register interest

Professor Adrianus Dondorp

Research Area: Global Health
Scientific Themes: Tropical Medicine & Global Health
Web Links:
Difference in parasite clearance rates after oral artesunate in patients with uncomplicated falciparum malaria in Northwestern Thailand (Wang Pha) compared to Western Cambodia (Pailin)
(N Engl J Med. 2009 Jul 30;361(5):455-67)

Difference in parasite clearance rates after oral artesunate in patients with uncomplicated ...

Survival curves of (mainly adult) patients with severe malaria treated with intravenous quinine versus artesunate (SEAQUAMAT trial; n=1461; Lancet 2005 Aug;366(9487):717-25).

Survival curves of (mainly adult) patients with severe malaria treated with intravenous quinine ...

Relation between the calculated total body Plasmodium falciparum biomass and severity of disease. Parasite biomass was derived from the plasma PfHRP2 concentration, a protein released in discrete amounts at the moment of schizont rupture (PLoS Med 2005 Aug;2(8):e204)

Relation between the calculated total body Plasmodium falciparum biomass and severity of disease. ...

Since November 2000, I work at the Mahidol-Oxford Tropical Medicine Research Unit in Bangkok, Thailand, where I am currently the Deputy Director and Head of Malaria Research.

Our research unit studies a diversity of aspects of infectious (and other) diseases that are causing significant morbidity and mortality in the tropics. For this purpose we have a large network of clinical sites in both Africa and Asia, including Thailand, Bangladesh, India, Cambodia, Mozambique and elsewhere.

My main research interests include the pathophysiology and treatment of severe malaria, antimalarial drug resistance and improvement of intensive care practice in developing countries. We showed in a large multinational trial in SE Asia in 1461 mainly adult patients that parenteral artesunate is superior to quinine in preventing death; an even larger trial comparing the same drugs in African children with severe malaria is under way. Studies on the microcirculation in patients with severe malaria have revealed the likely contribution of red cell rigidity to the impairment of microcirculatory flow. We were the first to apply a new method to visualise obstruction of the microcirculation in vivo in patients with severe malaria. We also developed a method to calculate the sequestered parasite biomass from plasma PfHRP2, a protein released by the parasite and showed that yet to be identified organic acids other than lactate are an important contributor to the metabolic acidosis in severe malaria and have strong prognostic significance. Our team was the first to perform a comparative trial which unambiguously showed resistance of P. falciparum to artemisinins in Western Cambodia and in collaboration with other international groups are now working on further characterization of this resistance phenotype and identify molecular determinants of artemisinin resistance. We are also involved in finding new treatments for these patients and in the containment efforts against this serious threat to global malaria control.

In addition to this focus on malaria, we are coordinating a dedicated effort to improve intensive care medicine in developing countries in Asia, through teaching as well as the systematic implementation of cost-effective interventions and their assessment. My vision is that true collaborations with enthousiastic and like minded doctors and researchers from all countries where we are active help to identify important research questions, improves the quality of research, and builds capacity as an inherent side effect. Our unit has unique expertise and possibilities to conduct research from bench to bedside, and is in the centre of an extensive network enabling to conduct large multinational trials when definite answers are sought for important research questions aimed at improving treatment and reducing morbidity and mortality of malaria and other diseases.

There are no collaborations listed for this principal investigator.

Thwaites CL, Lundeg G, Dondorp AM, sepsis in resource-limited settings–expert consensus recommendations group of the European Society of Intensive Care Medicine (ESICM) and the Mahidol-Oxford Research Unit (MORU) in Bangkok, Thailand. 2016. Recommendations for infection management in patients with sepsis and septic shock in resource-limited settings. Intensive Care Med, pp. 1-3. | Read more

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.

Beane A, Stephens T, Silva AP, Welch J, Sigera C, De Alwis S, Athapattu P, Dharmagunawardene D et al. 2016. A sustainable approach to training nurses in acute care skills in a resource limited setting (Network for Intensive Care Skills Training, NICST). Resuscitation, 101 pp. e1-e2. | 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.

Leang R, Canavati SE, Khim N, Vestergaard LS, Borghini Fuhrer I, Kim S, Denis MB, Heng P et al. 2016. Efficacy and Safety of Pyronaridine-Artesunate for Treatment of Uncomplicated Plasmodium falciparum Malaria in Western Cambodia. Antimicrob Agents Chemother, 60 (7), pp. 3884-3890. | Show Abstract | Read more

Pyronaridine-artesunate efficacy for the treatment of uncomplicated Plasmodium falciparum malaria was assessed in an area of artemisinin resistance in western Cambodia. This nonrandomized, single-arm, observational study was conducted between 2014 and 2015. Eligible patients were adults or children with microscopically confirmed P. falciparum infection and fever. Patients received pyronaridine-artesunate once daily for 3 days, dosed according to body weight. The primary outcome was an adequate clinical and parasitological response (ACPR) on day 42, estimated by using Kaplan-Meier analysis, PCR adjusted to exclude reinfection. One hundred twenty-three patients were enrolled. Day 42 PCR-crude ACPRs were 87.2% (95% confidence interval [CI], 79.7 to 92.6%) for the overall study, 89.8% (95% CI, 78.8 to 95.3%) for Pursat, and 82.1% (95% CI, 68.4 to 90.2%) for Pailin. Day 42 PCR-adjusted ACPRs were 87.9% (95% CI, 80.6 to 93.2%) for the overall study, 89.8% (95% CI, 78.8 to 95.3%) for Pursat, and 84.0% (95% CI, 70.6 to 91.7%) for Pailin (P = 0.353 by a log rank test). Day 28 PCR-crude and -adjusted ACPRs were 93.2% (95% CI, 82.9 to 97.4%) and 88.1% (95% CI, 75.3 to 94.5%) for Pursat and Pailin, respectively. A significantly lower proportion of patients achieved day 3 parasite clearance in Pailin (56.4% [95% CI, 43.9 to 69.6%]) than in Pursat (86.7% [95% CI, 76.8 to 93.8%]; P = 0.0019). Fever clearance was also extended at Pailin versus Pursat (P < 0.0001). Most patients (95.9% [116/121]) harbored P. falciparum kelch13 C580Y mutant parasites. Pyronaridine-artesunate was well tolerated; mild increases in hepatic transaminase levels were consistent with data from previous reports. Pyronaridine-artesunate efficacy was below the World Health Organization-recommended threshold at day 42 for medicines with a long half-life (90%) for first-line treatment of P. falciparum malaria in western Cambodia despite high efficacy elsewhere in Asia and Africa. (This study has been registered at ClinicalTrials.gov under registration number NCT02389439.).

Dondorp AM, Iyer SS, Schultz MJ. 2016. Critical Care in Resource-Restricted Settings. JAMA, 315 (8), pp. 753-754. | Read more

Sigera PC, Tunpattu TM, Jayashantha TP, De Silva AP, Athapattu PL, Dondorp A, Haniffa R. 2016. National Profile of Physical Therapists in Critical Care Units of Sri Lanka: Lower Middle-Income Country. Phys Ther, 96 (7), pp. 933-939. | Show Abstract | Read more

BACKGROUND: The availability and role of physical therapists in critical care is variable in resource-poor settings, including lower middle-income countries. OBJECTIVE: The aim of this study was to determine: (1) the availability of critical care physical therapist services, (2) the equipment and techniques used and needed, and (3) the training and continuous professional development of physical therapists. METHODS: All physical therapists working in critical care units (CCUs) of state hospitals in Sri Lanka were contacted. The study tool used was an interviewer-administered telephone questionnaire. RESULTS: The response rate was 100% (N=213). Sixty-one percent of the physical therapists were men. Ninety-four percent of the respondents were at least diploma holders in physical therapy, and 6% had non-physical therapy degrees. Most (n=145, 68%) had engaged in some continuous professional development in the past year. The majority (n=119, 56%) attended to patients after referral from medical staff. Seventy-seven percent, 98%, and 96% worked at nights, on weekends, and on public holidays, respectively. Physical therapists commonly perform manual hyperinflation, breathing exercises, manual airway clearance techniques, limb exercises, mobilization, positioning, and postural drainage in the CCUs. Lack of specialist training, lack of adequate physical therapy staff numbers, a heavy workload, and perceived lack of infection control in CCUs were the main difficulties they identified. LIMITATIONS: Details on the proportions of time spent by the physical therapists in the CCUs, wards, or medical departments were not collected. CONCLUSIONS: The availability of physical therapist services in CCUs in Sri Lanka, a lower middle-income country, was comparable to that in high-income countries, as per available literature, in terms of service availability and staffing, although the density of physical therapists remained very low, critical care training was limited, and resource limitations to physical therapy practices were evident.

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.

Herdman MT, Maude RJ, Chowdhury MS, Kingston HW, Jeeyapant A, Samad R, Karim R, Dondorp AM, Hossain MA. 2016. The Relationship between Poverty and Healthcare Seeking among Patients Hospitalized with Acute Febrile Illnesses in Chittagong, Bangladesh. PLoS One, 11 (4), pp. e0152965. | Show Abstract | Read more

Delays in seeking appropriate healthcare can increase the case fatality of acute febrile illnesses, and circuitous routes of care-seeking can have a catastrophic financial impact upon patients in low-income settings. To investigate the relationship between poverty and pre-hospital delays for patients with acute febrile illnesses, we recruited a cross-sectional, convenience sample of 527 acutely ill adults and children aged over 6 months, with a documented fever ≥38.0°C and symptoms of up to 14 days' duration, presenting to a tertiary referral hospital in Chittagong, Bangladesh, over the course of one year from September 2011 to September 2012. Participants were classified according to the socioeconomic status of their households, defined by the Oxford Poverty and Human Development Initiative's multidimensional poverty index (MPI). 51% of participants were classified as multidimensionally poor (MPI>0.33). Median time from onset of any symptoms to arrival at hospital was 22 hours longer for MPI poor adults compared to non-poor adults (123 vs. 101 hours) rising to a difference of 26 hours with adjustment in a multivariate regression model (95% confidence interval 7 to 46 hours; P = 0.009). There was no difference in delays for children from poor and non-poor households (97 vs. 119 hours; P = 0.394). Case fatality was 5.9% vs. 0.8% in poor and non-poor individuals respectively (P = 0.001)-5.1% vs. 0.0% for poor and non-poor adults (P = 0.010) and 6.4% vs. 1.8% for poor and non-poor children (P = 0.083). Deaths were attributed to central nervous system infection (11), malaria (3), urinary tract infection (2), gastrointestinal infection (1) and undifferentiated sepsis (1). Both poor and non-poor households relied predominantly upon the (often informal) private sector for medical advice before reaching the referral hospital, but MPI poor participants were less likely to have consulted a qualified doctor. Poor participants were more likely to attribute delays in decision-making and travel to a lack of money (P<0.001), and more likely to face catastrophic expenditure of more than 25% of monthly household income (P<0.001). We conclude that multidimensional poverty is associated with greater pre-hospital delays and expenditure in this setting. Closer links between health and development agendas could address these consequences of poverty and streamline access to adequate healthcare.

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.

Canavati SE, Lawford HL, Fatunmbi BS, Lek D, Top-Samphor N, Leang R, Dondorp AM, Huy R, Kazadi WM. 2016. Establishing research priorities for malaria elimination in the context of the emergency response to artemisinin resistance framework-the Cambodian approach. Malar J, 15 (1), pp. 120. | Show Abstract | Read more

BACKGROUND: Countries of the greater Mekong subregion have made a transition from malaria control to an aim for falciparum and vivax malaria elimination. The elimination of falciparum malaria will have to be achieved against a background of increasing artemisinin and multi-drug resistance. This ambitious goal requires an operational research (OR) agenda that addresses the dynamic challenges encountered on the path to elimination, which will need to be flexible and developed in close relation with the cambodian national programme for parasitology, entomology and malaria control (CNM). In Cambodia, a number of meetings with stakeholders were convened by the CNM and emergency response to artemisinin resistance (ERAR) hub, producing an initial list of priority OR topics. The process and outcome of these meetings are described, which could serve as a template for other countries in the region. METHODS: A landscaping exercise was conducted to gather all past, on-going and planned malaria focussed OR activities conducted by the cambodian research consortium in Cambodia and categorized according to research theme. The six themes included (1) malaria epidemiology, surveillance and response, (2) malaria case management, (3) malaria vector control, (4) malaria behavioural issues, (5) malaria clinical studies, and (6) other vector-borne diseases (dengue, neglected tropical diseases, soil-transmitted helminths). The different themes were discussed in small focus groups, which made an initial prioritization list which was then presented to a plenary group for further discussion. This produced a list of research questions ranked according to priority. RESULTS: OR priorities produced by the thematic groups were discussed in the plenary meeting and given a priority score by group voting. A list of 17 OR questions were developed, finalized and listed, which included questions on surveillance, active case detection and treatment efficacy. CONCLUSION: This paper describes ERAR's work on supporting Cambodia's transition to malaria elimination by identifying national operational research priorities. ERAR has initiated and currently plays a critical role in the development of country specific research agendas for malaria elimination. The first example of this has been the described exercise in Cambodia, which could serve a template for setting OR priorities in the wider region.

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.

Canavati SE, Lawford HL, Fatunmbi BS, Lek D, Leang R, Top Samphor N, Dondorp AM, Huy R, Kazadi WM. 2016. The Cambodia Research Consortium: expediting research for malaria elimination with the emergency response to artemisinin resistance framework. Malar J, 15 (1), pp. 5. | Show Abstract | Read more

This commentary offers insight into how to best address barriers that may hinder the translation of malaria research findings into policy. It also proposes viable methods of implementing these policies in Cambodia. Currently, a wide range of malaria research is being conducted by in-country stakeholders, including Cambodia's National Programme for Parasitology, Entomology and Malaria Control's (CNM), non-governmental organizations, and academic institutions. Coordinating research amongst these partners, as well as within the Ministry of Health, is a challenge. Results are rarely disseminated widely and seldom inform programme and policy decisions. CNM and its research partners have severely limited access to each other's databases. This lack of accessibility, timeliness, engagement and cooperation between CNM and its partners greatly impacts overall research efficiency in this field, and is stifling innovation both within and beyond CNM. Cambodia has set a goal to eradicate all forms of malaria by 2030. As countries approach the elimination phase, there is a greater need for sharing research-generated evidence amongst partners, in order to ensure that appropriate and impactful activities are conducted. The Cambodian Research Consortium was established to serve as a framework for partners, stakeholders and researchers to share research projects, information and results, and to promote the goals of CNM. The sharing of malaria research results will help to inform prevention, control and elimination activities in the country. It will also determine and address the country's operational research needs, and could potentially become a framework model to be used in other countries aiming to transition from malaria control to elimination.

