeki reminds us that clinical care of patients with H5N1 entails much more than antiviral treatment. These issues may also apply to therapy of patients hospitalized with severe disease due to novel swine-origin influenza A (H1N1) virus infection.

Chotivanich K, Sattabongkot J, Choi YK, Park JS, Sritabal J, Lim CS, Udomsangpetch R, White NJ, Lee WJ. 2009. Antimalarial drug susceptibility of Plasmodium vivax in the Republic of Korea. Am J Trop Med Hyg, 80 (6), pp. 902-904. | Show Abstract

The antimalarial susceptibility of ring stage (> 80%) Plasmodium vivax from the Republic of Korea, where long incubation-period strains are prevalent, was evaluated using the schizont maturation inhibition technique. During 2005-2007, susceptibility to seven antimalarial drugs was evaluated with 24 fresh isolates. The geometric mean (95% confidence interval) 50% inhibition concentration (IC(50)) were quinine 60 (54-75) ng/mL, chloroquine 39 (22-282) ng/mL, piperaquine 27 (17-58) ng/mL, mefloquine 39 (35-67) ng/mL, pyrimethamine 138 (89-280) ng/mL, artesunate 0.6 (0.5-0.8) ng/mL, and primaquine 122 (98-232) ng/mL. Positive correlations were found between quinine and mefloquine (r = 0.6, P = 0.004), piperaquine and chloroquine (r = 0.6, P = 0.008), and piperaquine and primaquine IC(50) values (r = 0.5, P = 0.01). Compared with P. vivax in Thailand, P. vivax in the Republic of Korea was more sensitive to quinine and mefloquine, but equally sensitive to chloroquine and artesunate.

Maude RJ, Pontavornpinyo W, Saralamba S, Dondorp AM, Day NP, White NJ, White LJ. 2009. The role of mathematical modelling in malaria elimination and eradication (Comment on: Can malaria be eliminated?). Trans R Soc Trop Med Hyg, 103 (6), pp. 643-644. | Read more

Lindegardh N, Tarning J, Toi PV, Hien TT, Farrar J, Singhasivanon P, White NJ, Ashton M, Day NP. 2009. Quantification of artemisinin in human plasma using liquid chromatography coupled to tandem mass spectrometry. J Pharm Biomed Anal, 49 (3), pp. 768-773. | Show Abstract | Read more

A liquid chromatographic tandem mass spectroscopy method for the quantification of artemisinin in human heparinised plasma has been developed and validated. The method uses Oasis HLB mu-elution solid phase extraction 96-well plates to facilitate a high throughput of 192 samples a day. Artesunate (internal standard) in a plasma-water solution was added to plasma (50 microL) before solid phase extraction. Artemisinin and its internal standard artesunate were analysed by liquid chromatography and MS/MS detection on a Hypersil Gold C18 (100 mm x 2.1 mm, 5 microm) column using a mobile phase containing acetonitrile-ammonium acetate 10mM pH 3.5 (50:50, v/v) at a flow rate of 0.5 mL/min. The method has been validated according to published FDA guidelines and showed excellent performance. The within-day, between-day and total precisions expressed as R.S.D., were lower than 8% at all tested quality control levels including the upper and lower limit of quantification. The limit of detection was 0.257 ng/mL for artemisinin and the calibration range was 1.03-762 ng/mL using 50 microL plasma. The method was free from matrix effects as demonstrated both graphically and quantitatively.

Hanpithakpong W, Kamanikom B, Singhasivanon P, White NJ, Day NP, Lindegardh N. 2009. A liquid chromatographic-tandem mass spectrometric method for determination of artemether and its metabolite dihydroartemisinin in human plasma. Bioanalysis, 1 (1), pp. 37-46. | Show Abstract | Read more

BACKGROUND: Artemether-lumefantrine is the most widely recommended artemisinin-based combination treatment for falciparum malaria. Quantification of artemether and its metabolite dihydroartemisinin in biological matrices has traditionally been difficult, with sensitivity being an issue. RESULTS: A high-throughput bioanalytical method for the analysis of artemether and its metabolite dihydroartemisinin in human plasma using solid-phase extraction in the 96-well plate format and liquid chromatography coupled to positive ion mode tandem mass spectroscopy has been developed and validated according to US FDA guidelines. The method uses 50 µl plasma and covers the calibration range 1.43-500 ng/ml with a limit of detection at 0.36 ng/ml. CONCLUSIONS: The developed liquid chromatography-tandem mass spectrometry assay is more sensitive than all previous methods despite using a lower plasma volume (50 µl) and is highly suitable for clinical studies where plasma volumes are limited, such as pediatric trials.

Preechapornkul P, Imwong M, Chotivanich K, Pongtavornpinyo W, Dondorp AM, Day NP, White NJ, Pukrittayakamee S. 2009. Plasmodium falciparum pfmdr1 amplification, mefloquine resistance, and parasite fitness. Antimicrob Agents Chemother, 53 (4), pp. 1509-1515. | Show Abstract | Read more

Mefloquine is widely used in combination with artemisinin derivatives for the treatment of falciparum malaria. Mefloquine resistance in Plasmodium falciparum has been related to increased copy numbers of multidrug-resistant gene 1 (pfmdr1). We studied the ex vivo dynamics of pfmdr1 gene amplification in culture-adapted P. falciparum in relation to mefloquine resistance and parasite fitness. A Thai P. falciparum isolate (isolate TM036) was assessed by the use of multiple genetic markers as a single genotype. Resistance was selected by exposure to stepwise increasing concentrations of mefloquine up to 30 ng/ml in continuous culture. The pfmdr1 gene copy numbers increased as susceptibility to mefloquine declined (P = 0.03). No codon mutations at positions 86, 184, 1034, 1042, and 1246 in the pfmdr1 gene were detected. Two subclones of selected parasites (average copy numbers, 2.3 and 3.1, respectively) showed a fitness disadvantage when they were grown together with the original parasites containing a single pfmdr1 gene copy in the absence of mefloquine; the multiplication rates were 6.3% and 8.7% lower, respectively (P < 0.01). Modeling of the dynamics of the pfmdr1 copy numbers over time in relation to the relative fitness of the parasites suggested that net pfmdr1 gene amplification from one to two copies occurs once in every 10(8) parasites and that amplification from two to three copies occurs once in every 10(3) parasites. pfmdr1 gene amplification in P. falciparum is a frequent event and confers mefloquine resistance. Parasites with multiple copies of the pfmdr1 gene have decreased survival fitness in the absence of drug pressure.

Bicanic T, Brouwer AE, Meintjes G, Rebe K, Limmathurotsakul D, Chierakul W, Teparrakkul P, Loyse A et al. 2009. Relationship of cerebrospinal fluid pressure, fungal burden and outcome in patients with cryptococcal meningitis undergoing serial lumbar punctures. AIDS, 23 (6), pp. 701-706. | Show Abstract | Read more

OBJECTIVES: To assess impact of serial lumbar punctures on association between cerebrospinal fluid (CSF) opening pressure and prognosis in HIV-associated cryptococcal meningitis; to explore time course and relationship of opening pressure with neurological findings, CSF fungal burden, immune response, and CD4 cell count. DESIGN: Evaluation of 163 HIV-positive ART-naive patients enrolled in three trials of amphotericin B-based therapy for cryptococcal meningitis in Thailand and South Africa. METHODS: Study protocols required four lumbar punctures with measurements of opening pressure over the first 2 weeks of treatment and additional lumbar punctures if opening pressure raised. Fungal burden and clearance, CSF immune parameters, CD4 cell count, neurological symptoms and signs, and outcome at 2 and 10 weeks were compared between groups categorized by opening pressure at cryptococcal meningitis diagnosis. RESULTS: Patients with higher baseline fungal burden had higher baseline opening pressure. High fungal burden appeared necessary but not sufficient for development of high pressure. Baseline opening pressure was not associated with CD4 cell count, CSF pro-inflammatory cytokines, or altered mental status. Day 14 opening pressure was associated with day 14 fungal burden. Overall mortality was 12% (20/162) at 2 weeks and 26% (42/160) at 10 weeks, with no significant differences between opening pressure groups. CONCLUSION: Studies are needed to define factors, in addition to fungal burden, associated with raised opening pressure. Aggressive management of raised opening pressure through repeated CSF drainage appeared to prevent any adverse impact of raised opening pressure on outcome in patients with cryptococcal meningitis. The results support increasing access to manometers in resource-poor settings and routine management of opening pressure in patients with cryptococcal meningitis.

Newton PN, Lee SJ, Goodman C, Fernández FM, Yeung S, Phanouvong S, Kaur H, Amin AA et al. 2009. Guidelines for field surveys of the quality of medicines: a proposal. PLoS Med, 6 (3), pp. e52. | Read more

Lubell Y, Yeung S, Dondorp AM, Day NP, Nosten F, Tjitra E, Abul Faiz M, Yunus EB et al. 2009. Cost-effectiveness of artesunate for the treatment of severe malaria. Trop Med Int Health, 14 (3), pp. 332-337. | Show Abstract | Read more

OBJECTIVE: To explore the cost-effectiveness of artesunate against quinine based principally on the findings of a large multi-centre trial carried out in Southeast Asia. METHODS: Trial data were used to compare mortality of patients with severe malaria, treated with either artesunate or quinine. This was combined with retrospectively collected cost data to estimate the incremental cost per death averted with the use of artesunate instead of quinine. RESULTS: The incremental cost per death averted using artesunate was approximately 140 USD. Artesunate maintained this high level of cost-effectiveness also when allowing for the uncertainty surrounding the cost and effectiveness assessments. CONCLUSION: This analysis confirms the vast superiority of artesunate for treatment of severe malaria from an economic as well as a clinical perspective.