Wihokhoen B, Dondorp AM, Turner P, Woodrow CJ, Imwong M. 2016. Use of Blood Smears and Dried Blood Spots for Polymerase Chain Reaction-Based Detection and Quantification of Bacterial Infection and Plasmodium falciparum in Severely Ill Febrile African Children. Am J Trop Med Hyg, 94 (2), pp. 322-326. | Show Abstract | Read more

Molecular approaches offer a means of testing archived samples stored as dried blood spots in settings where standard blood cultures are not possible. Peripheral blood films are one suggested source of material, although the sensitivity of this approach has not been well defined. Thin blood smears and dried blood spots from a severe pediatric malaria study were assessed using specific polymerase chain reaction (PCR) primers to detect non-typhoidal Salmonella (NTS; MisL gene), Streptococcus pneumoniae (lytA), and Plasmodium falciparum (18S rRNA). Of 16 cases of NTS and S. pneumoniae confirmed on blood culture, none were positive by PCR using DNA extracts from blood films or dried blood spots. In contrast, four of 36 dried blood spots and two of 178 plasma samples were PCR positive for S. pneumoniae, despite negative bacterial blood cultures, suggesting false positives. Quantitative assessment revealed that the effective concentration of P. falciparum DNA in blood films was three log orders of magnitude lower than for dried blood spots. The P. falciparum kelch13 gene could not be amplified from blood films. These findings question the value of blood PCR-based approaches for detection of NTS and S. pneumoniae, and show that stored blood films are an inefficient method of studying P. falciparum.

Govindasamy G, Barber BE, Ghani SA, William T, Grigg MJ, Borooah S, Dhillon B, Dondorp AM, Yeo TW, Anstey NM, Maude RJ. 2016. Retinal Changes in Uncomplicated and Severe Plasmodium knowlesi Malaria. J Infect Dis, 213 (9), pp. 1476-1482. | Show Abstract | Read more

BACKGROUND: Plasmodium knowlesicauses severe malaria, but its pathogenesis is poorly understood. Retinal changes provide insights into falciparum malaria pathogenesis but have not been studied in knowlesi malaria. METHODS: An observational study was conducted in Malaysian adults hospitalized with severe (n = 20) and nonsevere (n = 24) knowlesi malaria using indirect ophthalmoscopy (n = 44) and fundus photography (n = 29). RESULTS: The patients' median age was 44 years (range, 18-74 years). No coma or deaths occurred. Photography detected retinal changes in 11 of 12 patients (92%) with severe and 14 of 17 (82%) with nonsevere knowlesi malaria. Nonspecific retinal whitening occurred in 3 (35%) and 5 (29%) patients with severe and nonsevere disease, respectively; hemorrhages in 2 (17%) and 3 (18%); loss of retinal pigment epithelium in 1 (8%) and 4 (24%); and drusen in 9 (71%) and 12 (75%). All changes were mild, with no significant differences between severe and nonsevere disease. Patients with retinal hemorrhages had lower platelet counts than those without (median, 22 vs 43 × 10(9)/L;P= .04). CONCLUSIONS: The paucity of specific retinal findings associated with disease severity in knowlesi malaria contrasts with the retinopathy of severe adult falciparum malaria with and without coma, suggesting that falciparum-like microvascular sequestration in the brain is not a major component in severe knowlesi malaria pathogenesis.

de Haan F, Onyamboko MA, Fanello CI, Woodrow CJ, Lubell Y, Boon WP, Dondorp AM. 2015. Exploring health practitioners' acceptability of a prospective semi-quantitative pfHRP2 device to define severe malaria in the Democratic Republic of Congo. Malar J, 14 (1), pp. 503. | Show Abstract | Read more

BACKGROUND: A rapid diagnostic tool is being developed to discern severely ill children with severe malaria from children who are ill with alternative febrile diseases but have coincidental peripheral blood parasitaemia. The device semi-quantitatively measures plasma pfHRP2 and has the potential to reduce mortality in children with severe febrile illnesses by improving diagnosis. The aim of this study is to identify contributing and inhibiting factors that affect healthcare practitioners' acceptability of this prospective diagnostic device in a high malaria transmission setting in the Democratic Republic of Congo. METHODS: Data were collected qualitatively by conducting semi-structured interviews with a purposeful sample of health professionals in Kinshasa, capital of Democratic Republic of Congo. In total, 11 interviews were held with professionals at four different institutes. RESULTS: Four key findings emerged: (1) Congolese practitioners perceive the semi-quantitative pfHRP2 device as a welcome intervention as they recognize the limited reliability of their current diagnostic and therapeutic approaches to severe febrile illnesses; (2) compatibility of the semi-quantitative pfHRP2 device with clinical equipment and competences of Congolese health practitioners is considered to be limited, especially in rural settings; (3) a formal training programme is crucial for correct understanding and application of the semi-quantitative pfHRP2 device; and, (4) provision of evidence to practitioners, and support from health authorities would be important to establish confidence in the semi-quantitative pfHRP2 device. CONCLUSIONS: Congolese practitioners perceive the prospective semi-quantitative pfHRP2 device as a welcome addition to their clinical equipment. The device could improve current diagnostic work-up of severe febrile illness, which might consequently improve treatment choices. However, despite this recognized potential, several hurdles and drivers need to be taken into account when implementing this device in DR Congo.

Abreha T, Alemayehu B, Assefa A, Awab GR, Baird JK, Bezabih B, Cheah PY, Day NP et al. 2015. Improving the radical cure of vivax malaria (IMPROV): a study protocol for a multicentre randomised, placebo-controlled comparison of short and long course primaquine regimens BMC INFECTIOUS DISEASES, 15 (1), | Show Abstract | Read more

© 2015 The IMPROV Study Group.Background: Plasmodium vivax malaria is a major cause of morbidity and recognised as an important contributor to mortality in some endemic areas. The current recommended treatment regimen for the radical cure of P. vivax includes a schizontocidal antimalarial, usually chloroquine, combined with a 14 day regimen of primaquine. The long treatment course frequently results in poor adherence and effectiveness. Shorter courses of higher daily doses of primaquine have the potential to improve adherence and thus effectiveness without compromising safety. The proposed multicentre randomised clinical trial aims to provide evidence across a variety of endemic settings on the safety and efficacy of high dose short course primaquine in glucose-6-phosphate-dehydrogenase (G6PD) normal patients. Design: This study is designed as a placebo controlled, double blinded, randomized trial in four countries: Indonesia, Vietnam, Afghanistan and Ethiopia. G6PD normal patients diagnosed with vivax malaria are randomized to receive either 7 or 14 days high dose primaquine or placebo. G6PD deficient (G6PDd) patients are allocated to weekly primaquine doses for 8 weeks. All treatment is directly observed and recurrent episodes are treated with the same treatment than allocated at the enrolment episode. Patients are followed daily until completion of treatment, weekly until 8 weeks and then monthly until 1 year after initiation of the treatment. The primary endpoint is the incidence rate (per person year) of symptomatic recurrent P. vivax parasitaemia over 12 months of follow-up, for all individuals, controlling for site, comparing the 7 versus 14-day primaquine treatment arms. Secondary endpoints are other efficacy measures such as incidence risk at different time points. Further endpoints are risks of haemolysis and severe adverse events. Discussion: This study has been approved by relevant institutional ethics committees in the UK and Australia, and all participating countries. Results will be disseminated to inform P. vivax malaria treatment policy through peer-reviewed publications and academic presentations. Findings will contribute to a better understanding of the risks and benefits of primaquine which is crucial in persuading policy makers as well as clinicians of the importance of radical cure of vivax malaria, contributing to decreased transmission and a reduce parasite reservoir. Trial registration: ClinicalTrials.gov Identifier: NCT01814683. Registered March 18, 2013

Plewes K, Maude RJ, Ghose A, Dondorp AM. 2015. Severe falciparum malaria complicated by prolonged haemolysis and rhinomaxillary mucormycosis after parasite clearance: a case report. BMC Infect Dis, 15 (1), pp. 555. | Show Abstract | Read more

BACKGROUND: Severe falciparum malaria may be complicated by prolonged haemolysis and recurrent fever after parasite clearance. However, their respective etiologies are unclear and challenging to diagnose. We report the first case of severe falciparum malaria followed by prolonged haemolytic anaemia and rhinomaxillary mucormycosis in a previously healthy adult male. CASE PRESENTATION: A 30-year old Bangladeshi man was admitted with severe falciparum malaria complicated by hyperlactataemia and haemoglobinuria. Prior to admission he was treated with intravenous quinine and upon admission received intravenous artesunate and empiric ceftriaxone. Thirty hours later the peripheral parasitaemia cleared with resolution of fever and haemoglobinuria. Despite parasite clearance, on day 3 the patient developed recurrent fever and acute haemolytic anaemia requiring seven blood transfusions over six days with no improvement of his haemoglobin or haemoglobinuria. On day 10, he was treated with high-dose dexamethasone and meropenem with discontinuation of the ceftriaxone. Two days later the haemoglobinuria resolved. Ceftriaxone-induced haemolysis was the suspected final diagnosis. On day 16, the patient had progressively worsening right-sided facial pain and swelling; a necrotic ulceration of the hard palate was observed. Rhinomaxillary mucormycosis was diagnosed supported by microscopy findings. The patient initially responded to treatment with urgent surgical debridement, itraconazole, followed by two weeks of amphotericin B deoxycholate, however was subsequently lost to follow up. CONCLUSIONS: This case highlights the range of potential alternative aetiologies of acute, prolonged haemolysis and recurrent fever following parasite clearance in severe falciparum malaria. It emphasizes the importance of a high degree of suspicion for alternative causes of haemolysis in order to avoid unnecessary treatments, including blood transfusion and steroids. It is critical to consider and identify common invasive bacterial and rare opportunistic co-infections as a cause of fever in severe malaria patients remaining febrile after parasite clearance to promote antimicrobial stewardship and prompt emergency care.

Stephens T, Beane A, De Silva AP, Welch J, Sigera C, De Alwis S, Athapattu P, Peiris L et al. 2015. Capacity building for critical care skills training provision in resource limited settings: the nursing intensive care skills training (nicst) project. Intensive Care Med Exp, 3 (Suppl 1), pp. A444. | Read more

Beane A, Stephens T, De Silva AP, Adikaram M, De Alwis S, Athapattu P, Sigera C, Peiris L et al. 2015. A collaborative approach to training ward nurses in acute care skills in resource limited settings: the nursing intensive care skills training (nicts) project. Intensive Care Med Exp, 3 (Suppl 1), pp. A445. | Read more

Imwong M, Jindakhad T, Kunasol C, Sutawong K, Vejakama P, Dondorp AM. 2015. An outbreak of artemisinin resistant falciparum malaria in Eastern Thailand. Sci Rep, 5 pp. 17412. | Show Abstract | Read more

Artemisinin resistant falciparum malaria is an increasing problem in Southeast Asia, but has not been associated with increased transmission of the disease, yet. During a recent outbreak in 2014 in Ubon Ratchatani, Eastern Thailand, parasites from 101 patients with falciparum malaria were genotyped for antimalarial drug resistance markers. Mutations in the Kelch13 marker for artemisinin resistance were present in 93% of samples, mainly C580Y from 2 major clusters as identified by microsatellite typing. Resistance markers for antifolates and chloroquine were also highly prevalent. Most strains (91%) carried single copy number PfMDR1, suggesting sustained sensitivity to mefloquine, the partner drug in the local first-line artemisinin combination therapy (ACT). The high prevalence of artemisinin resistance in this recent malaria outbreak suggests but does not prove a causative role in increased transmission. Careful monitoring of ACT efficacy and additional genetic epidemiological studies are warranted to guide the public health response to the outbreak.

Cheeseman IH, Miller B, Tan JC, Tan A, Nair S, Nkhoma SC, De Donato M, Rodulfo H et al. 2016. Population Structure Shapes Copy Number Variation in Malaria Parasites. Mol Biol Evol, 33 (3), pp. 603-620. | Show Abstract | Read more

If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen.

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.

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.

Abdulla S, Ashley EA, Bassat Q, Bethell D, Bjorkman A, Borrmann S, D'Alessandro U, Dahal P et al. 2015. Baseline data of parasite clearance in patients with falciparum malaria treated with an artemisinin derivative: an individual patient data meta-analysis MALARIA JOURNAL, 14 (1), | Read more

Sinha I, Ekapirat N, Dondorp AM, Woodrow CJ. 2015. Use of a rapid test to assess plasma Plasmodium falciparum HRP2 and guide management of severe febrile illness. Malar J, 14 (1), pp. 362. | Show Abstract | Read more

BACKGROUND: Plasma Plasmodium falciparum histidine-rich protein-2 (PfHRP2) is the most accurate biomarker for severe malaria, but its measurement by ELISA has been considered too unwieldy to incorporate into clinical management. METHODS: Plasma samples covering a wide range of PfHRP2 concentrations were applied to rapid diagnostic tests (RDTs). RDTs were read by eye and digital capture, and PfHRP2 concentrations were measured via serial dilution with results compared to ELISA readings. RESULTS: The Paracheck (®) brand showed the strongest correlation (r(2) = 0.963) as well as the lowest inter-observer variability (combined kappa across band intensities for three observers = 0.938). Plasma PfHRP2 measurement via serial dilution showed minimal bias compared to ELISA and acceptable limits of agreement. Three different dilutions of a well characterized set of admission samples from uncomplicated and severe malaria patients studied in a low transmission setting gave an area under the receiver operator characteristic curve of 0.844 in terms of identifying severe malaria. CONCLUSIONS: These studies show that plasma PfHRP2 can be assessed via a single RDT, with application of a plasma dilution of 1:5 or 1:10 providing useful diagnostic information to assist in patient management or clinical trial inclusion.

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.

Tanner M, Greenwood B, Whitty CJ, Ansah EK, Price RN, Dondorp AM, von Seidlein L, Baird JK et al. 2015. Malaria eradication and elimination: views on how to translate a vision into reality. BMC Med, 13 (1), pp. 167. | Show Abstract | Read more

Although global efforts in the past decade have halved the number of deaths due to malaria, there are still an estimated 219 million cases of malaria a year, causing more than half a million deaths. In this forum article, we asked experts working in malaria research and control to discuss the ways in which malaria might eventually be eradicated. Their collective views highlight the challenges and opportunities, and explain how multi-factorial and integrated processes could eventually make malaria eradication a reality.

Guyant P, Corbel V, Guérin PJ, Lautissier A, Nosten F, Boyer S, Coosemans M, Dondorp AM, Sinou V, Yeung S, White N. 2015. Past and new challenges for malaria control and elimination: the role of operational research for innovation in designing interventions Malaria Journal, 14 (1), | Show Abstract | Read more

© 2015 Guyant et al.This meeting report presents the outcomes of a workshop held in Bangkok on December 1st 2014, where the following challenges were discussed: the threat of resistance to artemisinin and artemisinin-based combination therapy in the Greater Mekong Sub-region (GMS) and in Africa; access to treatment for most at risk and hard to reach population; insecticide resistance, residual and outdoors transmission. The role of operational research and the interactions between research institutions, National Malaria Control Programmes, Civil Society Organizations, and of financial and technical partners to address those challenges and to accelerate translation of research into policies and programmes were debated. The threat and the emergency of the artemisinin resistance spread and independent emergence in the GMS was intensely debated as it is now close to the border of India. The need for key messages, based on scientific evidence and information available and disseminated without delay, was highlighted as crucial for an effective and urgent response.

Parry CM, Thieu NTV, Dolecek C, Karkey A, Gupta R, Turner P, Dance D, Maude RR et al. 2015. Clinically and Microbiologically Derived Azithromycin Susceptibility Breakpoints for Salmonella enterica Serovars Typhi and Paratyphi A (vol 59, pg 2756, 2015) ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 59 (7), pp. 4364-4364. | Read more

Parry CM, Thieu NT, Dolecek C, Karkey A, Gupta R, Turner P, Dance D, Maude RR et al. 2015. Erratum for Parry et al., Clinically and microbiologically derived azithromycin susceptibility breakpoints for Salmonella enterica serovars Typhi and Paratyphi A. Antimicrob Agents Chemother, 59 (7), pp. 4364. | Read more

Garcia-Laorden MI, Blok DC, Kager LM, Hoogendijk AJ, van Mierlo GJ, Lede IO, Rahman W, Afroz R et al. 2015. Increased intra- and extracellular granzyme expression in patients with tuberculosis. Tuberculosis (Edinb), 95 (5), pp. 575-580. | Show Abstract | Read more

Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Granzymes (gzms) are proteases mainly found in cytotoxic lymphocytes, but also extracellularly. While the role of gzms in target cell death has been widely characterized, considerable evidence points towards broader roles related to infectious and inflammatory responses. To investigate the expression of the gzms in TB, intracellular gzms A, B and K were measured by flow cytometry in lymphocyte populations from peripheral blood mononuclear cells from 18 TB patients and 12 healthy donors from Bangladesh, and extracellular levels of gzmA and B were measured in serum from 58 TB patients and 31 healthy controls. TB patients showed increased expression of gzmA in CD8(+) T, CD4(+) T and CD56(+) T, but not NK, cells, and of gzmB in CD8(+) T cells, when compared to controls. GzmK expression was not altered in TB patients in any lymphocyte subset. The extracellular levels of gzmA and, to a lesser extent, of gzmB, were increased in TB patients, but did not correlate with intracellular gzm expression in lymphocyte subsets. Our results reveal enhanced intra- and extracellular expression of gzmA and B in patients with pulmonary TB, suggesting that gzms are part of the host response to tuberculosis.

Maude RR, de Jong HK, Wijedoru L, Fukushima M, Ghose A, Samad R, Hossain MA, Karim MR, Faiz MA, Parry CM, CMCH Typhoid Study Group. 2015. The diagnostic accuracy of three rapid diagnostic tests for typhoid fever at Chittagong Medical College Hospital, Chittagong, Bangladesh. Trop Med Int Health, 20 (10), pp. 1376-1384. | Show Abstract | Read more

OBJECTIVE: To determine the diagnostic accuracy of three rapid diagnostic tests (RDTs) for typhoid fever in febrile hospitalised patients in Bangladesh. METHODS: Febrile adults and children admitted to Chittagong Medical College Hospital, Bangladesh, were investigated with Bact/Alert(®) blood cultures and real-time PCR to detect Salmonella enterica Typhi and Paratyphi A and assays for Rickettsia, leptospirosis and dengue fever. Acute serum samples were examined with the LifeAssay (LA) Test-it™ Typhoid IgM lateral flow assay detecting IgM antibodies against S. Typhi O antigen, CTKBiotech Onsite Typhoid IgG/IgM Combo Rapid-test cassette lateral flow assay detecting IgG and IgM antibodies against S. Typhi O and H antigens and SD Bioline line assay for IgG and IgM antibodies against S. Typhi proteins. RESULTS: In 300 malaria smear-negative febrile patients [median (IQR) age of 13.5 (5-31) years], 34 (11.3%) had confirmed typhoid fever: 19 positive by blood culture for S. Typhi (three blood PCR positive) and 15 blood culture negative but PCR positive for S. Typhi in blood. The respective sensitivity and specificity of the three RDTs in patients using a composite reference standard of blood culture and/or PCR-confirmed typhoid fever were 59% and 61% for LifeAssay, 59% and 74% for the CTK IgM and/or IgG, and 24% and 96% for the SD Bioline RDT IgM and/or IgG. The LifeAssay RDT had a sensitivity of 63% and a specificity of 91% when modified with a positive cut-off of ≥2+ and analysed using a Bayesian latent class model. CONCLUSIONS: These typhoid RDTs demonstrated moderate diagnostic accuracies, and better tests are needed.

Plewes K, Haider MS, Kingston HWF, Yeo TW, Ghose A, Hossain MA, Dondorp AM, Turner GDH, Anstey NM. 2015. Severe falciparum malaria treated with artesunate complicated by delayed onset haemolysis and acute kidney injury Malaria Journal, 14 (1), | Show Abstract | Read more

© 2015 Plewes et al.Background: Severe falciparum malaria may be complicated by haemolysis after parasite clearance, however the mechanisms remain unclear. Recent reports describe a pattern of delayed onset haemolysis among non-immune travellers with hyperparasitaemia treated with intravenous artesunate, termed post-artesunate delayed haemolysis (PADH). The occurrence and clinical impact of PADH following severe malaria infections in areas of unstable transmission are unknown. Case: A 45-year-old Bangladeshi male was initially admitted to a local hospital with severe falciparum malaria complicated by hyperparasitaemia and treated with intravenous artesunate. Twenty days from his first presentation he was readmitted with delayed onset haemolytic anaemia and acute kidney injury. Multiple blood transfusions and haemodialysis were required. Renal biopsy revealed acute tubular injury and haem pigment nephropathy. His haemoglobin and renal function recovered to baseline after 62 days from his second admission. Discussion: This case highlights the differential diagnosis of post-malaria delayed onset haemolysis, including the recently described syndrome of post-artemisinin delayed haemolysis. The pathophysiology contributing to acute kidney injury in this patient and the limited treatment options are discussed. Conclusions: This report describes PADH complicated by acute kidney injury in an adult patient living in a malaria hypoendemic region who subsequently required blood transfusions and haemodialysis. This case emphasizes the importance of routine follow up of haemoglobin and renal function in artesunate-treated patients who have recovered from severe malaria.

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

Mbengue A, Bhattacharjee S, Pandharkar T, Liu H, Estiu G, Stahelin RV, Rizk SS, Njimoh DL et al. 2015. A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria. Nature, 520 (7549), pp. 683-687. | Show Abstract | Read more

Artemisinins are the cornerstone of anti-malarial drugs. Emergence and spread of resistance to them raises risk of wiping out recent gains achieved in reducing worldwide malaria burden and threatens future malaria control and elimination on a global level. Genome-wide association studies (GWAS) have revealed parasite genetic loci associated with artemisinin resistance. However, there is no consensus on biochemical targets of artemisinin. Whether and how these targets interact with genes identified by GWAS, remains unknown. Here we provide biochemical and cellular evidence that artemisinins are potent inhibitors of Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), revealing an unexpected mechanism of action. In resistant clinical strains, increased PfPI3K was associated with the C580Y mutation in P. falciparum Kelch13 (PfKelch13), a primary marker of artemisinin resistance. Polyubiquitination of PfPI3K and its binding to PfKelch13 were reduced by the PfKelch13 mutation, which limited proteolysis of PfPI3K and thus increased levels of the kinase, as well as its lipid product phosphatidylinositol-3-phosphate (PI3P). We find PI3P levels to be predictive of artemisinin resistance in both clinical and engineered laboratory parasites as well as across non-isogenic strains. Elevated PI3P induced artemisinin resistance in absence of PfKelch13 mutations, but remained responsive to regulation by PfKelch13. Evidence is presented for PI3P-dependent signalling in which transgenic expression of an additional kinase confers resistance. Together these data present PI3P as the key mediator of artemisinin resistance and the sole PfPI3K as an important target for malaria elimination.

Dogovski C, Xie SC, Burgio G, Bridgford J, Mok S, McCaw JM, Chotivanich K, Kenny S et al. 2015. Targeting the cell stress response of Plasmodium falciparum to overcome artemisinin resistance. PLoS Biol, 13 (4), pp. e1002132. | Show Abstract | Read more

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.

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.

von Seidlein L, Dondorp A. 2015. Fighting fire with fire: mass antimalarial drug administrations in an era of antimalarial resistance. Expert Rev Anti Infect Ther, 13 (6), pp. 715-730. | Show Abstract | Read more

The emergence and spread of antimalarial resistance has been a major liability for malaria control. The spread of chloroquine-resistant Plasmodium falciparum strains had catastrophic consequences for people in malaria-endemic regions, particularly in sub-Saharan Africa. The recent emergence of artemisinin-resistant P. falciparum strains is of highest concern. Current efforts to contain artemisinin resistance have yet to show success. In the absence of more promising plans, it has been suggested to eliminate falciparum malaria from foci of artemisinin resistance using a multipronged approach, including mass drug administrations. The use of mass drug administrations is controversial as it increases drug pressure. Based on current knowledge it is difficult to conceptualize how targeted malaria elimination could contribute to artemisinin resistance, provided a full treatment course is ensured.

De Silva AP, Stephens T, Welch J, Sigera C, De Alwis S, Athapattu P, Dharmagunawardene D, Olupeliyawa A et al. 2015. Nursing intensive care skills training: A nurse led, short, structured, and practical training program, developed and tested in a resource-limited setting JOURNAL OF CRITICAL CARE, 30 (2), | Read more

Parry CM, Thieu NT, Dolecek C, Karkey A, Gupta R, Turner P, Dance D, Maude RR et al. 2015. Clinically and microbiologically derived azithromycin susceptibility breakpoints for Salmonella enterica serovars Typhi and Paratyphi A. Antimicrob Agents Chemother, 59 (5), pp. 2756-2764. | Show Abstract | Read more

Azithromycin is an effective treatment for uncomplicated infections with Salmonella enterica serovar Typhi and serovar Paratyphi A (enteric fever), but there are no clinically validated MIC and disk zone size interpretative guidelines. We studied individual patient data from three randomized controlled trials (RCTs) of antimicrobial treatment in enteric fever in Vietnam, with azithromycin used in one treatment arm, to determine the relationship between azithromycin treatment response and the azithromycin MIC of the infecting isolate. We additionally compared the azithromycin MIC and the disk susceptibility zone sizes of 1,640 S. Typhi and S. Paratyphi A clinical isolates collected from seven Asian countries. In the RCTs, 214 patients who were treated with azithromycin at a dose of 10 to 20 mg/ml for 5 to 7 days were analyzed. Treatment was successful in 195 of 214 (91%) patients, with no significant difference in response (cure rate, fever clearance time) with MICs ranging from 4 to 16 μg/ml. The proportion of Asian enteric fever isolates with an MIC of ≤ 16 μg/ml was 1,452/1,460 (99.5%; 95% confidence interval [CI], 98.9 to 99.7) for S. Typhi and 207/240 (86.3%; 95% CI, 81.2 to 90.3) (P < 0.001) for S. Paratyphi A. A zone size of ≥ 13 mm to a 5-μg azithromycin disk identified S. Typhi isolates with an MIC of ≤ 16 μg/ml with a sensitivity of 99.7%. An azithromycin MIC of ≤ 16 μg/ml or disk inhibition zone size of ≥ 13 mm enabled the detection of susceptible S. Typhi isolates that respond to azithromycin treatment. Further work is needed to define the response to treatment in S. Typhi isolates with an azithromycin MIC of >16 μg/ml and to determine MIC and disk breakpoints for S. Paratyphi A.

Blok DC, Kager LM, Hoogendijk AJ, Lede IO, Rahman W, Afroz R, Bresser P, van der Zee JS et al. 2015. Expression of inhibitory regulators of innate immunity in patients with active tuberculosis. BMC Infect Dis, 15 (1), pp. 98. | Show Abstract | Read more

BACKGROUND: Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Toll-like-receptors (TLRs) are important for the recognition of the causative agent Mycobacterium tuberculosis. Negative regulation of TLRs is necessary to control deleterious inflammatory damage, but could provide a means of immune evasion by M. tuberculosis as well. METHODS: To obtain insight in the extent of expression of inhibitory regulators of immunity in patients with active TB, peripheral-blood-mononuclear-cells (PBMCs) and plasma were obtained from 54 TB patients and 29 healthy blood donors from Chittagong, Bangladesh. Bilateral alveolar macrophages were obtained from an infected versus a contralateral normal lung segment of 9 patients. Statistical analyses were performed using Mann-Whitney U and Wilcoxon matched pairs testing. Correlations were calculated using the Spearman rho test. RESULTS: PBMCs harvested from TB patients demonstrated increased mRNA expression of IL-1-receptor-associated-kinase-M, suppressor-of-cytokine-signalling-3 and Toll-interacting-protein. Flow cytometry revealed enhanced expression of IL-1-receptor-like-1 (ST2) on lymphocytes. Plasma soluble ST2 was elevated in patients with TB and correlated with established TB biomarkers, most strongly with soluble interleukin-2 receptor subunit α and interleukin-8. Alveolar macrophage mRNA expression of negative TLR regulators did not differ between the infected and contralateral lung side. CONCLUSION: These results show enhanced expression of distinct negative regulators of innate immunity in PBMCs of patients with TB and identify plasma soluble ST2 as a potential novel biomarker for TB disease activity.

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.

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.

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.

Hoogendijk AJ, Blok DC, Garcia Laorden MI, Kager LM, Lede IO, Rahman W, Afroz R, Bresser P et al. 2015. Soluble and cell-associated triggering receptor expressed on myeloid cells-1 and -2 in patients with pulmonary tuberculosis. J Infect, 71 (6), pp. 706-709. | Read more

Garcia-Laorden MI, Blok DC, Kager LM, Hoogendijk AJ, Van Mierlo GJ, Lede IO, Rahman W, Afroz R et al. 2015. Increased intra- and extracellular granzyme expression in patients with tuberculosis Tuberculosis, 95 (5), pp. 575-580. | Show Abstract | Read more

© 2015 Elsevier Ltd.Summary Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Granzymes (gzms) are proteases mainly found in cytotoxic lymphocytes, but also extracellularly. While the role of gzms in target cell death has been widely characterized, considerable evidence points towards broader roles related to infectious and inflammatory responses. To investigate the expression of the gzms in TB, intracellular gzms A, B and K were measured by flow cytometry in lymphocyte populations from peripheral blood mononuclear cells from 18 TB patients and 12 healthy donors from Bangladesh, and extracellular levels of gzmA and B were measured in serum from 58 TB patients and 31 healthy controls. TB patients showed increased expression of gzmA in CD8+ T, CD4+ T and CD56+ T, but not NK, cells, and of gzmB in CD8+ T cells, when compared to controls. GzmK expression was not altered in TB patients in any lymphocyte subset. The extracellular levels of gzmA and, to a lesser extent, of gzmB, were increased in TB patients, but did not correlate with intracellular gzm expression in lymphocyte subsets. Our results reveal enhanced intra- and extracellular expression of gzmA and B in patients with pulmonary TB, suggesting that gzms are part of the host response to tuberculosis.