Wattanagoon Y, Stepniewska K, Lindegårdh N, Pukrittayakamee S, Silachamroon U, Piyaphanee W, Singtoroj T, Hanpithakpong W et al. 2009. Pharmacokinetics of high-dose oseltamivir in healthy volunteers. Antimicrob Agents Chemother, 53 (3), pp. 945-952. | Show Abstract | Read more

The effects of loading doses and probenecid coadministration on oseltamivir pharmacokinetics at four increasing dose levels in groups of eight healthy adult Thai volunteers (125 individual series) were evaluated. Doses of up to 675 mg were well-tolerated. The pharmacokinetics were dose linear. Oseltamivir phosphate (OS) was rapidly and completely absorbed and converted (median conversion level, 93%) to the active carboxylate metabolite. Median elimination half-lives (and 95% confidence intervals [CI]) were 1.0 h (0.9 to 1.1 h) for OS and 5.1 h (4.7 to 5.7 h) for oseltamivir carboxylate (OC). One subject repeatedly showed markedly reduced OS-to-OC conversion, indicating constitutionally impaired carboxylesterase activity. The coadministration of probenecid resulted in a mean contraction in the apparent volume of distribution of OC of 40% (95% CI, 37 to 44%) and a reduction in the renal elimination of OC of 61% (95% CI, 58 to 62%), thereby increasing the median area under the concentration-time curve (AUC) for OC by 154% (range, 71 to 278%). The AUC increase for OC in saliva was approximately three times less than the AUC increase for OC in plasma. A loading dose 1.25 times the maintenance dose should be given for severe influenza pneumonia. Probenecid coadministration may allow considerable dose saving for oseltamivir, but more information on OC penetration into respiratory secretions is needed to devise appropriate dose regimens.

Gomes MF, Faiz MA, Gyapong JO, Warsame M, Agbenyega T, Babiker A, Baiden F, Yunus EB et al. 2009. Pre-referral rectal artesunate to prevent death and disability in severe malaria: a placebo-controlled trial. Lancet, 373 (9663), pp. 557-566. | Show Abstract | Read more

BACKGROUND: Most malaria deaths occur in rural areas. Rapid progression from illness to death can be interrupted by prompt, effective medication. Antimalarial treatment cannot rescue terminally ill patients but could be effective if given earlier. If patients who cannot be treated orally are several hours from facilities for injections, rectal artesunate can be given before referral and acts rapidly on parasites. We investigated whether this intervention reduced mortality and permanent disability. METHODS: In Bangladesh, Ghana, and Tanzania, patients with suspected severe malaria who could not be treated orally were allocated randomly to a single artesunate (n=8954) or placebo (n=8872) suppository by taking the next numbered box, then referred to clinics at which injections could be given. Those with antimalarial injections or negative blood smears before randomisation were excluded, leaving 12 068 patients (6072 artesunate, 5996 placebo) for analysis. Primary endpoints were mortality, assessed 7-30 days later, and permanent disability, reassessed periodically. All investigators were masked to group assignment. Analysis was by intention to treat. This study is registered in all three countries, numbers ISRCTN83979018, 46343627, and 76987662. RESULTS: Mortality was 154 of 6072 artesunate versus 177 of 5996 placebo (2.5%vs 3.0%, p=0.1). Two versus 13 (0.03%vs 0.22%, p=0.0020) were permanently disabled; total dead or disabled: 156 versus 190 (2.6%vs 3.2%, p=0.0484). There was no reduction in early mortality (56 vs 51 deaths within 6 h; median 2 h). In patients reaching clinic within 6 h (median 3 h), pre-referral artesunate had no significant effect on death after 6 h or permanent disability (71/4450 [1.6%] vs 82/4426 [1.9%], risk ratio 0.86 [95% CI 0.63-1.18], p=0.35). In patients still not in clinic after more than 6 h, however, half were still not there after more than 15 h, and pre-referral rectal artesunate significantly reduced death or permanent disability (29/1566 [1.9%] vs 57/1519 [3.8%], risk ratio 0.49 [95% CI 0.32-0.77], p=0.0013). INTERPRETATION: If patients with severe malaria cannot be treated orally and access to injections will take several hours, a single inexpensive artesunate suppository at the time of referral substantially reduces the risk of death or permanent disability. FUNDING: UNICEF/UNDP/World Bank Special Programme for Research and Training in Tropical Diseases (WHO/TDR); WHO Global Malaria Programme (WHO/GMP); Sall Family Foundation; the European Union (QLRT-2000-01430); the UK Medical Research Council; USAID; Irish Aid; the Karolinska Institute; and the University of Oxford Clinical Trial Service Unit (CTSU).

Charunwatthana P, Abul Faiz M, Ruangveerayut R, Maude RJ, Rahman MR, Roberts LJ, Moore K, Bin Yunus E et al. 2009. N-acetylcysteine as adjunctive treatment in severe malaria: a randomized, double-blinded placebo-controlled clinical trial. Crit Care Med, 37 (2), pp. 516-522. | Show Abstract | Read more

OBJECTIVE: Markers of oxidative stress are reported to be increased in severe malaria. It has been suggested that the antioxidant N-acetylcysteine (NAC) may be beneficial in treatment. We studied the efficacy and safety of parenteral NAC as an adjunct to artesunate treatment of severe falciparum malaria. DESIGN: A randomized, double-blind, placebo-controlled trial on the use of high-dose intravenous NAC as adjunctive treatment to artesunate. SETTING: A provincial hospital in Western Thailand and a tertiary referral hospital in Chittagong, Bangladesh. PATIENTS: One hundred eight adult patients with severe falciparum malaria. INTERVENTIONS: Patients were randomized to receive NAC or placebo as an adjunctive treatment to intravenous artesunate. MEASUREMENTS AND MAIN RESULTS: A total of 56 patients were treated with NAC and 52 received placebo. NAC had no significant effect on mortality, lactate clearance times (p = 0.74), or coma recovery times (p = 0.46). Parasite clearance time was increased from 30 hours (range, 6-144 hours) to 36 hours (range, 6-120 hours) (p = 0.03), but this could be explained by differences in admission parasitemia. Urinary F2-isoprostane metabolites, measured as a marker of oxidative stress, were increased in severe malaria compared with patients with uncomplicated malaria and healthy volunteers. Admission red cell rigidity correlated with mortality, but did not improve with NAC. CONCLUSION: Systemic oxidative stress is increased in severe malaria. Treatment with NAC had no effect on outcome in patients with severe falciparum malaria in this setting.

Pongtavornpinyo W, Hastings IM, Dondorp A, White LJ, Maude RJ, Saralamba S, Day NP, White NJ, Boni MF. 2009. Probability of emergence of antimalarial resistance in different stages of the parasite life cycle. Evol Appl, 2 (1), pp. 52-61. | Show Abstract | Read more

Understanding the evolution of drug resistance in malaria is a central area of study at the intersection of evolution and medicine. Antimalarial drug resistance is a major threat to malaria control and directly related to trends in malaria attributable mortality. Artemisinin combination therapies (ACT) are now recommended worldwide as first line treatment for uncomplicated malaria, and losing them to resistance would be a disaster for malaria control. Understanding the emergence and spread of antimalarial drug resistance in the context of different scenarios of antimalarial drug use is essential for the development of strategies protecting ACTs. In this study, we review the basic mechanisms of resistance emergence and describe several simple equations that can be used to estimate the probabilities of de novo resistance mutations at three stages of the parasite life cycle: sporozoite, hepatic merozoite and asexual blood stages; we discuss the factors that affect parasite survival in a single host in the context of different levels of antimalarial drug use, immunity and parasitaemia. We show that in the absence of drug effects, and despite very different parasite numbers, the probability of resistance emerging at each stage is very low and similar in all stages (for example per-infection probability of 10(-10)-10(-9) if the per-parasite chance of mutation is 10(-10) per asexual division). However, under the selective pressure provided by antimalarial treatment and particularly in the presence of hyperparasitaemia, the probability of resistance emerging in the blood stage of the parasite can be approximately five orders of magnitude higher than in the absence of drugs. Detailed models built upon these basic methods should allow us to assess the relative probabilities of resistance emergence in the different phases of the parasite life cycle.