Imwong M, Tun KM, Hlaing TM, Grist EP, Guerin P, Smithuis F, Dondorp AM, Day NP, Nosten F, White N, Woodrow CJ. 2015. Artemisinin resistance in Myanmar--Authors' reply. Lancet Infect Dis, 15 (9), pp. 1002-1003. | Read more

Ringwald P, Dondorp AM. 2015. Severe Malaria Not Responsive to Artemisinin Derivatives in Man Returning from Angola to Vietnam. Emerg Infect Dis, 21 (7), pp. 1264-1265. | Read more

Plewes K, Haider MS, Kingston HW, Yeo TW, Ghose A, Hossain MA, Dondorp AM, Turner GD, Anstey NM. 2015. Severe falciparum malaria treated with artesunate complicated by delayed onset haemolysis and acute kidney injury. Malar J, 14 (1), pp. 246. | Show Abstract | Read more

BACKGROUND: Severe falciparum malaria may be complicated by haemolysis after parasite clearance, however the mechanisms remain unclear. Recent reports describe a pattern of delayed onset haemolysis among non-immune travellers with hyperparasitaemia treated with intravenous artesunate, termed post-artesunate delayed haemolysis (PADH). The occurrence and clinical impact of PADH following severe malaria infections in areas of unstable transmission are unknown. CASE: A 45-year-old Bangladeshi male was initially admitted to a local hospital with severe falciparum malaria complicated by hyperparasitaemia and treated with intravenous artesunate. Twenty days from his first presentation he was readmitted with delayed onset haemolytic anaemia and acute kidney injury. Multiple blood transfusions and haemodialysis were required. Renal biopsy revealed acute tubular injury and haem pigment nephropathy. His haemoglobin and renal function recovered to baseline after 62 days from his second admission. DISCUSSION: This case highlights the differential diagnosis of post-malaria delayed onset haemolysis, including the recently described syndrome of post-artemisinin delayed haemolysis. The pathophysiology contributing to acute kidney injury in this patient and the limited treatment options are discussed. CONCLUSIONS: This report describes PADH complicated by acute kidney injury in an adult patient living in a malaria hypoendemic region who subsequently required blood transfusions and haemodialysis. This case emphasizes the importance of routine follow up of haemoglobin and renal function in artesunate-treated patients who have recovered from severe malaria.

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.

Vellinga NA, Boerma EC, Koopmans M, Donati A, Dubin A, Shapiro NI, Pearse RM, Machado FR et al. 2015. International study on microcirculatory shock occurrence in acutely ill patients. Crit Care Med, 43 (1), pp. 48-56. | Show Abstract | Read more

OBJECTIVES: Microcirculatory alterations are associated with adverse outcome in subsets of critically ill patients. The prevalence and significance of microcirculatory alterations in the general ICU population are unknown. We studied the prevalence of microcirculatory alterations in a heterogeneous ICU population and its predictive value in an integrative model of macro- and microcirculatory variables. DESIGN: Multicenter observational point prevalence study. SETTING: The Microcirculatory Shock Occurrence in Acutely ill Patients study was conducted in 36 ICUs worldwide. PATIENTS: A heterogeneous ICU population consisting of 501 patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Demographic, hemodynamic, and laboratory data were collected in all ICU patients who were 18 years old or older. Sublingual Sidestream Dark Field imaging was performed to determine the prevalence of an abnormal capillary microvascular flow index (< 2.6) and its additional value in predicting hospital mortality. In 501 patients with a median Acute Physiology and Chronic Health Evaluation II score of 15 (10-21), a Sequential Organ Failure Assessment score of 5 (2-8), and a hospital mortality of 28.4%, 17% exhibited an abnormal capillary microvascular flow index. Tachycardia (heart rate > 90 beats/min) (odds ratio, 2.71; 95% CI, 1.67-4.39; p < 0.001), mean arterial pressure (odds ratio, 0.979; 95% CI, 0.963-0.996; p = 0.013), vasopressor use (odds ratio, 1.84; 95% CI, 1.11-3.07; p = 0.019), and lactate level more than 1.5 mEq/L (odds ratio, 2.15; 95% CI, 1.28-3.62; p = 0.004) were independent risk factors for hospital mortality, but not abnormal microvascular flow index. In reference to microvascular flow index, a significant interaction was observed with tachycardia. In patients with tachycardia, the presence of an abnormal microvascular flow index was an independent, additive predictor for in-hospital mortality (odds ratio, 3.24; 95% CI, 1.30-8.06; p = 0.011). This was not true for nontachycardic patients nor for the total group of patients. CONCLUSIONS: In a heterogeneous ICU population, an abnormal microvascular flow index was present in 17% of patients. This was not associated with mortality. However, in patients with tachycardia, an abnormal microvascular flow index was independently associated with an increased risk of hospital death.

Kager LM, Blok DC, Lede IO, Rahman W, Afroz R, Bresser P, van der Zee JS, Ghose A et al. 2015. Pulmonary tuberculosis induces a systemic hypercoagulable state Journal of Infection, 70 (4), pp. 324-334. | Show Abstract | Read more

© 2014 The British Infection Association.Objectives: Human tuberculosis (TB) remains an important cause of death globally. Bangladesh is one of the most affected countries. We aimed to investigate the impact of pulmonary TB on pro- and anticoagulant mechanisms. Methods: This prospective study was conducted in Chittagong, Bangladesh. We performed an in-depth analysis of coagulation activation and inhibition in plasma obtained from 64 patients with primary lung TB and 11 patients with recurrent lung TB and compared these with 37 healthy controls. Additionally, in nine patients coagulation activation was studied in bronchoalveolar lavage fluid (BALF) harvested from the site of infection and compared with BALF from a contralateral unaffected lung subsegment. Results: Relative to uninfected controls, primary and recurrent TB were associated with a systemic net procoagulant state, as indicated by enhanced activation of coagulation (elevated plasma levels of thrombin-antithrombin complexes, D-dimer and fibrinogen) together with impaired anticoagulant mechanisms (reduced plasma levels of antithrombin, protein C activity, free protein S, and protein C inhibitor). Activation of coagulation did not correlate with plasma concentrations of established TB biomarkers. Coagulation activation could not be detected at the primary site of infection in a subset of TB patients. Conclusions: Pulmonary TB is associated with a systemic hypercoagulable state.

Brown TS, Jacob CG, Silva JC, Takala-Harrison S, Djimdé A, Dondorp AM, Fukuda M, Noedl H et al. 2015. Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype. Infect Genet Evol, 30 pp. 318-322. | Show Abstract | Read more

Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives.

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.

Kager LM, Blok DC, Lede IO, Rahman W, Afroz R, Bresser P, van der Zee JS, Ghose A et al. 2015. Pulmonary tuberculosis induces a systemic hypercoagulable state. J Infect, 70 (4), pp. 324-334. | Show Abstract | Read more

OBJECTIVES: Human tuberculosis (TB) remains an important cause of death globally. Bangladesh is one of the most affected countries. We aimed to investigate the impact of pulmonary TB on pro- and anticoagulant mechanisms. METHODS: This prospective study was conducted in Chittagong, Bangladesh. We performed an in-depth analysis of coagulation activation and inhibition in plasma obtained from 64 patients with primary lung TB and 11 patients with recurrent lung TB and compared these with 37 healthy controls. Additionally, in nine patients coagulation activation was studied in bronchoalveolar lavage fluid (BALF) harvested from the site of infection and compared with BALF from a contralateral unaffected lung subsegment. RESULTS: Relative to uninfected controls, primary and recurrent TB were associated with a systemic net procoagulant state, as indicated by enhanced activation of coagulation (elevated plasma levels of thrombin-antithrombin complexes, D-dimer and fibrinogen) together with impaired anticoagulant mechanisms (reduced plasma levels of antithrombin, protein C activity, free protein S, and protein C inhibitor). Activation of coagulation did not correlate with plasma concentrations of established TB biomarkers. Coagulation activation could not be detected at the primary site of infection in a subset of TB patients. CONCLUSIONS: Pulmonary TB is associated with a systemic hypercoagulable state.

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.

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.

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.

Maude RR, Maude RJ, Ghose A, Amin MR, Islam MB, Ali M, Bari MS, Majumder MI et al. 2014. Serosurveillance of Orientia tsutsugamushi and Rickettsia typhi in Bangladesh. Am J Trop Med Hyg, 91 (3), pp. 580-583. | Show Abstract | Read more

Scrub and murine typhus infections are under-diagnosed causes of febrile illness across the tropics, and it is not known how common they are in Bangladesh. We conducted a prospective seroepidemiologic survey across six major teaching hospitals in Bangladesh by using an IgM enzyme-linked immunosorbent assay. Results indicated recent exposure (287 of 1,209, 23.7% seropositive for Orientia tsutsugamushi and 805 of 1,209, 66.6% seropositive for Rickettsia typhi). Seropositive rates were different in each region. However, there was no geographic clustering of seropositive results for both organisms. There was no difference between those from rural or urban areas. Rickettsia typhi seroreactivity was positively correlated with age. Scrub typhus and murine typhus should be considered as possible causes of infection in Bangladesh.

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.

Dondorp AM, Haniffa R. 2014. Critical care and severe sepsis in resource poor settings. Trans R Soc Trop Med Hyg, 108 (8), pp. 453-454. | Read more

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.).

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.

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.

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.

Maude RR, Amir Hossain M, Hassan MU, Osbourne S, Langan K, Sayeed A, Karim MR, Samad R et al. 2014. Correction: Transorbital Sonographic Evaluation of Normal Optic Nerve Sheath Diameter in Healthy Volunteers in Bangladesh PLoS ONE, 9 (1), | Read more

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.

Sathpathi S, Mohanty AK, Satpathi P, Mishra SK, Behera PK, Patel G, Dondorp AM. 2014. Comparing Leishman and Giemsa staining for the assessment of peripheral blood smear preparations in a malaria-endemic region in India. Malar J, 13 (1), pp. 512. | Show Abstract | Read more

BACKGROUND: Microscopy of peripheral blood thin and thick films remains the reference for malaria diagnosis. Although Giemsa staining is most commonly used, the Leishman staining method provides better visualization of the nuclear chromatin pattern of cells. It is less well known whether accuracy of parasitaemia assessment is equally accurate with the latter method. METHODS: Peripheral blood thin and thick smears from consecutive febrile patients admitted to Ispat General hospital, Rourkela, Odhisa, India, were stained with Giemsa and Leishman stain. Methods were compared for species identification, parasite quantification, and ability for identification of alternative diagnoses. RESULTS: Blood films from 1,180 fever patients were compared according to staining method, of which 111 were identified as parasitaemic using Giemsa and 110 with Leishman staining. The Kappa value as a measure of agreement between methods was 0.995 (p < 0.001), and the log10parasitaemia between methods were strongly correlated (r2 = 0.9981). In parasite negative patients, thin smear assessment contributed to making a diagnosis in 276/1,180 (23%) of cases. These assessments were better made in Leishman-stained preparations, especially for the assessment of morphological changes in red and white cells. CONCLUSION: Leishman's staining method for thin and thick smears is a good alternative to Giemsa's stain for identifying Plasmodium parasites. The Leishman method is superior for visualization of red and white blood cell morphology.

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.

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.

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.

Maude RJ, Ahmed BU, Rahman AH, Rahman R, Majumder MI, Menezes DB, Abu Sayeed A, Hughes L et al. 2014. Retinal changes in visceral leishmaniasis by retinal photography. BMC Infect Dis, 14 (1), pp. 527. | Show Abstract | Read more

BACKGROUND: In visceral leishmaniasis (VL), retinal changes have previously been noted but not described in detail and their clinical and pathological significance are unknown. A prospective observational study was undertaken in Mymensingh, Bangladesh aiming to describe in detail visible changes in the retina in unselected patients with VL. METHODS: Patients underwent assessment of visual function, indirect and direct ophthalmoscopy and portable retinal photography. The photographs were assessed by masked observers including assessment for vessel tortuosity using a semi-automated system. RESULTS: 30 patients with VL were enrolled, of whom 6 (20%) had abnormalities. These included 5 with focal retinal whitening, 2 with cotton wool spots, 2 with haemorrhages, as well as increased vessel tortuosity. Visual function was preserved. CONCLUSIONS: These changes suggest a previously unrecognized retinal vasculopathy. An inflammatory aetiology is plausible such as a subclinical retinal vasculitis, possibly with altered local microvascular autoregulation, and warrants further investigation.

Haniffa R, De Silva AP, Iddagoda S, Batawalage H, De Silva STGR, Mahipala PG, Dondorp A, de Keizer N, Jayasinghe S. 2014. A cross-sectional survey of critical care services in Sri Lanka: A lower middle-income country Journal of Critical Care, 29 (5), pp. 764-768. | Show Abstract | Read more

Purpose: To describe the extent and variation of critical care services in Sri Lanka as a first step towards the development of a nationwide critical care unit (CCU) registry. Materials and Methods: A cross-sectional survey was conducted in all state CCUs by telephone or by visits to determine administration, infrastructure, equipment, staffing, and overall patient outcomes. Results: There were 99 CCUs with 2.5 CCU beds per 100. 000 population and 13 CCU beds per 1 000 hospital beds. The median number of beds per CCU was 5. The overall admissions were 194 per 100. 000 population per year. The overall bed turnover was 76.5 per unit per year, with CCU mortality being 17%.Most CCUs were headed by an anesthetist. There were a total of 790 doctors (1.6 per bed), 1 989 nurses (3.9 per bed), and 626 health care assistants (1.2 per bed). Majority (87.9%) had 1:1 nurse-to-patient ratio, although few (11.4%) nurses had received formal intensive care unit training. All CCUs had basic infrastructure (electricity, running water, piped oxygen) and basic equipment (such as electronic monitoring and infusion pumps). Conclusion: Sri Lanka, a lower middle-income country has an extensive network of critical care facilities but with inequalities in its distribution and facilities. © 2014 Elsevier Inc.

Woodrow CJ, Dondorp AM. 2014. Parasite burden and severity of malaria in Tanzanian children. N Engl J Med, 371 (5), pp. 481-482. | Read more

Haniffa R, De Silva AP, Iddagoda S, Batawalage H, De Silva ST, Mahipala PG, Dondorp A, de Keizer N, Jayasinghe S. 2014. A cross-sectional survey of critical care services in Sri Lanka: a lower middle-income country. J Crit Care, 29 (5), pp. 764-768. | Show Abstract | Read more

PURPOSE: To describe the extent and variation of critical care services in Sri Lanka as a first step towards the development of a nationwide critical care unit (CCU) registry. MATERIALS AND METHODS: A cross-sectional survey was conducted in all state CCUs by telephone or by visits to determine administration, infrastructure, equipment, staffing, and overall patient outcomes. RESULTS: There were 99 CCUs with 2.5 CCU beds per 100000 population and 13 CCU beds per 1 000 hospital beds. The median number of beds per CCU was 5. The overall admissions were 194 per 100000 population per year. The overall bed turnover was 76.5 per unit per year, with CCU mortality being 17%. Most CCUs were headed by an anesthetist. There were a total of 790 doctors (1.6 per bed), 1,989 nurses (3.9 per bed), and 626 health care assistants (1.2 per bed). Majority (87.9%) had 1:1 nurse-to-patient ratio, although few (11.4%) nurses had received formal intensive care unit training. All CCUs had basic infrastructure (electricity, running water, piped oxygen) and basic equipment (such as electronic monitoring and infusion pumps). CONCLUSION: Sri Lanka, a lower middle-income country has an extensive network of critical care facilities but with inequalities in its distribution and facilities.

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.

Plewes K, Royakkers AA, Hanson J, Hasan MM, Alam S, Ghose A, Maude RJ, Stassen PM et al. 2014. Correlation of biomarkers for parasite burden and immune activation with acute kidney injury in severe falciparum malaria. Malar J, 13 (1), pp. 91. | Show Abstract | Read more

BACKGROUND: Acute kidney injury (AKI) complicating severe Plasmodium falciparum malaria occurs in up to 40% of adult patients. The case fatality rate reaches 75% in the absence of renal replacement therapy (RRT). The precise pathophysiology of AKI in falciparum malaria remains unclear. Histopathology shows acute tubular necrosis with localization of host monocytes and parasitized red blood cells in the microvasculature. This study explored the relationship of plasma soluble urokinase-type plasminogen activator receptor (suPAR), as a proxy-measure of mononuclear cell activation, and plasma P. falciparum histidine rich protein 2 (PfHRP2), as a measure of sequestered parasite burden, with AKI in severe malaria. METHODS: Admission plasma suPAR and PfHRP2 concentrations were assessed in Bangladeshi adults with severe falciparum malaria (n=137). Patients were stratified according to AKI severity based on admission creatinine clearance. RESULTS: A total of 106 (77%) patients had AKI; 32 (23%), 42 (31%) and 32 (23%) were classified into 'mild, 'moderate' and 'severe' AKI groups, respectively. Plasma suPAR and PfHRP2 concentrations increased with AKI severity (test-for-trend P <0.0001) and correlated with other markers of renal dysfunction. Admission plasma suPAR and PfHRP2 concentrations were higher in patients who later required RRT (P <0.0001 and P=0.0004, respectively). In a multivariate analysis, both increasing suPAR and PfHRP2 were independently associated with increasing urine neutrophil gelatinase-associated lipocalin concentration, a marker of acute tubular necrosis (β=16.54 (95% CI 6.36-26.71) and β=0.07 (0.02-0.11), respectively). CONCLUSIONS: Both sequestered parasite burden and immune activation contribute to the pathogenesis of AKI in severe falciparum malaria.

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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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.

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.

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.

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.

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.

Kager LM, Weehuizen TA, Wiersinga WJ, Roelofs JJ, Meijers JC, Dondorp AM, van 't Veer C, van der Poll T. 2013. Endogenous α2-antiplasmin is protective during severe gram-negative sepsis (melioidosis). Am J Respir Crit Care Med, 188 (8), pp. 967-975. | Show Abstract | Read more

RATIONALE: α2-Antiplasmin (A2AP) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit plasmin. Although the fibrinolytic system is strongly affected by infection, the functional role of A2AP in the host response to sepsis is unknown. OBJECTIVES: To study the role of A2AP in melioidosis, a common form of community-acquired sepsis in Southeast Asia and Northern Australia caused by the gram-negative bacterium Burkholderia pseudomallei. METHODS: In a single-center observational study A2AP was measured in patients with culture-proven septic melioidosis. Wild-type and A2AP-deficient (A2AP(-/-)) mice were intranasally infected with B. pseudomallei to induce severe pneumosepsis (melioidosis). Parameters of inflammation and coagulation were measured, and survival studies were performed. MEASUREMENTS AND MAIN RESULTS: Patients with melioidosis showed elevated A2AP plasma levels. Likewise, A2AP levels in plasma and lung homogenates were elevated in mice infected with B. pseudomallei. A2AP-deficient (A2AP(-/-)) mice had a strongly disturbed host response during experimental melioidosis as reflected by enhanced bacterial growth at the primary site of infection accompanied by increased dissemination to distant organs. In addition, A2AP(-/-) mice showed more severe lung pathology and injury together with an increased accumulation of neutrophils and higher cytokine levels in lung tissue. A2AP deficiency further was associated with exaggerated systemic inflammation and coagulation, increased distant organ injury, and enhanced lethality. CONCLUSIONS: This study is the first to identify A2AP as a protective mediator during gram-negative (pneumo)sepsis by limiting bacterial growth, inflammation, tissue injury, and coagulation.

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.

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.

Cited:

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Scopus

Dondorp AM, Ringwald P. 2013. Artemisinin resistance is a clear and present danger Trends in Parasitology, 29 (8), pp. 359-360. | Read more

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.

Kager LM, Schouten M, Wiersinga WJ, de Boer JD, Lattenist LC, Roelofs JJ, Meijers JC, Levi M et al. 2013. Overexpression of the endothelial protein C receptor is detrimental during pneumonia-derived gram-negative sepsis (Melioidosis). PLoS Negl Trop Dis, 7 (7), pp. e2306. | Show Abstract | Read more

BACKGROUND: The endothelial protein C receptor (EPCR) enhances anticoagulation by accelerating activation of protein C to activated protein C (APC) and mediates anti-inflammatory effects by facilitating APC-mediated signaling via protease activated receptor-1. We studied the role of EPCR in the host response during pneumonia-derived sepsis instigated by Burkholderia (B.) pseudomallei, the causative agent of melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia. METHODOLOGY/PRINCIPAL FINDINGS: Soluble EPCR was measured in plasma of patients with septic culture-proven melioidosis and healthy controls. Experimental melioidosis was induced by intranasal inoculation of B. pseudomallei in wild-type (WT) mice and mice with either EPCR-overexpression (Tie2-EPCR) or EPCR-deficiency (EPCR(-/-)). Mice were sacrificed after 24, 48 or 72 hours. Organs and plasma were harvested to measure colony forming units, cellular influxes, cytokine levels and coagulation parameters. Plasma EPCR-levels were higher in melioidosis patients than in healthy controls and associated with an increased mortality. Tie2-EPCR mice demonstrated enhanced bacterial growth and dissemination to distant organs during experimental melioidosis, accompanied by increased lung damage, neutrophil influx and cytokine production, and attenuated coagulation activation. EPCR(-/-) mice had an unremarkable response to B. pseudomallei infection as compared to WT mice, except for a difference in coagulation activation in plasma. CONCLUSION/SIGNIFICANCE: Increased EPCR-levels correlate with accelerated mortality in patients with melioidosis. In mice, transgenic overexpression of EPCR aggravates outcome during Gram-negative pneumonia-derived sepsis caused by B. pseudomallei, while endogenous EPCR does not impact on the host response. These results add to a better understanding of the regulation of coagulation during severe (pneumo)sepsis.

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.

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.

Maude RR, Hossain MA, Hassan MU, Osbourne S, Sayeed KL, Karim MR, Samad R, Borooah S et al. 2013. Transorbital sonographic evaluation of normal optic nerve sheath diameter in healthy volunteers in Bangladesh. PLoS One, 8 (12), pp. e81013. | Show Abstract | Read more

INTRODUCTION: Measurement of optic nerve sheath diameter (ONSD) by ultrasound is increasingly used as a marker to detect raised intracranial pressure (ICP). ONSD varies with age and there is no clear consensus between studies for an upper limit of normal. Knowledge of normal ONSD in a healthy population is essential to interpret this measurement. METHODS: In a prospective observational study, ONSD was measured using a 15 MHz ultrasound probe in healthy volunteers in Chittagong, Bangladesh. The aims were to determine the normal range of ONSD in healthy Bangladeshi adults and children, compare measurements in males and females, horizontal and vertical beam orientations and left and right eyes in the same individual and to determine whether ONSD varies with head circumference independent of age. RESULTS: 136 subjects were enrolled, 12.5% of whom were age 16 or under. Median ONSD was 4.41 mm with 95% of subjects in the range 4.25-4.75 mm. ONSD was bimodally distributed. There was no relationship between ONSD and age (≥4 years), gender, head circumference, and no difference in left vs right eye or horizontal vs vertical beam. CONCLUSIONS: Ultrasonographic ONSD in Bangladeshi healthy volunteers has a narrow bimodal distribution independent of age (≥4 years), gender and head circumference. ONSD >4.75 mm in this population should be considered abnormal.

Hanson J, Lam SW, Alam S, Pattnaik R, Mahanta KC, Uddin Hasan M, Mohanty S, Mishra S et al. 2013. The reliability of the physical examination to guide fluid therapy in adults with severe falciparum malaria: an observational study. Malar J, 12 (1), pp. 348. | Show Abstract | Read more

BACKGROUND: Adults with severe malaria frequently require intravenous fluid therapy to restore their circulating volume. However, fluid must be delivered judiciously as both under- and over-hydration increase the risk of complications and, potentially, death. As most patients will be cared for in a resource-poor setting, management guidelines necessarily recommend that physical examination should guide fluid resuscitation. However, the reliability of this strategy is uncertain. METHODS: To determine the ability of physical examination to identify hypovolaemia, volume responsiveness, and pulmonary oedema, clinical signs and invasive measures of volume status were collected independently during an observational study of 28 adults with severe malaria. RESULTS: The physical examination defined volume status poorly. Jugular venous pressure (JVP) did not correlate with intravascular volume as determined by global end diastolic volume index (GEDVI; r(s) = 0.07, p = 0.19), neither did dry mucous membranes (p = 0.85), or dry axillae (p = 0.09). GEDVI was actually higher in patients with decreased tissue turgor (p < 0.001). Poor capillary return correlated with GEDVI, but was present infrequently (7% of observations) and, therefore, insensitive. Mean arterial pressure (MAP) correlated with GEDVI (rs = 0.16, p = 0.002), but even before resuscitation patients with a low GEDVI had a preserved MAP. Anuria on admission was unrelated to GEDVI and although liberal fluid resuscitation led to a median hourly urine output of 100 ml in 19 patients who were not anuric on admission, four (21%) developed clinical pulmonary oedema subsequently. MAP was unrelated to volume responsiveness (p = 0.71), while a low JVP, dry mucous membranes, dry axillae, increased tissue turgor, prolonged capillary refill, and tachycardia all had a positive predictive value for volume responsiveness of ≤50%. Extravascular lung water ≥11 ml/kg indicating pulmonary oedema was present on 99 of the 353 times that it was assessed during the study, but was identified on less than half these occasions by tachypnoea, chest auscultation, or an elevated JVP. A clear chest on auscultation and a respiratory rate <30 breaths/minute could exclude pulmonary oedema on 82% and 72% of occasions respectively. CONCLUSIONS: Findings on physical examination correlate poorly with true volume status in adults with severe malaria and must be used with caution to guide fluid therapy. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT00692627.

Dondorp AM, Ringwald P. 2013. Artemisinin resistance is a clear and present danger. Trends Parasitol, 29 (8), pp. 359-360. | Read more

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.

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.

Kolyva C, Kingston H, Tachtsidis I, Mohanty S, Mishra S, Patnaik R, Maude RJ, Dondorp AM, Elwell CE. 2013. Oscillations in cerebral haemodynamics in patients with falciparum malaria. Adv Exp Med Biol, 765 pp. 101-107. | Show Abstract | Read more

Spontaneous oscillations in cerebral haemodynamics studied with near-infrared spectroscopy (NIRS), become impaired in several pathological conditions. We assessed the spectral characteristics of these oscillations in 20 patients with falciparum malaria admitted to Ispat General Hospital, Rourkela, India. Monitoring included continuous frontal lobe NIRS recordings within 24 h of admission (Day 0), together with single measurements of a number of clinical and chemical markers recorded on admission. Seven patients returned for follow-up measurements on recovery (FU). A 2,048 sampling-point segment of oxygenated haemoglobin concentration ([ΔHbO(2)]) data was subjected to Fourier analysis per patient, and power spectral density was derived over the very low frequency (VLF: 0.02-0.04 Hz), low frequency (LF: 0.04-0.15 Hz) and high frequency (HF: 0.15-0.4 Hz) bands. At Day 0, VLF spectral power was 21.1 ± 16.4, LF power 7.2 ± 4.6 and HF power 2.6 ± 5.0, with VLF power being statistically significantly higher than LF and HF (P < 0.005). VLF power tended to decrease in the severely ill patients and correlated negatively with heart rate (r = 0.57, P < 0.01), while LF power correlated positively with aural body temperature (r = 0.49, P < 0.05). In all but one of the patients who returned for FU measurements, VLF power increased after recovery. This may be related to autonomic dysfunction in severe malaria, a topic of little research to date. The present study demonstrated that application of NIRS in a resource-poor setting is feasible and has potential as a research tool.

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.

Witkowski B, Khim N, Chim P, Kim S, Ke S, Kloeung N, Chy S, Duong S et al. 2013. Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia. Antimicrob Agents Chemother, 57 (2), pp. 914-923. | Show Abstract | Read more

The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA.

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.

Dondorp AM. 2013. Editorial commentary: single-dose primaquine as gametocytocidal treatment in patients with uncomplicated falciparum malaria. Clin Infect Dis, 56 (5), pp. 694-696. | Read more

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

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.

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

Fairhurst RM, Nayyar GM, Breman JG, Hallett R, Vennerstrom JL, Duong S, Ringwald P, Wellems TE, Plowe CV, Dondorp AM. 2012. Artemisinin-resistant malaria: research challenges, opportunities, and public health implications. Am J Trop Med Hyg, 87 (2), pp. 231-241. | Show Abstract | Read more

Artemisinin-based combination therapies are the most effective drugs to treat Plasmodium falciparum malaria. Reduced sensitivity to artemisinin monotherapy, coupled with the emergence of parasite resistance to all partner drugs, threaten to place millions of patients at risk of inadequate treatment of malaria. Recognizing the significance and immediacy of this possibility, the Fogarty International Center and the National Institute of Allergy and Infectious Diseases of the U.S. National Institutes of Health convened a conference in November 2010 to bring together the diverse array of stakeholders responding to the growing threat of artemisinin resistance, including scientists from malarious countries in peril. This conference encouraged and enabled experts to share their recent unpublished data from studies that may improve our understanding of artemisinin resistance. Conference sessions addressed research priorities to forestall artemisinin resistance and fostered collaborations between field- and laboratory-based researchers and international programs, with the aim of translating new scientific evidence into public health solutions. Inspired by this conference, this review summarizes novel findings and perspectives on artemisinin resistance, approaches for translating research data into relevant public health information, and opportunities for interdisciplinary collaboration to combat artemisinin resistance.

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.