Carrara VI, Zwang J, Ashley EA, Price RN, Stepniewska K, Barends M, Brockman A, Anderson T et al. 2009. Changes in the treatment responses to artesunate-mefloquine on the northwestern border of Thailand during 13 years of continuous deployment. PLoS One, 4 (2), pp. e4551. | Show Abstract | Read more

BACKGROUND: Artemisinin combination treatments (ACT) are recommended as first line treatment for falciparum malaria throughout the malaria affected world. We reviewed the efficacy of a 3-day regimen of mefloquine and artesunate regimen (MAS(3)), over a 13 year period of continuous deployment as first-line treatment in camps for displaced persons and in clinics for migrant population along the Thai-Myanmar border. METHODS AND FINDINGS: 3,264 patients were enrolled in prospective treatment trials between 1995 and 2007 and treated with MAS(3). The proportion of patients with parasitaemia persisting on day-2 increased significantly from 4.5% before 2001 to 21.9% since 2002 (p<0.001). Delayed parasite clearance was associated with increased risk of developing gametocytaemia (AOR = 2.29; 95% CI, 2.00-2.69, p = 0.002). Gametocytaemia on admission and carriage also increased over the years (p = 0.001, test for trend, for both). MAS(3) efficacy has declined slightly but significantly (Hazards ratio 1.13; 95% CI, 1.07-1.19, p<0.001), although efficacy in 2007 remained well within acceptable limits: 96.5% (95% CI, 91.0-98.7). The in vitro susceptibility of P. falciparum to artesunate increased significantly until 2002, but thereafter declined to levels close to those of 13 years ago (geometric mean in 2007: 4.2 nM/l; 95% CI, 3.2-5.5). The proportion of infections caused by parasites with increased pfmdr1 copy number rose from 30% (12/40) in 1996 to 53% (24/45) in 2006 (p = 0.012, test for trend). CONCLUSION: Artesunate-mefloquine remains a highly efficacious antimalarial treatment in this area despite 13 years of widespread intense deployment, but there is evidence of a modest increase in resistance. Of particular concern is the slowing of parasitological response to artesunate and the associated increase in gametocyte carriage.

Maude RJ, Plewes K, Faiz MA, Hanson J, Charunwatthana P, Lee SJ, Tärning J, Yunus EB et al. 2009. Does artesunate prolong the electrocardiograph QT interval in patients with severe malaria? Am J Trop Med Hyg, 80 (1), pp. 126-132. | Show Abstract

Several antimalarials can cause significant prolongation of the electrocardiograph QT interval, which can be associated with an increased risk of potentially lethal ventricular arrhythmias. High doses of artemether and artemotil have been associated with QT prolongation in dogs, raising the possibility of a class effect with the artemisinin derivatives. Serial electrocardiograms were recorded, and QTc interval was calculated before and after administration of artesunate by intravenous injection in patients with severe falciparum malaria in Bangladesh. Of 21 adult patients with severe malaria enrolled, 8 (38%) died. The mean QTc interval was unaffected by bolus intravenous artesunate (2.4 mg/kg). In two patients, the QTc interval exceeded 0.5 seconds, but in both cases, an alternative explanation was plausible. No effect was observed on the JTc or PR interval, QRS width, blood pressure, or heart rate. Intravenous artesunate does not have significant cardiovascular effects in patients with severe falciparum malaria.

McGready R, Stepniewska K, Lindegardh N, Ashley EA, La Y, Singhasivanon P, White NJ, Nosten F. 2009. Erratum:The pharmacokinetics of artemether and lumefantrine in pregnant women with uncomplicated falciparum malaria(European Journal of Clinical Pharmacology (2006) 62 (1021-1031) DOI: 10.1007/s00228-006-0199-7) European Journal of Clinical Pharmacology, 65 (8), pp. 847. | Read more

Medana IM, Day NP, Hien TT, White NJ, Turner GD. 2009. Erythropoietin and its receptors in the brainstem of adults with fatal falciparum malaria. Malar J, 8 (1), pp. 261. | Show Abstract | Read more

BACKGROUND: Facilitation of endogenous neuroprotective pathways, such as the erythropoietin (Epo) pathway, has been proposed as adjuvant treatment strategies in cerebral malaria. Whether different endogenous protein expression levels of Epo or differences in the abundance of its receptor components could account for the extent of structural neuropathological changes or neurological complications in adults with severe malaria was investigated. METHODS: High sensitivity immunohistochemistry was used to assess the frequency, distribution and concordance of Epo and components of its homodimeric and heteromeric receptors, Epo receptor and CD131, within the brainstem of adults who died of severe malaria. The following relationships with Epo and its receptor components were also defined: (i) sequestration and indicators of hypoxia; (ii) vascular damage in the form of plasma protein leakage and haemorrhage; (iii) clinical complications and neuropathological features of severe malaria disease. Brainstems of patients dying in the UK from unrelated non-infectious causes were examined for comparison. RESULTS: The incidence of endogenous Epo in parenchymal brain cells did not greatly differ between severe malaria and non-neurological UK controls at the time of death. However, EpoR and CD131 labelling of neurons was greater in severe malaria compared with non-neurological controls (P = .009). EpoR labelling of vessels was positively correlated with admission peripheral parasite count (P = .01) and cerebral sequestration (P < .0001). There was a strong negative correlation between arterial oxygen saturation and EpoR labelling of glia (P = .001). There were no significant correlations with indicators of vascular damage, neuronal chromatolysis, axonal swelling or vital organ failure. CONCLUSION: Cells within the brainstem of severe malaria patients showed protein expression of Epo and its receptor components. However, the incidence of endogeneous expression did not reflect protection from vascular or neuronal injury, and/or clinical manifestations, such as coma. These findings do not provide support for Epo as an adjuvant neuroprotective agent in adults with severe malaria.

White NJ, Pongtavornpinyo W, Maude RJ, Saralamba S, Aguas R, Stepniewska K, Lee SJ, Dondorp AM, White LJ, Day NP. 2009. Hyperparasitaemia and low dosing are an important source of anti-malarial drug resistance. Malar J, 8 (1), pp. 253. | Show Abstract | Read more

BACKGROUND: Preventing the emergence of anti-malarial drug resistance is critical for the success of current malaria elimination efforts. Prevention strategies have focused predominantly on qualitative factors, such as choice of drugs, use of combinations and deployment of multiple first-line treatments. The importance of anti-malarial treatment dosing has been underappreciated. Treatment recommendations are often for the lowest doses that produce "satisfactory" results. METHODS: The probability of de-novo resistant malaria parasites surviving and transmitting depends on the relationship between their degree of resistance and the blood concentration profiles of the anti-malarial drug to which they are exposed. The conditions required for the in-vivo selection of de-novo emergent resistant malaria parasites were examined and relative probabilities assessed. RESULTS: Recrudescence is essential for the transmission of de-novo resistance. For rapidly eliminated anti-malarials high-grade resistance can arise from a single drug exposure, but low-grade resistance can arise only from repeated inadequate treatments. Resistance to artemisinins is, therefore, unlikely to emerge with single drug exposures. Hyperparasitaemic patients are an important source of de-novo anti-malarial drug resistance. Their parasite populations are larger, their control of the infection insufficient, and their rates of recrudescence following anti-malarial treatment are high. As use of substandard drugs, poor adherence, unusual pharmacokinetics, and inadequate immune responses are host characteristics, likely to pertain to each recurrence of infection, a small subgroup of patients provides the particular circumstances conducive to de-novo resistance selection and transmission. CONCLUSION: Current dosing recommendations provide a resistance selection opportunity in those patients with low drug levels and high parasite burdens (often children or pregnant women). Patients with hyperparasitaemia who receive outpatient treatments provide the greatest risk of selecting de-novo resistant parasites. This emphasizes the importance of ensuring that only quality-assured anti-malarial combinations are used, that treatment doses are optimized on the basis of pharmacodynamic and pharmacokinetic assessments in the target populations, and that patients with heavy parasite burdens are identified and receive sufficient treatment to prevent recrudescence.

Hanson J, Hossain A, Charunwatthana P, Hassan MU, Davis TM, Lam SW, Chubb SA, Maude RJ et al. 2009. Hyponatremia in severe malaria: evidence for an appropriate anti-diuretic hormone response to hypovolemia. Am J Trop Med Hyg, 80 (1), pp. 141-145. | Show Abstract

Although hyponatremia occurs in most patients with severe malaria, its pathogenesis, prognostic significance, and optimal management have not been established. Clinical and biochemical data were prospectively collected from 171 consecutive Bangladeshi adults with severe malaria. On admission, 57% of patients were hyponatremic. Plasma sodium and Glasgow Coma Score were inversely related (r(s) = -0.36, P < 0.0001). Plasma antidiuretic hormone concentrations were similar in hyponatremic and normonatremic patients (median, range: 6.1, 2.3-85.3 versus 32.7, 3.0-56.4 pmol/L; P = 0.19). Mortality was lower in hyponatremic than normonatremic patients (31.6% versus 51.4%; odds ratio [95% confidence interval]: 0.44 [0.23-0.82]; P = 0.01 by univariate analysis). Plasma sodium normalized with crystalloid rehydration from (median, range) 127 (123-140) mmol/L on admission to 136 (128-149) mmol/L at 24 hours (P = 0.01). Hyponatremia in adults with severe malaria is common and associated with preserved consciousness and decreased mortality. It likely reflects continued oral hypotonic fluid intake in the setting of hypovolemia and requires no therapy beyond rehydration.

Stepniewska K, Taylor W, Sirima SB, Ouedraogo EB, Ouedraogo A, Gansané A, Simpson JA, Morgan CC, White NJ, Kiechel JR. 2009. Population pharmacokinetics of artesunate and amodiaquine in African children. Malar J, 8 (1), pp. 200. | Show Abstract | Read more

BACKGROUND: Pharmacokinetic (PK) data on amodiaquine (AQ) and artesunate (AS) are limited in children, an important risk group for malaria. The aim of this study was to evaluate the PK properties of a newly developed and registered fixed dose combination (FDC) of artesunate and amodiaquine. METHODS: A prospective population pharmacokinetic study of AS and AQ was conducted in children aged six months to five years. Participants were randomized to receive the new artesunate and amodiaquine FDC or the same drugs given in separate tablets. Children were divided into two groups of 70 (35 in each treatment arm) to evaluate the pharmacokinetic properties of AS and AQ, respectively. Population pharmacokinetic models for dihydroartemisinin (DHA) and desethylamodiaquine (DeAq), the principal pharmacologically active metabolites of AS and AQ, respectively, and total artemisinin anti-malarial activity, defined as the sum of the molar equivalent plasma concentrations of DHA and artesunate, were constructed using the non-linear mixed effects approach. Relative bioavailability between products was compared by estimating the ratios (and 95% CI) between the areas under the plasma concentration-time curves (AUC). RESULTS: The two regimens had similar PK properties in young children with acute malaria. The ratio of loose formulation to fixed co-formulation AUCs, was estimated as 1.043 (95% CI: 0.956 to 1.138) for DeAq. For DHA and total anti-malarial activity AUCs were estimated to be the same. Artesunate was rapidly absorbed, hydrolysed to DHA, and eliminated. Plasma concentrations were significantly higher following the first dose, when patients were acutely ill, than after subsequent doses when patients were usually afebrile and clinically improved. Amodiaquine was converted rapidly to DeAq, which was then eliminated with an estimated median (range) elimination half-life of 9 (7 to 12) days. Efficacy was similar in the two treatments groups, with cure rates of 0.946 (95% CI: 0.840-0.982) in the AS+AQ group and 0.892 (95% CI: 0.787 - 0.947) in the AS/AQ group. Four out of five patients with PCR confirmed recrudescences received AQ doses < 10 mg/kg. Both regimens were well tolerated. No child developed severe, post treatment neutropaenia (<1,000/muL). There was no evidence of AQ dose related hepatotoxicity, but one patient developed an asymptomatic rise in liver enzymes that was resolving by Day-28. CONCLUSION: The bioavailability of the co-formulated AS-AQ FDC was similar to that of the separate tablets for desethylamodiaquine, DHA and the total anti-malarial activity. These data support the use this new AS-AQ FDC in children with acute uncomplicated falciparum malaria.

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Zwang J, Ashley EA, Karema C, D'Alessandro U, Smithuis F, Dorsey G, Janssens B, Mayxay M et al. 2009. Safety and efficacy of dihydroartemisinin-piperaquine in falciparum malaria: A prospective multi-centre individual patient data analysis PLoS ONE, 4 (7), | Show Abstract | Read more

Background: The fixed dose antimalarial combination of dihydroartemisinin-piperaquine (DP) is a promising new artemisinin-based combination therapy (ACT). We present an individual patient data analysis of efficacy and tolerability in acute uncomplicated falciparum malaria, from seven published randomized clinical trials conducted in Africa and South East Asia using a predefined in-vivo protocol. Comparator drugs were mefloquine-artesunate (MAS3) in Thailand, Myanmar, Laos and Cambodia; artemether-lumefantrine in Uganda; and amodiaquine+sulfadoxine-pyrimethamine and artesunate+amodiaquine in Rwanda. Methods and Findings: In total 3,547 patients were enrolled: 1,814 patients (32% children under five years) received DP and 1,733 received a comparator antimalarial at 12 different sites and were followed for 28-63 days. There was no significant heterogeneity between trials. DP was well tolerated with 1.7% early vomiting. There were less adverse events with DP in children and adults compared to MAS3 except for diarrhea; ORs (95%CI) 2.74 (2.13 to 3.51) and 3.11 (2.31 to 4.18), respectively. DP treatment resulted in a rapid clearance of fever and parasitaemia. The PCR genotype corrected efficacy at Day 28 of DP assessed by survival analysis was 98.7% (95%CI 97.6-99.8). DP was superior to the comparator drugs in protecting against both P.falciparum recurrence and recrudescence (P = 0.001, weighted by site). There was no difference between DP and MAS3 in treating P. vivax co-infections and in suppressing the first relapse (median interval to P. vivax recurrence: 6 weeks). Children under 5 y were at higher risk of recurrence for both infections. The proportion of patients developing gametocytaemia (P = 0.002, weighted by site) and the subsequent gametocyte carriage rates were higher with DP (11/1000 person gametocyte week, PGW) than MAS3 (6/ 1000 PGW, P = 0.001, weighted by site). Conclusions: DP proved a safe, well tolerated, and highly effective treatment of P.falciparum malaria in Asia and Africa, but the effect on gametocyte carriage was inferior to that of MAS3. © 2009 Zwang et al.

Nickerson EK, Hongsuwan M, Limmathurotsakul D, Wuthiekanun V, Shah KR, Srisomang P, Mahavanakul W, Wacharaprechasgul T et al. 2009. Staphylococcus aureus bacteraemia in a tropical setting: patient outcome and impact of antibiotic resistance. PLoS One, 4 (1), pp. e4308. | Show Abstract | Read more

BACKGROUND: Most information on invasive Staphylococcus aureus infections comes from temperate countries. There are considerable knowledge gaps in epidemiology, treatment, drug resistance and outcome of invasive S. aureus infection in the tropics. METHODS: A prospective, observational study of S. aureus bacteraemia was conducted in a 1000-bed regional hospital in northeast Thailand over 1 year. Detailed clinical data were collected and final outcomes determined at 12 weeks, and correlated with antimicrobial susceptibility profiles of infecting isolates. PRINCIPAL FINDINGS: Ninety-eight patients with S. aureus bacteraemia were recruited. The range of clinical manifestations was similar to that reported from temperate countries. The prevalence of endocarditis was 14%. The disease burden was highest at both extremes of age, whilst mortality increased with age. The all-cause mortality rate was 52%, with a mortality attributable to S. aureus of 44%. Methicillin-resistant S. aureus (MRSA) was responsible for 28% of infections, all of which were healthcare-associated. Mortality rates for MRSA and methicillin-susceptible S. aureus (MSSA) were 67% (18/27) and 46% (33/71), respectively (p = 0.11). MRSA isolates were multidrug resistant. Only vancomycin or fusidic acid would be suitable as empirical treatment options for suspected MRSA infection. CONCLUSIONS: S. aureus is a significant pathogen in northeast Thailand, with comparable clinical manifestations and a similar endocarditis prevalence but higher mortality than industrialised countries. S. aureus bacteraemia is frequently associated with exposure to healthcare settings with MRSA causing a considerable burden of disease. Further studies are required to define setting-specific strategies to reduce mortality from S. aureus bacteraemia, prevent MRSA transmission, and to define the burden of S. aureus disease and emergence of drug resistance throughout the developing world.

Dondorp AM, Nosten F, White NJ. 2009. Peginterferon Alfa-2b or Alfa-2a with Ribavirin for Hepatitis C New England Journal of Medicine, 361 (18), pp. 1808-1809. | Read more

Maude RJ, Pontavornpinyo W, Saralamba S, Aguas R, Yeung S, Dondorp AM, Day NP, White NJ, White LJ. 2009. The last man standing is the most resistant: eliminating artemisinin-resistant malaria in Cambodia. Malar J, 8 (1), pp. 31. | Show Abstract | Read more

BACKGROUND: Artemisinin combination therapy (ACT) is now the recommended first-line treatment for falciparum malaria throughout the world. Initiatives to eliminate malaria are critically dependent on its efficacy. There is recent worrying evidence that artemisinin resistance has arisen on the Thai-Cambodian border. Urgent containment interventions are planned and about to be executed. Mathematical modeling approaches to intervention design are now integrated into the field of malaria epidemiology and control. The use of such an approach to investigate the likely effectiveness of different containment measures with the ultimate aim of eliminating artemisinin-resistant malaria is described. METHODS: A population dynamic mathematical modeling framework was developed to explore the relative effectiveness of a variety of containment interventions in eliminating artemisinin-resistant malaria in western Cambodia. RESULTS: The most effective intervention to eliminate artemisinin-resistant malaria was a switch of treatment from artemisinin monotherapy to ACT (mean time to elimination 3.42 years (95% CI 3.32-3.60 years). However, with this approach it is predicted that elimination of artemisinin-resistant malaria using ACT can be achieved only by elimination of all malaria. This is because the various forms of ACT are more effective against infections with artemisinin-sensitive parasites, leaving the more resistant infections as an increasing proportion of the dwindling parasite population. CONCLUSION: Containment of artemisinin-resistant malaria can be achieved by elimination of malaria from western Cambodia using ACT. The "last man standing" is the most resistant and thus this strategy must be sustained until elimination is truly achieved.

White LJ, Maude RJ, Pongtavornpinyo W, Saralamba S, Aguas R, Van Effelterre T, Day NP, White NJ. 2009. The role of simple mathematical models in malaria elimination strategy design. Malar J, 8 (1), pp. 212. | Show Abstract | Read more

BACKGROUND: Malaria has recently been identified as a candidate for global eradication. This process will take the form of a series of national eliminations. Key issues must be considered specifically for elimination strategy when compared to the control of disease. Namely the spread of drug resistance, data scarcity and the adverse effects of failed elimination attempts. Mathematical models of various levels of complexity have been produced to consider the control and elimination of malaria infection. If available, detailed data on malaria transmission (such as the vector life cycle and behaviour, human population behaviour, the acquisition and decay of immunity, heterogeneities in transmission intensity, age profiles of clinical and subclinical infection) can be used to populate complex transmission models that can then be used to design control strategy. However, in many malaria countries reliable data are not available and policy must be formed based on information like an estimate of the average parasite prevalence. METHODS: A simple deterministic model, that requires data in the form of a single estimate of parasite prevalence as an input, is developed for the purpose of comparison with other more complex models. The model is designed to include key aspects of malaria transmission and integrated control. RESULTS: The simple model is shown to have similar short-term dynamic behaviour to three complex models. The model is used to demonstrate the potential of alternative methods of delivery of controls. The adverse effects on clinical infection and spread of resistance are predicted for failed elimination attempts. Since elimination strategies present an increased risk of the spread of drug resistance, the model is used to demonstrate the population level protective effect of multiple controls against this very serious threat. CONCLUSION: A simple model structure for the elimination of malaria is suitable for situations where data are sparse yet strategy design requirements are urgent with the caveat that more complex models, populated with new data, would provide more information, especially in the long-term.