Duenser MW, Festic E, Dondorp A, Kissoon N, Ganbat T, Kwizera A, Haniffa R, Baker T, Schultz MJ, Med ESIC. 2012. Point of care ultrasound for sepsis management in resource-limited settings: response to Via et al. INTENSIVE CARE MEDICINE, 38 (8), pp. 1408-1409. | Read more

Maude RR, Maude RJ, Ghose A, Amin MR, Islam MB, Ali M, Bari MS, Majumder MI et al. 2012. Seroepidemiological surveillance of Burkholderia pseudomallei in Bangladesh. Trans R Soc Trop Med Hyg, 106 (9), pp. 576-578. | Show Abstract | Read more

Melioidosis (Burkholderia pseudomallei infection) has yet to be demonstrated systematically in Bangladesh. A prospective, cross-sectional serological survey was conducted in 2010 at six Bangladeshi hospitals. Age, gender, occupation and residential address were recorded. Of 1244 patients, 359 (28.9%) were positive for B. pseudomallei by indirect haemagglutination assay. Farmers had an increased risk of seropositivity (risk ratio=1.4, 95% CI 1.0-1.8; p=0.03). There was no clear geographic clustering of seropositives. Melioidosis should be considered as a possible cause of febrile illness in Bangladesh. Further studies are needed to establish the incidence of clinical disease and distribution of environmental risk.

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.

Dondorp A. 2012. Artemisinin resistance in falciparum malaria INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E43-E43. | Read more

Witkowski B, Sokunmalis K, Kim S, Pheaktra C, Sopheakvatey K, Kloeung N, Khim N, Duong S et al. 2012. In vitro phenotype of reduced susceptibility to artemisinin in Plasmodium falciparum isolates from western Cambodia INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E178-E178. | Read more

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.

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.

Dünser MW, Festic E, Dondorp A, Kissoon N, Ganbat T, Kwizera A, Haniffa R, Baker T, Schultz MJ, Global Intensive Care Working Group of European Society of Intensive Care Medicine. 2012. Recommendations for sepsis management in resource-limited settings. Intensive Care Med, 38 (4), pp. 557-574. | Show Abstract | Read more

PURPOSE: To provide clinicians practicing in resource-limited settings with a framework to improve the diagnosis and treatment of pediatric and adult patients with sepsis. METHODS: The medical literature on sepsis management was reviewed. Specific attention was paid to identify clinical evidence on sepsis management from resource-limited settings. RESULTS: Recommendations are grouped into acute and post-acute interventions. Acute interventions include liberal fluid resuscitation to achieve adequate tissue perfusion, normal heart rate and arterial blood pressure, use of epinephrine or dopamine for inadequate tissue perfusion despite fluid resuscitation, frequent measurement of arterial blood pressure in hemodynamically unstable patients, administration of hydrocortisone or prednisolone to patients requiring catecholamines, oxygen administration to achieve an oxygen saturation >90%, semi-recumbent and/or lateral position, non-invasive ventilation for increased work of breathing or hypoxemia despite oxygen therapy, timely administration of adequate antimicrobials, thorough clinical investigation for infectious source identification, fluid/tissue sampling and microbiological work-up, removal, drainage or debridement of the infectious source. Post-acute interventions include regular re-assessment of antimicrobial therapy, administration of antimicrobials for an adequate but not prolonged duration, avoidance of hypoglycemia, pharmacological or mechanical deep vein thrombosis prophylaxis, resumption of oral food intake after resuscitation and regaining of consciousness, careful use of opioids and sedatives, early mobilization, and active weaning of invasive support. Specific considerations for malaria, puerperal sepsis and HIV/AIDS patients with sepsis are included. CONCLUSION: Only scarce evidence exists for the management of pediatric and adult sepsis in resource-limited settings. The presented recommendations may help to improve sepsis management in middle- and low-income countries.

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.

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.

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.

Smith M, Campino S, Gu Y, Clark TG, Otto TD, Maslen G, Manske M, Imwong M et al. 2012. An In-Solution Hybridisation Method for the Isolation of Pathogen DNA from Human DNA-rich Clinical Samples for Analysis by NGS. Open Genomics J, 5 (1), pp. 18-29. | Show Abstract | Read more

Studies on DNA from pathogenic organisms, within clinical samples, are often complicated by the presence of large amounts of host, e.g., human DNA. Isolation of pathogen DNA from these samples would improve the efficiency of next-generation sequencing (NGS) and pathogen identification. Here we describe a solution-based hybridisation method for isolation of pathogen DNA from a mixed population. This straightforward and inexpensive technique uses probes made from whole-genome DNA and off-the-shelf reagents. In this study, Escherichia coli DNA was successfully enriched from a mixture of E.coli and human DNA. After enrichment, genome coverage following NGS was significantly higher and the evenness of coverage and GC content were unaffected. This technique was also applied to samples containing a mixture of human and Plasmodium falciparum DNA. The P.falciparum genome is particularly difficult to sequence due to its high AT content (80.6%) and repetitive nature. Post enrichment, a bias in the recovered DNA was observed, with a poorer representation of the AT-rich non-coding regions. This uneven coverage was also observed in pre-enrichment samples, but to a lesser degree. Despite the coverage bias in enriched samples, SNP (single-nucleotide polymorphism) calling in coding regions was unaffected and the majority of samples had over 90% of their coding region covered at 5× depth. This technique shows significant promise as an effective method to enrich pathogen DNA from samples with heavy human contamination, particularly when applied to GC-neutral genomes.

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131

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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.

Paris DH, Chansamouth V, Nawtaisong P, Löwenberg EC, Phetsouvanh R, Blacksell SD, Lee SJ, Dondorp AM et al. 2012. Coagulation and inflammation in scrub typhus and murine typhus-a prospective comparative study from Laos Clinical Microbiology and Infection, 18 (12), pp. 1221-1228. | Show Abstract | Read more

Scrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited. This study compared human in vivo changes in 15 coagulation, inflammation and endothelial activation markers in prospectively collected admission and follow-up samples of 121 patients (55 scrub typhus, 55 murine typhus, and 11 typhus-like illness) and 51 healthy controls from Laos. As compared with controls, all but one of the markers assessed were significantly affected in typhus patients; however, the activation patterns differed significantly between scrub and murine typhus patients. The levels of markers of coagulation activation and all inflammatory cytokines, except for interleukin-12, were significantly higher in patients with scrub typhus than in those with murine typhus. In patients with murine typhus, however, the levels of endothelium-derived markers were significantly higher. Anticoagulant factors were inhibited in both typhus patient groups. This is the first study demonstrating that, in scrub typhus, in vivo coagulation activation is prominent and is related to a strong proinflammatory response, whereas in murine typhus, changes in coagulant and fibrinolytic pathways are suggestive of endothelial cell perturbation. These data suggest that, although late-stage endothelial infection is common in both diseases, the in vivo pathogenic mechanisms of R. typhi and O. tsutsugamushi could differ in the early phase of infection and may contribute to disease differentiation. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Wongtanachai J, Silamut K, Day NP, Dondorp A, Chaisri U. 2012. Effects of antimalarial drugs on movement of Plasmodium falciparum. Southeast Asian J Trop Med Public Health, 43 (1), pp. 1-9. | Show Abstract

In vitro antimalarial drug susceptibility is conventionally assessed by the concentration dependent growth inhibition of Plasmodium in an in vitro culture system. Inhibition of the kinetic properties of the parasites could provide an alternative method to assess in vitro antimalarial drugs sensitivity. In this study we used a novel real time microscopic technique, which does not require fixation and staining of the parasite, to study the effects of antimalarial drugs on the intracellular movement of Plasmodium (P.) falciparum trophozoites. Using real time microscopy movement of P. falciparum pigment within erythrocytes was investigated before and after antimalarial drugs exposure (artesunate, quinine, and piperaquine). For artesunate, the 50% inhibition concentration (IC50) at which movement in half of the trophozoites was abolished was estimated by sigmoid curve fitting. Intra- and inter-observer agreements were also assessed. Healthy unexposed P. falciparum trophozoites in culture showed very active movement of malaria pigment. Quinine and piperaquine had no effect but artesunate did reduce pigment movement which started after 2.5 hours exposure to the drug. The mean (SD) IC50 for artesunate regarding abolishment of pigment movement was 54 (14) ng/ml. Assessments of intra- and inter-rater agreement showed good reproducibility of the technique (Kappa value 0.82 to 0.91). Abolishment of active movement of malaria pigment is an alternative approach to assess drug sensitivity for artesunate. Malaria pigment movement is abolished by artesunate early after exposure, but at concentrations higher than those inhibiting growth.

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356

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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.

Duenser MW, Festic E, Dondorp A, Kissoon N, Ganbat T, Kwizera A, Haniffa R, Baker T, Schultz MJ, Med ESIC. 2012. Erratum to: Recommendations for sepsis management in resource-limited settings (vol 38, pg 557, 2012) INTENSIVE CARE MEDICINE, 38 (4), pp. 575-576. | Read more

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.

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.

Maude RR, Maude RJ, Ghose A, Amin MR, Islam MB, Ali M, Bari MS, Majumder MI et al. 2012. Seroepidemiological surveillance of Burkholderia pseudomallei in Bangladesh Transactions of the Royal Society of Tropical Medicine and Hygiene, 106 (9), pp. 576-578. | Show Abstract | Read more

Melioidosis (. Burkholderia pseudomallei infection) has yet to be demonstrated systematically in Bangladesh. A prospective, cross-sectional serological survey was conducted in 2010 at six Bangladeshi hospitals. Age, gender, occupation and residential address were recorded. Of 1244 patients, 359 (28.9%) were positive for . B. pseudomallei by indirect haemagglutination assay. Farmers had an increased risk of seropositivity (risk ratio. =. 1.4, 95% CI 1.0-1.8; p. =. 0.03). There was no clear geographic clustering of seropositives. Melioidosis should be considered as a possible cause of febrile illness in Bangladesh. Further studies are needed to establish the incidence of clinical disease and distribution of environmental risk. © 2012 Royal Society of Tropical Medicine and Hygiene.

Maude R, Abu Sayeed A, Beare N, Charunwatthana P, Faiz MA, Hossain A, Bin Yunus E, Hoque MG et al. 2011. MALARIAL RETINOPATHY IN ADULTS JOURNAL OF INFECTION, 63 (6), pp. 494-495. | Read more

Maude R, Hoque MG, Hasan MMU, Abu Sayeed M, Akter S, Samad R, Alam B, Bin Yunus E et al. 2011. TIMING OF ENTERAL FEEDING IN CEREBRAL MALARIA IN RESOURCE-POOR SETTINGS: A RANDOMIZED TRIALCATEGORY: SCIENTIFIC FREE PAPER JOURNAL OF INFECTION, 63 (6), pp. E101-E101.

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.

Paris DH, Chansamouth V, Nawtaisong P, Löwenberg EC, Phetsouvanh R, Blacksell SD, Lee SJ, Dondorp AM et al. 2012. Coagulation and inflammation in scrub typhus and murine typhus--a prospective comparative study from Laos. Clin Microbiol Infect, 18 (12), pp. 1221-1228. | Show Abstract | Read more

Scrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited. This study compared human in vivo changes in 15 coagulation, inflammation and endothelial activation markers in prospectively collected admission and follow-up samples of 121 patients (55 scrub typhus, 55 murine typhus, and 11 typhus-like illness) and 51 healthy controls from Laos. As compared with controls, all but one of the markers assessed were significantly affected in typhus patients; however, the activation patterns differed significantly between scrub and murine typhus patients. The levels of markers of coagulation activation and all inflammatory cytokines, except for interleukin-12, were significantly higher in patients with scrub typhus than in those with murine typhus. In patients with murine typhus, however, the levels of endothelium-derived markers were significantly higher. Anticoagulant factors were inhibited in both typhus patient groups. This is the first study demonstrating that, in scrub typhus, in vivo coagulation activation is prominent and is related to a strong proinflammatory response, whereas in murine typhus, changes in coagulant and fibrinolytic pathways are suggestive of endothelial cell perturbation. These data suggest that, although late-stage endothelial infection is common in both diseases, the in vivo pathogenic mechanisms of R. typhi and O. tsutsugamushi could differ in the early phase of infection and may contribute to disease differentiation.

Mtove G, Amos B, Nadjm B, Hendriksen IC, Dondorp AM, Mwambuli A, Kim DR, Ochiai RL et al. 2011. Decreasing incidence of severe malaria and community-acquired bacteraemia among hospitalized children in Muheza, north-eastern Tanzania, 2006-2010. Malar J, 10 (1), pp. 320. | Show Abstract | Read more

BACKGROUND: The annual incidence and temporal trend of severe malaria and community-acquired bacteraemia during a four-year period in Muheza, Tanzania was assessed. METHODS: Data on severely ill febrile children aged 2 months to 14 years from three prospective studies conducted at Muheza District Hospital from 2006 to 2010 was pooled and analysed. On admission, each enrolled child had a thin and thick blood film and at least one rapid diagnostic test for falciparum malaria, as well as a blood culture. The annual incidence of bacteraemia and severe malaria among children coming from Muheza was calculated and their temporal trend was assessed. RESULTS: Overall, 1, 898 severe falciparum malaria and 684 bacteraemia cases were included. Of these, 1, 356 (71%) and 482 (71%), respectively, were from the referral population of Muheza. The incidence of falciparum malaria and all-cause bacteraemia in Muheza decreased five-fold and three-fold, respectively, from the first to the fourth year of surveillance (p < 0.0001). During this period, the median ages of children from Muheza admitted with severe malaria increased from 1.7 to 2.5 years (p < 0.0001). The reduction in all-cause bacteraemia was mainly driven by the 11-fold decline in the incidence of non-typhoidal salmonellosis. The annual incidences of Haemophilus influenzae and pneumococcal invasive bacterial infections decreased as well but were much fewer in number. CONCLUSIONS: These results add to the growing evidence of the decline in malaria associated with a decrease in non-typhoidal salmonellosis and possibly other bacteraemias. Malarial prevention and control strategies may provide a greater benefit than the mere reduction of malaria alone.

Dondorp AM, Fairhurst RM, Slutsker L, Macarthur JR, Breman JG, Guerin PJ, Wellems TE, Ringwald P, Newman RD, Plowe CV. 2011. The threat of artemisinin-resistant malaria. N Engl J Med, 365 (12), pp. 1073-1075. | Read more

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.

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.

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, 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.

Maude RJ, Plewes K, Dimock J, Dondorp AM. 2011. Low-cost portable fluorescein angiography. Br J Ophthalmol, 95 (9), pp. 1213-1215. | Show Abstract | Read more

Fundus fluorescein angiography has great potential as a unique non-invasive tool to investigate in vivo the microvascular pathogenesis of a wide variety of diseases affecting the central nervous system. However, because it requires a bulky and expensive tabletop retinal camera, it is normally limited to cooperative and alert seated patients in well-resourced settings. Recently completed and ongoing studies of the pathogenesis of severe malaria are using fluorescein angiography to examine in detail the postulated central role of microvascular obstruction. We describe a novel method of fluorescein angiography with a portable retinal camera that can be adapted at very low cost for use in sick patients at the bedside. This method greatly expands the scope of potential studies utilising fluorescein angiography.

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.

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.

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.