Simpson JA, Jamsen KM, Price RN, White NJ, Lindegardh N, Tarning J, Duffull SB. 2009. Towards optimal design of anti-malarial pharmacokinetic studies. Malar J, 8 (1), pp. 189. | Show Abstract | Read more

BACKGROUND: Characterization of anti-malarial drug concentration profiles is necessary to optimize dosing, and thereby optimize cure rates and reduce both toxicity and the emergence of resistance. Population pharmacokinetic studies determine the drug concentration time profiles in the target patient populations, including children who have limited sampling options. Currently, population pharmacokinetic studies of anti-malarial drugs are designed based on logistical, financial and ethical constraints, and prior knowledge of the drug concentration time profile. Although these factors are important, the proposed design may be unable to determine the desired pharmacokinetic profile because there was no formal consideration of the complex statistical models used to analyse the drug concentration data. METHODS: Optimal design methods incorporate prior knowledge of the pharmacokinetic profile of the drug, the statistical methods used to analyse data from population pharmacokinetic studies, and also the practical constraints of sampling the patient population. The methods determine the statistical efficiency of the design by evaluating the information of the candidate study design prior to the pharmacokinetic study being conducted. RESULTS: In a hypothetical population pharmacokinetic study of intravenous artesunate, where the number of patients and blood samples to be assayed was constrained to be 50 and 200 respectively, an evaluation of varying elementary designs using optimal design methods found that the designs with more patients and less samples per patient improved the precision of the pharmacokinetic parameters and inter-patient variability, and the overall statistical efficiency by at least 50%. CONCLUSION: Optimal design methods ensure that the proposed study designs for population pharmacokinetic studies are robust and efficient. It is unethical to continue conducting population pharmacokinetic studies when the sampling schedule may be insufficient to estimate precisely the pharmacokinetic profile.

Lindegardh N, Hanpithakpong W, Phakdeeraj A, Singhasivanon P, Farrar J, Hien TT, White NJ, Day NP. 2008. Development and validation of a high-throughput zwitterionic hydrophilic interaction liquid chromatography solid-phase extraction-liquid chromatography-tandem mass spectrometry method for determination of the anti-influenza drug peramivir in plasma. J Chromatogr A, 1215 (1-2), pp. 145-151. | Show Abstract | Read more

An assay for the analysis for the quantification of the anti-influenza drug peramivir in human plasma using high-throughput zwitterionic (ZIC) hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction (SPE) in a 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The ZIC-HILIC SPE efficiently removed sources of interference present in the supernatant after protein precipitation of plasma proteins. The main advantage of the ZIC-HILIC SPE sample preparation step was that it allowed load and elution conditions to be optimised to extract only peramivir and minimize co-extraction of lipophilic phospholipids. The method was validated according to published US Food and Drugs Administration guidelines and showed excellent performance. The assay was validated over two calibration ranges (0.952-500 and 50-50,000 ng/mL) to support analysis of peramivir after intra-venous administration. The lower limit of quantification for peramivir in plasma was 1 ng/mL and the upper limit of quantification was 50,000 ng/mL. The within-day and between-day precisions expressed as RSD, were lower than 8% at all tested quality control concentrations and below 11% at the lower limit of quantification. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range.

McGready R, Tan SO, Ashley EA, Pimanpanarak M, Viladpai-Nguen J, Phaiphun L, Wüstefeld K, Barends M et al. 2008. A randomised controlled trial of artemether-lumefantrine versus artesunate for uncomplicated plasmodium falciparum treatment in pregnancy. PLoS Med, 5 (12), pp. e253. | Show Abstract | Read more

BACKGROUND: To date no comparative trials have been done, to our knowledge, of fixed-dose artemisinin combination therapies (ACTs) for the treatment of Plasmodium falciparum malaria in pregnancy. Evidence on the safety and efficacy of ACTs in pregnancy is needed as these drugs are being used increasingly throughout the malaria-affected world. The objective of this study was to compare the efficacy, tolerability, and safety of artemether-lumefantrine, the most widely used fixed ACT, with 7 d artesunate monotherapy in the second and third trimesters of pregnancy. METHODS AND FINDINGS: An open-label randomised controlled trial comparing directly observed treatment with artemether-lumefantrine 3 d (AL) or artesunate monotherapy 7 d (AS7) was conducted in Karen women in the border area of northwestern Thailand who had uncomplicated P. falciparum malaria in the second and third trimesters of pregnancy. The primary endpoint was efficacy defined as the P. falciparum PCR-adjusted cure rates assessed at delivery or by day 42 if this occurred later than delivery, as estimated by Kaplan-Meier survival analysis. Infants were assessed at birth and followed until 1 y of life. Blood sampling was performed to characterise the pharmacokinetics of lumefantrine in pregnancy. Both regimens were very well tolerated. The cure rates (95% confidence interval) for the intention to treat (ITT) population were: AS7 89.2% (82.3%-96.1%) and AL 82.0% (74.8%-89.3%), p = 0.054 (ITT); and AS7 89.7% (82.6%-96.8%) and AL 81.2% (73.6%-88.8%), p = 0.031 (per-protocol population). One-third of the PCR-confirmed recrudescent cases occurred after 42 d of follow-up. Birth outcomes and infant (up to age 1 y) outcomes did not differ significantly between the two groups. The pharmacokinetic study indicated that low concentrations of artemether and lumefantrine were the main contributors to the poor efficacy of AL. CONCLUSION: The current standard six-dose artemether-lumefantrine regimen was well tolerated and safe in pregnant Karen women with uncomplicated falciparum malaria, but efficacy was inferior to 7 d artesunate monotherapy and was unsatisfactory for general deployment in this geographic area. Reduced efficacy probably results from low drug concentrations in later pregnancy. A longer or more frequent AL dose regimen may be needed to treat pregnant women effectively and should now be evaluated. Parasitological endpoints in clinical trials of any antimalarial drug treatment in pregnancy should be extended to delivery or day 42 if it comes later. TRIAL REGISTRATION: Current Controlled Trials ISRCTN86353884.

Stepniewska K, Price RN, Sutherland CJ, Drakeley CJ, von Seidlein L, Nosten F, White NJ. 2008. Plasmodium falciparum gametocyte dynamics in areas of different malaria endemicity. Malar J, 7 (1), pp. 249. | Show Abstract | Read more

BACKGROUND: The aim of this study was to identify and compare factors associated with Plasmodium falciparum gametocyte carriage in three regions of differing malaria endemicity. METHODS: Retrospective data from Thailand, The Gambia and Tanzania were used. The data came from large prospective field-based clinical trials, which investigated gametocyte carriage after different anti-malarial drug treatments. RESULTS: Gametocytaemia was detected during the observation period in 12% of patients (931 out of 7548) in Thailand, 34% (683 out of 2020) in The Gambia, and 31% (430 out of 1400) in Tanzania (p < 0.001). Approximately one third (33%, 680/2044) of the patients with gametocytaemia during the observation period, already had patent gametocytaemia at enrolment (day 0 or day 1): 35% (318/931) in Thailand, 37% (250/683) in The Gambia, 26% (112/430) in Tanzania. Maximum gametocytaemia was usually observed on or before the seventh day after starting treatment (93% in Thailand, 70% in Tanzania and 78% in The Gambia). Lowest gametocyte carriage rates were observed following treatment with artemisinin derivatives, while sulphadoxine-pyrimethamine (SP) was associated with significantly greater development of gametocytaemia than other drug treatments (p < 0.001). The duration of gametocyte carriage was shorter in Thailand by 86% and Tanzania by 65% than in The Gambia. Gametocyte carriage was 27% longer among people presenting with anaemia, and was shorter in duration among patients who received artemisinin derivatives, by 27% in Thailand and by 71% in Tanzania and The Gambia. CONCLUSION: This study confirms the independent association of gametocytaemia with anaemia, and the significantly lower prevalence and duration of gametocyte carriage following treatment with an artemisinin derivative. The large differences in gametocyte carriage rates between regions with different levels of malaria transmission suggest that drug interventions to prevent transmission will have different effects in different places.

Hanpithakpong W, Kamanikom B, Dondorp AM, Singhasivanon P, White NJ, Day NP, Lindegardh N. 2008. A liquid chromatographic-tandem mass spectrometric method for determination of artesunate and its metabolite dihydroartemisinin in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci, 876 (1), pp. 61-68. | Show Abstract | Read more

A bioanalytical method for the analysis of artesunate and its metabolite dihydroartemisinin in human plasma using high throughput solid-phase extraction in the 96-wellplate format and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as RSD, were lower than 7% at all tested concentrations including the lower limit of quantification. Using 50 microl plasma the calibration range was 1.19-728 ng/ml with a limit of detection at 0.5 ng/ml for artesunate and 1.96-2500 ng/ml with a limit of detection at 0.6 ng/ml for dihydroartemisinin. Using 250 microl of plasma sample the lower limit of quantification was decreased to 0.119 ng/ml for artesunate and 0.196 ng/ml dihydroartemisinin. Validation of over-curve samples in plasma ensured that accurate estimation would be possible with dilution if samples went outside the calibration range. The method was free from matrix effects as demonstrated both graphically and quantitatively.