Nguyen HP, Hanson J, Bethell D, Nguyen TH, Tran TH, Ly VC, Pham PL, Dinh XS et al. 2011. A retrospective analysis of the haemodynamic and metabolic effects of fluid resuscitation in Vietnamese adults with severe falciparum malaria. PLoS One, 6 (10), pp. e25523. | Show Abstract | Read more

BACKGROUND: Optimising the fluid resuscitation of patients with severe malaria is a simple and potentially cost-effective intervention. Current WHO guidelines recommend central venous pressure (CVP) guided, crystalloid based, resuscitation in adults. METHODS: Prospectively collected haemodynamic data from intervention trials in Vietnamese adults with severe malaria were analysed retrospectively to assess the responses to fluid resuscitation. RESULTS: 43 patients were studied of whom 24 received a fluid load. The fluid load resulted in an increase in cardiac index (mean increase: 0.75 L/min/m(2) (95% Confidence interval (CI): 0.41 to 1.1)), but no significant change in acid-base status post resuscitation (mean increase base deficit 0.6 mmol/L (95% CI: -0.1 to 1.3). The CVP and PAoP (pulmonary artery occlusion pressure) were highly inter-correlated (r(s) = 0.7, p<0.0001), but neither were correlated with acid-base status (arterial pH, serum bicarbonate, base deficit) or respiratory status (PaO(2)/FiO(2) ratio). There was no correlation between the oxygen delivery (DO(2)) and base deficit at the 63 time-points where they were assessed simultaneously (r(s) = -0.09, p = 0.46). CONCLUSIONS: In adults with severe falciparum malaria there was no observed improvement in patient outcomes or acid-base status with fluid loading. Neither CVP nor PAoP correlated with markers of end-organ perfusion or respiratory status, suggesting these measures are poor predictors of their fluid resuscitation needs.

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

Mohanty S, Mishra SK, Patnaik R, Dutt AK, Pradhan S, Das B, Patnaik J, Mohanty AK, Lee SJ, Dondorp AM. 2011. Brain swelling and mannitol therapy in adult cerebral malaria: a randomized trial. Clin Infect Dis, 53 (4), pp. 349-355. | Show Abstract | Read more

BACKGROUND: Coma is a frequent presentation of severe malaria in adults and an important cause of death. The role of cerebral swelling in its pathogenesis, and the possible benefit of intravenous mannitol therapy to treat this, is uncertain. METHODS: A computed tomographic (CT) scan of the cerebrum and lumbar puncture with measurement of cerebrospinal fluid (CSF) pressure were performed on admission for 126 consecutive adult Indian patients with cerebral malaria. Patients with brain swelling on CT scan were randomized to adjunctive treatment with intravenous mannitol (1.5 g/kg followed by 0.5 g/kg every 8 hours; n = 30) or no adjunctive therapy (n = 31). RESULTS: On CT scan 80 (63%) of 126 patients had cerebral swelling, of whom 36 (29%) had moderate or severe swelling. Extent of brain swelling was not related to coma depth or mortality. CSF pressures were elevated (≥200 mm H(2)O) in 43 (36%) of 120 patients and correlated with CT scan findings (P for trend = .001). Mortality with mannitol therapy was 9 (30%) of 30 versus 4 (13%) of 31 without adjunctive therapy (hazard ratio, 2.4 [95% confidence interval, 0.8-7.3]; P = .11). Median coma recovery time was 90 hours (range, 22-380 hours) with mannitol versus 32 hours (range, 5-168 hours) without (P = .02). CONCLUSIONS: Brain swelling on CT scan is a common finding in adult patients with cerebral malaria but is not related to coma depth or survival. Mannitol therapy as adjunctive treatment for brain swelling in adult cerebral malaria prolongs coma duration and may be harmful.

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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.

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.

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 Bulletin of the World Health Organization, 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.

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Mtove G, Amos B, Nadjm B, Hendriksen ICE, Dondorp AM, Mwambuli A, Kim DR, Ochiai RL et al. 2011. Decreasing incidence of severe malaria and community-acquired bacteraemia among hospitalized children in Muheza, north-eastern Tanzania, 2006-2010 Malaria Journal, 10 | Show Abstract | Read more

Background: The annual incidence and temporal trend of severe malaria and community-acquired bacteraemia during a four-year period in Muheza, Tanzania was assessed. Methods. Data on severely ill febrile children aged 2 months to 14 years from three prospective studies conducted at Muheza District Hospital from 2006 to 2010 was pooled and analysed. On admission, each enrolled child had a thin and thick blood film and at least one rapid diagnostic test for falciparum malaria, as well as a blood culture. The annual incidence of bacteraemia and severe malaria among children coming from Muheza was calculated and their temporal trend was assessed. Results: Overall, 1, 898 severe falciparum malaria and 684 bacteraemia cases were included. Of these, 1, 356 (71%) and 482 (71%), respectively, were from the referral population of Muheza. The incidence of falciparum malaria and all-cause bacteraemia in Muheza decreased five-fold and three-fold, respectively, from the first to the fourth year of surveillance (p < 0.0001). During this period, the median ages of children from Muheza admitted with severe malaria increased from 1.7 to 2.5 years (p < 0.0001). The reduction in all-cause bacteraemia was mainly driven by the 11-fold decline in the incidence of non-typhoidal salmonellosis. The annual incidences of Haemophilus influenzae and pneumococcal invasive bacterial infections decreased as well but were much fewer in number. Conclusions: These results add to the growing evidence of the decline in malaria associated with a decrease in non-typhoidal salmonellosis and possibly other bacteraemias. Malarial prevention and control strategies may provide a greater benefit than the mere reduction of malaria alone. © 2011Mtove et al; licensee BioMed Central Ltd.

Abu Sayeed A, Maude RJ, Hasan MU, Mohammed N, Hoque MG, Dondorp AM, Faiz MA. 2011. Malarial retinopathy in Bangladeshi adults. Am J Trop Med Hyg, 84 (1), pp. 141-147. | Show Abstract | Read more

To establish if assessment of malarial retinopathy in adult malaria using ophthalmoscopy by non-ophthalmologists has clinical and prognostic significance, 210 Bangladeshi adults were assessed by both direct and indirect ophthalmoscopy; 20 of 20 healthy subjects and 20 of 20 patients with vivax malaria showed no retinal changes, whereas in patients with falciparum malaria, indirect ophthalmoscopy revealed malarial retinopathy (predominantly retinal hemorrhages) in 18 of 21 (86%) fatal, 31 of 75 (41%) cerebral, 16 of 64 (25%) non-cerebral but severe, and 1 of 31 (3%) uncomplicated cases. Direct ophthalmoscopy missed retinopathy in one of these cases and found fewer retinal hemorrhages (mean difference = 3.09; 95% confidence interval = 1.50-4.68; P < 0.0001). Severity of retinopathy increased with severity of disease (P for trend < 0.0001), and renal failure, acidosis, and moderate/severe retinopathy were independent predictors of mortality by both ophthalmoscopic techniques. Direct ophthalmoscopy by non-ophthalmologists is an important clinical tool to aid diagnosis and prognosis in adults with severe malaria, and indirect ophthalmoscopy by non-ophthalmologists, although more sensitive, provides minimal additional prognostic information.

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.

Nguyen HP, Hanson J, Bethell D, Nguyen TH, Tran TH, Ly VC, Pham PL, Sinh DX et al. 2011. A retrospective analysis of the haemodynamic and metabolic effects of fluid resuscitation in vietnamese adults with severe falciparum malaria PLoS ONE, 6 (10), | Show Abstract | Read more

Background: Optimising the fluid resuscitation of patients with severe malaria is a simple and potentially cost-effective intervention. Current WHO guidelines recommend central venous pressure (CVP) guided, crystalloid based, resuscitation in adults. Methods: Prospectively collected haemodynamic data from intervention trials in Vietnamese adults with severe malaria were analysed retrospectively to assess the responses to fluid resuscitation. Results: 43 patients were studied of whom 24 received a fluid load. The fluid load resulted in an increase in cardiac index (mean increase: 0.75 L/min/m2 (95% Confidence interval (CI): 0.41 to 1.1)), but no significant change in acid-base status post resuscitation (mean increase base deficit 0.6 mmol/L (95% CI: -0.1 to 1.3). The CVP and PAoP (pulmonary artery occlusion pressure) were highly inter-correlated (rs = 0.7, p&0.0001), but neither were correlated with acid-base status (arterial pH, serum bicarbonate, base deficit) or respiratory status (PaO2/FiO2 ratio). There was no correlation between the oxygen delivery (DO2) and base deficit at the 63 time-points where they were assessed simultaneously (rs=-0.09, p=0.46). Conclusions: In adults with severe falciparum malaria there was no observed improvement in patient outcomes or acid-base status with fluid loading. Neither CVP nor PAoP correlated with markers of end-organ perfusion or respiratory status, suggesting these measures are poor predictors of their fluid resuscitation needs. © 2011 Phu et al.

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.

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.

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Dondorp AM, Yeung S, White L, Nguon C, Day NPJ, Socheat D, von Seidlein L. 2010. Artemisinin resistance: current status and scenarios for containment (vol 8, pg 272, 2010) NATURE REVIEWS MICROBIOLOGY, 8 (7), pp. 530-530. | Read more

Omodeo-Salè F, Cortelezzi L, Vommaro Z, Scaccabarozzi D, Dondorp AM. 2010. Dysregulation of L-arginine metabolism and bioavailability associated to free plasma heme. Am J Physiol Cell Physiol, 299 (1), pp. C148-C154. | Show Abstract | Read more

Severe Plasmodium falciparum malaria is associated with hypoargininemia, which contributes to impaired systemic and pulmonary nitric oxide (NO) production and endothelial dysfunction. Since intravascular hemolysis is an intrinsic feature of severe malaria, we investigated whether and by which mechanisms free heme [Fe(III)-protoporphyrin IX (FP)] might contribute to the dysregulation of L-arginine (L-Arg) metabolism and bioavailability. Carrier systems "y+" [or cationic amino acid transporter (CAT)] and "y+L" transport L-Arg into red blood cells (RBC), where it is hydrolyzed to ornithine and urea by arginase (isoform I) or converted to NO* and citrulline by endothelial nitric oxide synthase (eNOS). Our results show a significant and dose-dependent impairment of L-Arg transport into RBC pretreated with FP, with a strong inhibition of the system carrier y+L. Despite the impaired L-Arg influx, higher amounts of L-Arg-derived urea are produced by RBC preexposed to FP caused by activation of RBC arginase I. This activation appeared not to be mediated by oxidative modifications of the enzyme. We conclude that L-Arg transport across RBC membrane is impaired and arginase-mediated L-Arg consumption enhanced by free heme. This could contribute to reduced NO production in severe malaria.

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.

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.

Dondorp AM, Yeung S, White L, Nguon C, Day NP, Socheat D, von Seidlein L. 2010. Artemisinin resistance: current status and scenarios for containment. Nat Rev Microbiol, 8 (4), pp. 272-280. | Show Abstract | Read more

Artemisinin combination therapies are the first-line treatments for uncomplicated Plasmodium falciparum malaria in most malaria-endemic countries. Recently, partial artemisinin-resistant P. falciparum malaria has emerged on the Cambodia-Thailand border. Exposure of the parasite population to artemisinin monotherapies in subtherapeutic doses for over 30 years, and the availability of substandard artemisinins, have probably been the main driving force in the selection of the resistant phenotype in the region. A multifaceted containment programme has recently been launched, including early diagnosis and appropriate treatment, decreasing drug pressure, optimising vector control, targeting the mobile population, strengthening management and surveillance systems, and operational research. Mathematical modelling can be a useful tool to evaluate possible strategies for containment.

Wiersinga WJ, Kager LM, Hovius JW, van der Windt GJ, de Vos AF, Meijers JC, Roelofs JJ, Dondorp A et al. 2010. Urokinase receptor is necessary for bacterial defense against pneumonia-derived septic melioidosis by facilitating phagocytosis. J Immunol, 184 (6), pp. 3079-3086. | Show Abstract | Read more

Urokinase receptor (urokinase-type plasminogen activator receptor [uPAR], CD87), a GPI-anchored protein, is considered to play an important role in inflammation and fibrinolysis. The Gram-negative bacterium Burkholderia pseudomallei is able to survive and replicate within leukocytes and causes melioidosis, an important cause of pneumonia-derived community-acquired sepsis in Southeast Asia. In this study, we investigated the expression and function of uPAR both in patients with septic melioidosis and in a murine model of experimental melioidosis. uPAR mRNA and surface expression was increased in patients with septic melioidosis in/on both peripheral blood monocytes and granulocytes as well as in the pulmonary compartment during experimental pneumonia-derived melioidosis in mice. uPAR-deficient mice intranasally infected with B. pseudomallei showed an enhanced growth and dissemination of B. pseudomallei when compared with wild-type mice, corresponding with increased pulmonary and hepatic inflammation. uPAR knockout mice demonstrated significantly reduced neutrophil migration toward the pulmonary compartment after inoculation with B. pseudomallei. Further in vitro experiments showed that uPAR-deficient macrophages and granulocytes display a markedly impaired phagocytosis of B. pseudomallei. Additional studies showed that uPAR deficiency did not influence hemostatic and fibrinolytic responses during severe melioidosis. These data suggest that uPAR is crucially involved in the host defense against sepsis caused by B. pseudomallei by facilitating the migration of neutrophils toward the primary site of infection and subsequently facilitating the phagocytosis of B. pseudomallei.

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.

Maude RJ, Hassan MU, Ghose A, Douthwaite ST, Faiz MA, Dondorp AM. 2010. Studies on severe malaria are still possible and essential. Clin Infect Dis, 50 (2), pp. 281-282. | Read more

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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 The Lancet, 376 (9753), pp. 1647-1657. | Show Abstract | Read more

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. 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. 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. 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. The Wellcome Trust. © 2010 Elsevier Ltd.

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.

Rahman W, Chotivanich K, Silamut K, Tanomsing N, Hossain A, Faiz MA, Dondorp AM, Maude RJ. 2010. Plasmodium malariae in Bangladesh. Trans R Soc Trop Med Hyg, 104 (1), pp. 78-80. | Show Abstract | Read more

We describe a 32-year-old Bangladeshi male presenting with severe malaria caused by a mono-infection with Plasmodium malariae. Rosetting of infected and uninfected erythrocytes, a putative virulence factor in falciparum malaria, was observed in the blood slide. Severe disease caused by P. malariae is extremely rare. The patient made a rapid recovery with intravenous quinine treatment.