Lindegardh N, Hanpithakpong W, Kamanikom B, Singhasivanon P, Socheat D, Yi P, Dondorp AM, McGready R, Nosten F, White NJ, Day NP. 2008. Major pitfalls in the measurement of artemisinin derivatives in plasma in clinical studies. J Chromatogr B Analyt Technol Biomed Life Sci, 876 (1), pp. 54-60. | Show Abstract | Read more

A bioanalytical method for the analysis of artesunate (ARS) and its metabolite dihydroartemisinin (DHA) in human plasma using protein precipitation and liquid chromatography coupled to positive tandem mass spectroscopy was developed. The method was validated according to published US FDA-guidelines and showed excellent performance. However, when it was applied to clinical pharmacokinetic studies in malaria, variable degradation of the artemisinins introduced an unacceptable large source of error, rendering the assay useless. Haemolytic products related to sample collection and malaria infection degraded the compounds. Addition of organic solvents during sample processing and even low volume addition of the internal standard in an organic solvent caused degradation. A solid phase extraction method avoiding organic solvents eliminated problems arising from haemolysis induced degradation. Plasma esterases mediated only approximately 20% of ex vivo hydrolysis of ARS into DHA. There are multiple sources of major preventable error in measuring ARS and DHA in plasma samples from clinical trials. These various pitfalls have undoubtedly contributed to the large inter-subject variation in plasma concentration profiles and derived pharmacokinetic parameters for these important antimalarial drugs.

Boni MF, Buckee CO, White NJ. 2008. Mathematical models for a new era of malaria eradication. PLoS Med, 5 (11), pp. e231. | Read more

Newton PN, Hampton CY, Alter-Hall K, Teerwarakulpana T, Prakongpan S, Ruangveerayuth R, White NJ, Day NP, Tudino MB, Mancuso N, Fernández FM. 2008. Characterization of "Yaa Chud" Medicine on the Thailand-Myanmar border: selecting for drug-resistant malaria and threatening public health. Am J Trop Med Hyg, 79 (5), pp. 662-669. | Show Abstract

Multidrug-resistant Plasmodium falciparum malaria is a severe public health problem on the Thailand-Myanmar border. Many villagers buy packets of 4-5 mixed medicines ("yaa chud") from shops without medical assessment as their first-line malaria treatment. In 2000-2001 a local researcher purchased 50 yaa chud from 44 shops around Mae Sot, Thailand and Myawaddy, Myanmar (Burma), for his wife who was said to be pregnant with fever and drowsiness. The tablets/capsules were provisionally identified by appearance and active ingredients determined in a subset by using mass and atomic spectrometry. The most frequently detected active ingredients were acetaminophen (22%), chlorpheniramine (13.4%), chloroquine (12.6%), tetracycline/doxycycline (11.4%), and quinine (5.1%). Only seven bags contained potentially curative medicine for malaria. A total of 82% of the bags contained medicines contraindicated in pregnancy. Inappropriate, ineffective antimalarial drugs on the Thailand-Myanmar border are likely to increase malaria morbidity, mortality and health costs and engender the emergence and spread of antimalarial drug resistance.

Borrmann S, Peto T, Snow RW, Gutteridge W, White NJ. 2008. Revisiting the design of phase III clinical trials of antimalarial drugs for uncomplicated Plasmodium falciparum malaria. PLoS Med, 5 (11), pp. e227. | Read more

Bozdech Z, Mok S, Hu G, Imwong M, Jaidee A, Russell B, Ginsburg H, Nosten F et al. 2008. The transcriptome of Plasmodium vivax reveals divergence and diversity of transcriptional regulation in malaria parasites. Proc Natl Acad Sci U S A, 105 (42), pp. 16290-16295. | Show Abstract | Read more

Plasmodium vivax causes over 100 million clinical infections each year. Primarily because of the lack of a suitable culture system, our understanding of the biology of this parasite lags significantly behind that of the more deadly species P. falciparum. Here, we present the complete transcriptional profile throughout the 48-h intraerythrocytic cycle of three distinct P. vivax isolates. This approach identifies strain specific patterns of expression for subsets of genes predicted to encode proteins associated with virulence and host pathogen interactions. Comparison to P. falciparum revealed significant differences in the expression of genes involved in crucial cellular functions that underpin the biological differences between the two parasite species. These data provide insights into the biology of P. vivax and constitute an important resource for the development of therapeutic approaches.

Tan SO, McGready R, Zwang J, Pimanpanarak M, Sriprawat K, Thwai KL, Moo Y, Ashley EA et al. 2008. Thrombocytopaenia in pregnant women with malaria on the Thai-Burmese border. Malar J, 7 (1), pp. 209. | Show Abstract | Read more

BACKGROUND: Haematological changes associated with malaria in pregnancy are not well documented, and have focused predominantly on anaemia. Examined here is thrombocytopaenia in pregnant women infected with Plasmodium falciparum or Plasmodium vivax in a low transmission area on the north-western border of Thailand. METHODS: In this observational study we reviewed the platelet counts from routine complete blood count (CBC) in a cohort of healthy and malaria infected Karen pregnant women attending weekly antenatal clinics. A platelet count of 75,000/microL was the threshold at 2 standard deviations below the mean for healthy pregnant women used to indicate thrombocytopenia. Differences in platelet counts in non-pregnant and pregnant women were compared after matching for age, symptoms, malaria species and parasitaemia. RESULTS: In total 974 pregnant women had 1,558 CBC measurements between February 2004 and September 2006. The median platelet counts (/microL) were significantly lower in patients with an episode of falciparum 134,000 [11,000-690,000] (N = 694) or vivax malaria 184,000 [23,000-891,000] (N = 523) compared to healthy pregnant women 256,000 [64,000-781,000] (N = 255), P < 0.05 for both comparisons. Plasmodium falciparum and P. vivax caused a 34% (95% CI 24-47) and 22% (95% CI 8-36) reduction in platelet count, respectively. Pregnant compared to non pregnant women were at higher risk OR = 2.27 (95%CI 1.16-4.4) P = 0.017, for thrombocytopaenia. Platelets counts were higher in first compared with subsequent malaria infections within the same pregnancy. Malaria associated thrombocytopaenia had a median [range] time for recovery of 7 234567891011121314 days which did not differ by antimalarial treatment (P = 0.86), or species (P = 0.63) and was not associated with active bleeding. CONCLUSION: Pregnant women become more thrombocytopenic than non-pregnant women with acute uncomplicated malaria. Uncomplicated malaria associated thrombocytopaenia is seldom severe. Prompt antimalarial treatment resulted in normalization of platelet counts within a week.

Lee SJ, McGready R, Fernandez C, Stepniewska K, Paw MK, Viladpai-nguen SJ, Thwai KL, Villegas L et al. 2008. Chloroquine pharmacokinetics in pregnant and nonpregnant women with vivax malaria. Eur J Clin Pharmacol, 64 (10), pp. 987-992. | Show Abstract | Read more

PURPOSE: We compared the pharmacokinetics of chloroquine in pregnant and nonpregnant women treated for Plasmodium vivax malaria. METHODS: Twelve pregnant women and 15 nonpregnant women of child-bearing age with acute P. vivax malaria were treated with 25 mg chloroquine base/kg over 3 days on the northwestern border of Thailand. Blood concentrations of chloroquine and desethylchloroquine were measured using hydrophilic interaction liquid chromatography coupled with fluorescence detection. Twenty-five women completed the pharmacokinetic study. RESULTS: Although increasing gestational age was associated with reduced chloroquine AUC0-->infinity, there was no significant difference overall in the pharmacokinetics of chloroquine between pregnant and nonpregnant women. Fever was associated with lower chloroquine AUC0-->infinity values. Desethylchloroquine area under the curve (AUC) values were not significantly affected by pregnancy. CONCLUSIONS: Pregnancy did not significantly affect blood concentrations of chloroquine or its metabolite, desethylchloroquine, in women with P. vivax malaria.

Pukrittayakamee S, Imwong M, Singhasivanon P, Stepniewska K, Day NJ, White NJ. 2008. Effects of different antimalarial drugs on gametocyte carriage in P. vivax malaria. Am J Trop Med Hyg, 79 (3), pp. 378-384. | Show Abstract

The gametocytocidal and asexual stage activities of eight antimalarial and eight antibiotic-containing regimens were evaluated in 349 adult patients with P. vivax malaria. Gametocytemia was found in 63% of patients (22% before and 41% after treatment). The median (range) gametocyte clearance time was 24 hours (range, 2-504 hours) and correlated with asexual parasite clearance time (r = 0.52, P < 0.001). Gametocytemia in vivax malaria was more common in patients with admission parasitemia > 10,000/microL and after treatment with drugs which have weak antimalarial activity, and was also associated with an increased rate of vivax reappearance (29.4% versus 14.1%, P = 0.002). Sexual stage activities corresponded with asexual stage activity for all tested regimens. Treatment with potent antimalarial drugs reduces the transmission potential of P. vivax.

Tarning J, Lindegardh N, Sandberg S, Day NJ, White NJ, Ashton M. 2008. Pharmacokinetics and metabolism of the antimalarial piperaquine after intravenous and oral single doses to the rat. J Pharm Sci, 97 (8), pp. 3400-3410. | Show Abstract | Read more

This study aimed to evaluate the pharmacokinetic properties of piperaquine in the rat after intravenous and oral administration, and to identify and characterize the main piperaquine metabolites in rat plasma, urine, faeces and bile after intravenous administration. Male Sprague-Dawley rats were administered piperaquine as an emulsion orally or as a short-term intravenous infusion. Venous blood for pharmacokinetic evaluation was frequently withdrawn up to 90 h after dose. Urine, bile and faeces were collected after an infusion in rats kept in metabolic cages or in anesthetized rats. Pharmacokinetic characterization was done by compartmental modeling and non-compartmental analysis using WinNonlin. Piperaquine disposition was best described by a 3-compartment model with a rapid initial distribution phase after intravenous administration. The pharmacokinetics of piperaquine was characterized by a low clearance, a large volume of distribution and a long terminal half-life. Piperaquine displayed a low biliary clearance and less than 1% of the total dose was recovered in urine. The absolute oral bioavailability was approximately 50%. The main metabolite after intravenous administration of piperaquine was a carboxylic acid product identical to that reported in humans. The similarity with results in humans indicates the rat to be a suitable species for nonclinical in vivo piperaquine studies.

Dondorp AM, Lee SJ, Faiz MA, Mishra S, Price R, Tjitra E, Than M, Htut Y et al. 2008. The relationship between age and the manifestations of and mortality associated with severe malaria. Clin Infect Dis, 47 (2), pp. 151-157. | Show Abstract | Read more

BACKGROUND: The reported case-fatality rate associated with severe malaria varies widely. Whether age is an independent risk factor is uncertain. METHODS: In a large, multicenter treatment trial conducted in Asia, the presenting manifestations and outcome of severe malaria were analyzed in relation to age. RESULTS: Among 1050 patients with severe malaria, the mortality increased stepwise, from 6.1% in children (age, <10 years) to 36.5% in patients aged >50 years (P<0.001). Compared with adults aged 21-50 years, the decreased risk of death among children (adjusted odds ratio, 0.06; 95% confidence interval, 0.01-0.23; P<0.001) and the increased risk of death among patients aged >50 years (adjusted odds ratio, 1.88; 95% confidence interval, 1.01-3.52; P<0.001) was independent of the variation in presenting manifestations. The incidence of anemia and convulsions decreased with age, whereas the incidence of hyperparasitemia, jaundice, and renal insufficiency increased with age. Coma and metabolic acidosis did not vary with age and were the strongest predictors of a fatal outcome. The number of severity signs at hospital admission also had a strong prognostic value. CONCLUSION: Presenting syndromes in severe malaria depend on age, although the incidence and the strong prognostic significance of coma and acidosis are similar at all ages. Age is an independent risk factor for a fatal outcome of the disease.

Imwong M, Pukrittayakamee S, Pongtavornpinyo W, Nakeesathit S, Nair S, Newton P, Nosten F, Anderson TJ, Dondorp A, Day NP, White NJ. 2008. Gene amplification of the multidrug resistance 1 gene of Plasmodium vivax isolates from Thailand, Laos, and Myanmar. Antimicrob Agents Chemother, 52 (7), pp. 2657-2659. | Show Abstract | Read more

Plasmodium vivax mdr1 gene amplification, quantified by real-time PCR, was significantly more common on the western Thailand border (6 of 66 samples), where mefloquine pressure has been intense, than elsewhere in southeast Asia (3 of 149; P = 0.02). Five coding mutations in pvmdr1, independent of gene amplification, were also found.

Thwaites CL, Yen LM, Cordon SM, Thwaites GE, Loan HT, Thuy TT, White NJ, Soni N, Macdonald IA, Farrar JJ. 2008. Effect of magnesium sulphate on urinary catecholamine excretion in severe tetanus. Anaesthesia, 63 (7), pp. 719-725. | Show Abstract | Read more

Severe tetanus is characterised by muscle spasms and autonomic dysfunction. We recently reported the results of a randomised placebo controlled trial of magnesium sulphate infusions for the treatment of severe tetanus which showed magnesium was associated with improved muscle spasm and cardiovascular control. We hypothesised that magnesium controlled autonomic dysfunction by reducing catecholamine release and thus urinary excretion. Urinary adrenaline and noradrenaline concentrations were measured during the first 24 h of therapy in 180 adults with severe tetanus randomised to treatment with magnesium (n = 92) or placebo (n = 88). Magnesium therapy was associated with lower urinary adrenaline excretion and higher urinary noradrenaline excretion. High urinary adrenaline concentrations were associated with documented autonomic dysfunction. Patients given magnesium had significantly less autonomic dysfunction, required less cardiovascular stabilising drugs, and had lower urinary concentrations of adrenaline. These findings suggest adrenaline is important in the pathophysiology of severe tetanus and magnesium controls autonomic dysfunction by reducing adrenaline release.

Grais RF, Ashley EA, White NJ. 2008. The verbosity epidemic. Science, 320 (5884), pp. 1718. | Read more

White NJ, McGready RM, Nosten FH. 2008. New medicines for tropical diseases in pregnancy: catch-22. PLoS Med, 5 (6), pp. e133. | Read more

Yeung S, Van Damme W, Socheat D, White NJ, Mills A. 2008. Access to artemisinin combination therapy for malaria in remote areas of Cambodia. Malar J, 7 (1), pp. 96. | Show Abstract | Read more

BACKGROUND: Malaria-endemic countries are switching antimalarial drug policy to artemisinin combination therapies (ACTs) and the global community are considering the setting up of a global subsidy mechanism in order to make them accessible and affordable. However, specific interventions may be needed to reach remote at-risk communities and to ensure that they are used appropriately. This analysis documents the coverage with ACTs versus artemisinin monotherapies, and the effectiveness of malaria outreach teams (MOTs) and Village Malaria Workers (VMWs) in increasing access to appropriate diagnosis and treatment with ACTs in Cambodia, the first country to switch national antimalarial drug policy to an ACT of artesunate and mefloquine (A+M) in 2000. METHODS: A cross-sectional survey was carried out in three different types of intervention area: with VMWs, MOTs and no specific interventions. Individuals with a history of fever in the last three weeks were included in the study and completed a questionnaire on their treatment seeking and drug usage behaviour. Blood was taken for a rapid diagnostic test (RDT) and data on the household socio-economic status were also obtained. RESULTS: In areas without specific interventions, only 17% (42/251) of respondents received a biological diagnosis, 8% (17/206) of respondents who received modern drug did so from a public health facility, and only 8% of them (17/210) received A+M. Worryingly, 78% (102/131) of all artemisinin use in these areas was as a monotherapy. However, both the VMW scheme and MOT scheme significantly increased the likelihood of being seen by a trained provider (Adjusted Odds Ratios (AOR) of 148 and 4 respectively) and of receiving A+M (AORs of 2.7 and 7.7 respectively). CONCLUSION: The coverage rates of appropriate diagnosis and treatment of malaria were disappointingly low and the use of artemisinin monotherapy alarmingly high. This reflects the fragmented nature of Cambodia's health system in remote areas and the reliance placed by these communities on informal vendors from whom artemisinin monotherapies are widely available. However VMWs in particular are an effective means of improving access to malaria diagnosis and treatment. The VMW scheme and the social marketing of RDTS and blister-packaged artesunate and mefloquine have both been scaled up nationally. Case management in the public sector has also reportedly improved. Given recent concerns regarding the development of artemisinin drug resistance on the Thai-Cambodia border, the effectiveness of these measures in reducing the use of artemisinin monotherapy needs to be urgently re-evaluated.

Yeung S, Van Damme W, Socheat D, White NJ, Mills A. 2008. Cost of increasing access to artemisinin combination therapy: the Cambodian experience. Malar J, 7 (1), pp. 84. | Show Abstract | Read more

BACKGROUND: Malaria-endemic countries are switching antimalarial drug policy from cheap ineffective monotherapies to artemisinin combination therapies (ACTs) for the treatment of Plasmodium falciparum malaria and the global community are considering setting up a global subsidy to fund their purchase. However, in order to ensure that ACTs are correctly used and are accessible to the poor and remote communities who need them, specific interventions will be necessary and the additional costs need to be considered. METHODS: This paper presents an incremental cost analysis of some of these interventions in Cambodia, the first country to change national antimalarial drug policy to an ACT of artesunate and mefloquine. These costs include the cost of rapid diagnostic tests (RDTs), the cost of blister-packaging the drugs locally and the costs of increasing access to diagnosis and treatment to remote communities through malaria outreach teams (MOTs) and Village Malaria Workers (VMW). RESULTS: At optimum productive capacity, the cost of blister-packaging cost under $0.20 per package but in reality was significantly more than this because of the low rate of production. The annual fixed cost (exclusive of RDTs and drugs) per capita of the MOT and VMW schemes was $0.44 and $0.69 respectively. However because the VMW scheme achieved a higher rate of coverage than the MOT scheme, the cost per patient treated was substantially lower at $5.14 compared to $12.74 per falciparum malaria patient treated. The annual cost inclusive of the RDTs and drugs was $19.31 for the MOT scheme and $11.28 for the VMW scheme given similar RDT positivity rates of around 22% and good provider compliance to test results. CONCLUSION: In addition to the cost of ACTs themselves, substantial additional investments are required in order to ensure that they reach the targeted population via appropriate delivery systems and to ensure that they are used appropriately. In addition, differences in local conditions, in particular the prevalence of malaria and the pre-existing infrastructure, need to be considered in choosing appropriate diagnostic and delivery strategies.

Stepniewska K, White NJ. 2008. Pharmacokinetic determinants of the window of selection for antimalarial drug resistance. Antimicrob Agents Chemother, 52 (5), pp. 1589-1596. | Show Abstract | Read more

The selection and spread of antimalarial drug resistance pose enormous challenges to the health of people living in tropical countries. Most antimalarial drugs are slowly eliminated and so, following treatment in areas of endemicity, provide a gradient of concentrations to which newly acquired parasites are exposed. There is a variable period during which a new blood-stage infection with resistant malaria parasites can emerge from the liver and subsequently produce gametocyte densities sufficient for transmission while reinfection by sensitive parasites is still suppressed. This "window of selection" drives the spread of resistance. We have examined the factors which determine the duration of this window and, thus, the resistance selection pressure. The duration ranges from zero to several months and is dependent on the degree of parasite resistance, the slope of the concentration-effect relationship, and the elimination kinetics of the antimalarial drug. The time at which the window opens and the duration of opening are both linear functions of the terminal elimination half-life. Because of competition from sibling susceptible parasites, the greater risks of extinction with low starting numbers, and opening of the window only when blood concentrations have fallen below the MIC, the window of selection for de novo resistance is narrower than that for resistance acquired elsewhere. The windows were examined for the currently available antimalarials. Drugs with elimination half-lives of less than 1 day, such as the artemisinins and quinine, do not select for resistance during the elimination phase.

White NJ. 2008. Qinghaosu (artemisinin): the price of success. Science, 320 (5874), pp. 330-334. | Show Abstract | Read more

Artemisinin and its derivatives have become essential components of antimalarial treatment. These plant-derived peroxides are unique among antimalarial drugs in killing the young intraerythrocytic malaria parasites, thereby preventing their development to more pathological mature stages. This results in rapid clinical and parasitological responses to treatment and life-saving benefit in severe malaria. Artemisinin combination treatments (ACTs) are now first-line drugs for uncomplicated falciparum malaria, but access to ACTs is still limited in most malaria-endemic countries. Improved agricultural practices, selection of high-yielding hybrids, microbial production, and the development of synthetic peroxides will lower prices. A global subsidy would make these drugs more affordable and available. ACTs are central to current malaria elimination initiatives, but there are concerns that tolerance to artemisinins may be emerging in Cambodia.

White NJ, Stepniewska K, Barnes K, Price RN, Simpson J. 2008. Simplified antimalarial therapeutic monitoring: using the day-7 drug level? Trends Parasitol, 24 (4), pp. 159-163. | Show Abstract | Read more

The blood concentration profiles of most antimalarial drugs vary considerably between patients. The interpretation of antimalarial drug trials evaluating efficacy and effectiveness would be improved considerably if the exposure of the infecting parasite population to the antimalarial drug treatment could be measured. Artemisinin combination treatments are now recommended as first-line drugs for the treatment of falciparum malaria. Measurement of the blood, serum or plasma concentration of the slowly eliminated partner antimalarial drug on day 7 of follow-up is simpler and might be a better determinant of therapeutic response than the area under the concentration-time curve. Measurement of the day-7 drug level should be considered as a routine part of antimalarial drug trials.

Barnes KI, Watkins WM, White NJ. 2008. Antimalarial dosing regimens and drug resistance. Trends Parasitol, 24 (3), pp. 127-134. | Show Abstract | Read more

The contribution of underdosing to antimalarial treatment failure has been underappreciated. Most recommended dosage regimens are based on studies in non-pregnant adult patients. Young children and pregnant women, who bear the heaviest malaria burden, have the highest treatment failure rates. This has been attributed previously to lower immunity, although blood concentrations of many antimalarial drugs are significantly lower in pregnant women and young children than in non-pregnant adults. Nevertheless, there have been no studies of higher dosages. Sub-therapeutic concentrations will certainly contribute to poorer responses to treatment and will fuel the emergence and spread of antimalarial drug resistance. There is an urgent need for studies to optimise antimalarial dosage regimens in infants, young children and pregnant women, both to improve cure rates and to prolong the useful therapeutic lives of antimalarial drugs.

Sachanonta N, Medana IM, Roberts R, Jones M, Day NP, White NJ, Ferguson DJ, Turner GD, Pongponratn E. 2008. Host vascular endothelial growth factor is trophic for Plasmodium falciparum-infected red blood cells. Asian Pac J Allergy Immunol, 26 (1), pp. 37-45. | Show Abstract

Plasmodium falciparum, the protozoan parasite responsible for severe malaria infection, undergoes a complex life cycle. Infected red blood cells (iRBC) sequester in host cerebral microvessels, which underlies the pathology of cerebral malaria. Using immunohistochemistry on post mortem brain samples, we demonstrated positive staining for vascular endothelial growth factor (VEGF) on iRBC. Confocal microscopy of cultured iRBC revealed accumulation of VEGF within the parasitophorous vacuole, expression of host VEGF-receptor 1 and activated VEGF-receptor 2 on the surface of iRBC, but no accumulation of VEGF receptors within the iRBC. Addition of VEGF to parasite cultures had a trophic effect on parasite growth and also partially rescued growth of drug treated parasites. Both these effects were abrogated when parasites were grown in serum-free medium, suggesting a requirement for soluble VEGF receptor. We conclude that P. falciparum iRBC can bind host VEGF-R on the erythrocyte membrane and accumulate host VEGF within the parasitophorous vacuole, which may have a trophic effect on parasite growth.

Tarning J, Ashley EA, Lindegardh N, Stepniewska K, Phaiphun L, Day NP, McGready R, Ashton M, Nosten F, White NJ. 2008. Population pharmacokinetics of piperaquine after two different treatment regimens with dihydroartemisinin-piperaquine in patients with Plasmodium falciparum malaria in Thailand. Antimicrob Agents Chemother, 52 (3), pp. 1052-1061. | Show Abstract | Read more

The population pharmacokinetics of piperaquine in adults and children with uncomplicated Plasmodium falciparum malaria treated with two different dosage regimens of dihydroartemisinin-piperaquine were characterized. Piperaquine pharmacokinetics in 98 Burmese and Karen patients aged 3 to 55 years were described by a two-compartment disposition model with first-order absorption and interindividual random variability on all parameters and were similar with the three- and four-dose regimens. Children had a lower body weight-normalized oral clearance than adults, resulting in longer terminal elimination half-lives and higher total exposure to piperaquine (area under the concentration-time curve from 0 to 63 days [AUC day 0-63]). However, children had lower plasma concentrations in the therapeutically relevant posttreatment prophylactic period (AUC day 3-20) because of smaller body weight-normalized central volumes of distribution and shorter distribution half-lives. Our data lend further support to a simplified once-daily treatment regimen to improve treatment adherence and efficacy and indicate that weight-adjusted piperaquine doses in children may need to be higher than in adults.

Lindegardh N, Annerberg A, White NJ, Day NP. 2008. Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of piperaquine in plasma stable isotope labeled internal standard does not always compensate for matrix effects. J Chromatogr B Analyt Technol Biomed Life Sci, 862 (1-2), pp. 227-236. | Show Abstract | Read more

A bioanalytical method for the analysis of piperaquine in human plasma using off-line solid-phase extraction and liquid chromatography coupled to positive tandem mass spectroscopy has been developed and validated. It was found that a mobile phase with high pH (i.e. 10) led to better sensitivity than mobile phase combinations with low pH (i.e. 2.5-4.5) despite the use of positive electrospray and a basic analyte. The method was validated according to published FDA guidelines and showed excellent performance. The within-day and between-day precisions expressed as R.S.D., were lower than 7% at all tested concentrations (4.5, 20, 400 and 500ng/mL) and below 10% at the lower limit of quantification (LLOQ) (1.5ng/mL). The calibration range was 1.5-500ng/mL with a limit of detection (LOD) at 0.38ng/mL. Validation of over-curve samples ensured that it would be possible with dilution if samples went outside the calibration range. Matrix effects were thoroughly evaluated both graphically and quantitatively. Matrix effects originating from the sample clean-up (i.e. solid-phase extraction) procedure rather than the plasma background were responsible for the ion suppression seen in this study. Salts remaining from the buffers used in the solid-phase extraction suppressed the signals for both piperaquine and its deuterated internal standard. This had no effect on the quantification of piperaquine. Triethylamine residues remaining after evaporation of the solid-phase extraction eluate were found to suppress the signals for piperaquine and its deuterated internal standard differently. It was found that this could lead to an underestimation of the true concentration with 50% despite the use of a deuterated internal standard.

Chierakul W, Tientadakul P, Suputtamongkol Y, Wuthiekanun V, Phimda K, Limpaiboon R, Opartkiattikul N, White NJ, Peacock SJ, Day NP. 2008. Activation of the coagulation cascade in patients with leptospirosis. Clin Infect Dis, 46 (2), pp. 254-260. | Show Abstract | Read more

BACKGROUND: Disseminated intravascular coagulation (DIC) is common among patients with sepsis. Leptospirosis is an important cause of sepsis in tropical areas, and pulmonary hemorrhage associated with thrombocytopenia is the major cause of death, but the coagulopathy in severe leptospirosis has not been further characterized. The aim of this study was to evaluate coagulation factors and the presence of DIC in patients with leptospirosis in northeast Thailand. METHODS: We measured plasma concentrations of fibrinogen, D-dimer, thrombi