Löwenberg EC, Charunwatthana P, Cohen S, van den Born BJ, Meijers JC, Yunus EB, Hassan MU, Hoque G et al. 2010. Severe malaria is associated with a deficiency of von Willebrand factor cleaving protease, ADAMTS13. Thromb Haemost, 103 (1), pp. 181-187. | Show Abstract | Read more

Severe falciparum malaria remains a major killer in tropical countries. Central in the pathophysiology is mechanical obstruction in the microcirculation caused by cytoadherence and sequestration of parasitized erythrocytes. However, the pathogenesis of many features complicating severe malaria, including coma, renal failure and thrombocytopenia, remains incompletely understood. These disease manifestations are also key features of thrombotic thrombocytopenic purpura, a life-threatening disease strongly associated with a deficiency of the von Willebrand factor (VWF) cleaving protease, ADAMTS13. We measured plasma ADAMTS13 activity, VWF antigen and VWF propeptide levels in 30 patients with severe falciparum malaria, 12 patients with uncomplicated falciparum malaria and 14 healthy Bangladeshi controls. In patients with severe malaria ADAMTS13 activity levels were markedly decreased in comparison to normal controls (mean [95%CI]: 23% [20-26] vs. 64% [55-72]) and VWF antigen and propeptide concentrations were significantly elevated (VWF antigen: 439% [396-481] vs. 64% [46-83]; VWF propeptide: 576% [481-671] vs. 69% [59-78]). In uncomplicated malaria VWF levels were also increased compared to healthy controls but ADAMTS13 activity was normal. The results suggest that decreased ADAMTS13 activity in combination with increased VWF concentrations may contribute to the complications in severe malaria.

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.

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.

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 (vol 361, pg 455, 2009) NEW ENGLAND JOURNAL OF MEDICINE, 361 (17), pp. 1714-1714.

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.

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

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.

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.

Wiersinga WJ, van't Veer C, van den Pangaart PS, Dondorp AM, Day NP, Peacock SJ, van der Poll T. 2009. Immunosuppression associated with interleukin-1R-associated-kinase-M upregulation predicts mortality in Gram-negative sepsis (melioidosis). Crit Care Med, 37 (2), pp. 569-576. | Show Abstract | Read more

OBJECTIVES: Sepsis is associated with immunosuppression (characterized by a reduced capacity of circulating monocytes to release proinflammatory cytokines), which has been implicated in late mortality. Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast Asia with a mortality of up to 40%. Previous in vitro and murine studies have suggested a key role for the so-called negative regulators of the toll-like receptor (TLR) signaling pathway in immunosuppression. In this study, we investigated the expression of these negative TLR regulators in patients with septic melioidosis in association with the responsiveness of peripheral blood leukocytes of these patients to lipopolysaccharide and B. pseudomallei. DESIGN: Ex vivo study. SETTING: Academic research laboratory. PATIENTS: Thirty-two healthy controls and 34 patients with sepsis caused by B. pseudomallei. INTERVENTIONS: None. MEASUREMENTS: 1) Plasma cytokine levels; 2) ex vivo cytokine production capacity of whole blood; and 3) purified mononuclear cell-derived messenger RNA (mRNA) levels of key inhibitory molecules of the TLR-signaling cascade were investigated. MAIN RESULTS: In accordance with an immunosuppressed state, whole blood of patients demonstrated a strongly decreased capacity to release the proinflammatory cytokines tumor necrosis factor-[alpha], interleukin-1[beta], and the chemokine interleukin-8 after ex vivo stimulation with lipopolysaccharide or B. pseudomallei. Analysis of myeloid-differentiation-88-short, interleukin-1R-associated-kinase (IRAK)-M, IRAK-1, suppressor-of-cytokine signaling-3, Src-homology-2-domain-containing inositol-5-phosphatase-1, single-immunoglobulin-interleukin-1R-related-molecule, and A20 mRNA expression in purified mononuclear cells showed decreased IRAK-1 and elevated IRAK-M expression in patients with septic melioidosis. Immunosuppression was correlated with mortality; furthermore, patients who eventually died had higher IRAK-M mRNA levels on admission than the patients who survived. CONCLUSIONS: Immunosuppression in sepsis caused by B. pseudomallei is associated with an upregulation of IRAK-M and an indicator of poor outcome.

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.

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.

Steenkeste N, Dillies MA, Khim N, Sismeiro O, Chy S, Lim P, Crameri A, Bouchier C et al. 2009. FlexiChip package: an universal microarray with a dedicated analysis software for high-thoughput SNPs detection linked to anti-malarial drug resistance. Malar J, 8 (1), pp. 229. | Show Abstract | Read more

BACKGROUND: A number of molecular tools have been developed to monitor the emergence and spread of anti-malarial drug resistance to Plasmodium falciparum. One of the major obstacles to the wider implementation of these tools is the absence of practical methods enabling high throughput analysis. Here a new Zip-code array is described, called FlexiChip, linked to a dedicated software program, which largely overcomes this problem. METHODS: Previously published microarray probes detecting single-nucleotide polymorphisms (SNP) associated with parasite resistance to anti-malarial drugs (ResMalChip) were adapted for a universal microarray FlexiChip format. To evaluate the overall sensitivity of the FlexiChip package (microarray + software), the results of FlexiChip were compared to ResMalChip microarray, using the same extension probes and with the same PCR products. In both cases, sequence results were used as gold standard to calculate sensitivity and specificity. FlexiChip results obtained with a set of field isolates were then compared to those assessed in an independent reference laboratory. RESULTS: The FlexiChip package gave results identical to the ResMalChip results in 92.7% of samples (kappa coefficient 0.8491, with a standard error 0.021) and had a sensitivity of 95.88% and a specificity of 97.68% compared to the sequencing as the reference method. Moreover the method performed well compared to the results obtained in the reference laboratories, with 99.7% of identical results (kappa coefficient 0.9923, S.E. 0.0523). CONCLUSION: Microarrays could be employed to monitor P. falciparum drug resistance markers with greater cost effectiveness and the possibility for high throughput analysis. The FlexiChip package is a promising tool for use in poor resource settings of malaria endemic countries.

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.

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.

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.

Maude RJ, Dondorp AM, Faiz MA, Yunus EB, Samad R, Hossain A, Rahman MR. 2008. Malaria in southeast Bangladesh: a descriptive study. Bangladesh Med Res Counc Bull, 34 (3), pp. 87-89. | Show Abstract | Read more

Malaria in Asia is thought to be grossly under-reported and this is evident from previously published statistics from Bangladesh. Malaria screening data from four Upazillas was analysed alongside census data to assess the trends in malaria incidence over time and distribution of malaria by age and gender. Malaria incidence in this area has decreased by around two thirds since 2003, although control measures were not significantly increased until 2005. Malaria occurred in people of all ages with the highest incidence being in young adults. This is consistent with higher occupational exposure in this group. The probability of being screened for malaria decreased with age suggesting significant numbers of adults with malaria may be being missed.

Rahman MM, Dondorp AM, Day NP, Lindegardh N, Imwong M, Faiz MA, Bangali AM, Kamal AT, Karim J, Kaewkungwal J, Singhasivanon P. 2008. Adherence and efficacy of supervised versus non-supervised treatment with artemether/lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in Bangladesh: a randomised controlled trial. Trans R Soc Trop Med Hyg, 102 (9), pp. 861-867. | Show Abstract | Read more

As artemether/lumefantrine is now deployed as the first-line treatment for uncomplicated falciparum malaria in Bangladesh, information on its efficacy and adherence to its use is important. A randomised controlled non-inferiority trial comparing directly observed treatment (DOT) and non-directly observed treatment (NDOT) was conducted in 320 patients with uncomplicated falciparum malaria in Bandarban Hill Tract District, Bangladesh. Both regimens showed similar high levels of PCR-corrected 42-day parasitological and clinical cure rates (99.3% in the NDOT group and 100% in the DOT group; P=0.49). Survival analysis for the time to recurrence of infection showed no difference between treatment groups (log rank, P=0.98). Adherence, as assessed by counting remaining tablets and oral interviews, was 93% in the NDOT group and was confirmed by Day 7 lumefantrine concentrations. Adherence was independent of educational level. Patients with plasma lumefantrine concentrations < 280 ng/ml at Day 7 were at greater risk for re-infection (relative risk 5.62; P=0.027). The efficacy of artemether/lumefantrine for the treatment of uncomplicated falciparum malaria in Bangladesh is high and is similar for DOT and NDOT. Adherence to therapy is high.

Deen JL, von Seidlein L, Dondorp A. 2008. Therapy of uncomplicated malaria in children: a review of treatment principles, essential drugs and current recommendations. Trop Med Int Health, 13 (9), pp. 1111-1130. | Show Abstract | Read more

Understanding the optimal treatment of uncomplicated malaria in children is challenging because of the availability of new drugs and the shift to combination therapies. This is a review of the guiding principles for the treatment of uncomplicated malaria, the essential anti-malarial drugs for children, and the treatment regimens currently recommended.

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.

Nuchsongsin F, Chotivanich K, Charunwatthana P, Fausta OS, Taramelli D, Day NP, White NJ, Dondorp AM. 2008. Effects of malaria heme products on red blood cell deformability (vol 77, pg 617, 2007) AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 78 (5), pp. 847-847.

Dondorp AM. 2008. Clinical significance of sequestration in adults with severe malaria. Transfus Clin Biol, 15 (1-2), pp. 56-57. | Show Abstract | Read more

Reduced microcirculatory flow is a fundamental feature in the pathophysiology of severe Plasmodium falciparum malaria and sequestration of red blood cells containing mature parasites is considered a central cause of this. Direct microscopic observation of the microcirculation in the living patient with severe malaria has enabled us to quantify this phenomenon and link it to severity of disease, supporting the findings of pathology studies. Moreover, the sequestered parasite biomass, calculated from parasite derived plasma PfHRP2 concentrations, strongly correlates with disease severity. Artesunate prevents sequestration by killing ring form parasites, aborting their maturation, which can explain the mortality benefit of this drug compared to quinine in the treatment of adult severe malaria. Levamisole is currently tried as adjunctive treatment in severe malaria targeting sequestration.

Dondorp AM, Ince C, Charunwatthana P, Hanson J, van Kuijen A, Faiz MA, Rahman MR, Hasan M et al. 2008. Direct in vivo assessment of microcirculatory dysfunction in severe falciparum malaria. J Infect Dis, 197 (1), pp. 79-84. | Show Abstract | Read more

BACKGROUND: This study sought to describe and quantify microcirculatory changes in the mucosal surfaces of patients with severe malaria, by direct in vivo observation using orthogonal polarization spectral (OPS) imaging. METHODS: The microcirculation in the rectal mucosa of adult patients with severe malaria was assessed by use of OPS imaging, at admission and then daily. Comparison groups comprised patients with uncomplicated falciparum malaria, patients with bacterial sepsis, and healthy individuals. RESULTS: Erythrocyte velocities were measured directly in 43 adult patients with severe falciparum malaria, of whom 20 died. Microcirculatory blood flow was markedly disturbed, with heterogeneous obstruction that was proportional to severity of disease. Blocked capillaries were found in 29 patients (67%) and were associated with concurrent hyperdynamic blood flow (erythrocyte velocity, >750 mm/s) in adjacent vessels in 27 patients (93%). The proportion of blocked capillaries correlated with the base deficit in plasma and with the concentration of lactate. Abnormalities disappeared when the patients recovered. In healthy individuals and in patients with uncomplicated malaria or sepsis, no stagnant erythrocytes were detected, and, in patients with sepsis, hyperdynamic blood flow was prominent. CONCLUSION: Patients with severe falciparum malaria show extensive microvascular obstruction that is proportional to the severity of the disease. This finding underscores the prominent role that microvascular obstruction plays in the pathophysiology of severe malaria and illustrates the fundamental difference between the microvascular pathophysiology of malaria and that of bacterial sepsis.

Nantakomol D, Chimma P, Day NP, Dondorp AM, Combes V, Krudsood S, Looareesuwan S, White NJ, Pattanapanyasat K, Chotivanich K. 2008. Quantitation of cell-derived microparticles in plasma using flow rate based calibration Southeast Asian Journal of Tropical Medicine and Public Health, 39 (1), pp. 146-153. | Show Abstract

Activation of vascular endothelium and blood cells can result in the formation of microparticles (MPs), which are membrane vesicles with a diameter < 1 μm which can play a pathogenetic role in a variety of infectious and other diseases. In this study, we validated a modified quantitative method called "flow rate based calibration", to measure circulating MPs in plasma of healthy subjects and malaria patients using FACSCalibur flow cytometry. MPs counts obtained from "flow rate based calibration" correlated closely with the standard method (R2=0.9, p=0.001). The median (range) number of MPs in healthy subjects was 163/μl (81-375/μl). We demonstrated a flow rate based calibration for the quantitation of MPs in P. falciparum malaria-infected patients. The median (range) number of MPs was 2,051/μl (222-6,432/μl), n=28 in patients with falciparum malaria. The number of MPs in plasma from patients with severe falciparum malaria was significantly higher than in uncomplicated falciparum malaria (2,567/μl (366-6,432/μl), n=18 versus [1,947/μl (222-4,107/μl), n=10, p<0.01]. Cellular origin of MPs in malaria patients were mainly derived from red blood cells (35%), platelets (10%), and endothelial cells (5%). There was no significant correlation between the total number of MPs and parasitemia. Flow rate based calibration is a simple, reliable, reproducible method and more affordable to quantitate MPs.

Nantakomol D, Chimma P, Day NP, Dondorp AM, Combes V, Krudsood S, Looareesuwan S, White NJ, Pattanapanyasat K, Chotivanich K. 2008. Quantitation of cell-derived microparticles in plasma using flow rate based calibration. Southeast Asian J Trop Med Public Health, 39 (1), pp. 146-153. | Show Abstract

Activation of vascular endothelium and blood cells can result in the formation of microparticles (MPs), which are membrane vesicles with a diameter < 1 microm which can play a pathogenetic role in a variety of infectious and other diseases. In this study, we validated a modified quantitative method called "flow rate based calibration", to measure circulating MPs in plasma of healthy subjects and malaria patients using FACSCalibur flow cytometry. MPs counts obtained from "flow rate based calibration" correlated closely with the standard method (R2 = 0.9, p = 0.001). The median (range) number of MPs in healthy subjects was 163/microl (81-375/microl). We demonstrated a flow rate based calibration for the quantitation of MPs in P. falciparum malaria-infected patients. The median (range) number of MPs was 2,051/microl (222-6,432/microl), n = 28 in patients with falciparum malaria. The number of MPs in plasma from patients with severe falciparum malaria was significantly higher than in uncomplicated falciparum malaria (2,567/microl (366-6,432/microl), n = 18 versus [1,947/microl (222-4,107/microl), n = 10, p < 0.01]. Cellular origin of MPs in malaria patients were mainly derived from red blood cells (35%), platelets (10%), and endothelial cells (5%). There was no significant correlation between the total number of MPs and parasitemia. Flow rate based calibration is a simple, reliable, reproducible method and more affordable to quantitate MPs.

Pongtavornpinyo W, Yeung S, Hastings IM, Dondorp AM, Day NP, White NJ. 2008. Spread of anti-malarial drug resistance: mathematical model with implications for ACT drug policies. Malar J, 7 (1), pp. 229. | Show Abstract | Read more

BACKGROUND: Most malaria-endemic countries are implementing a change in anti-malarial drug policy to artemisinin-based combination therapy (ACT). The impact of different drug choices and implementation strategies is uncertain. Data from many epidemiological studies in different levels of malaria endemicity and in areas with the highest prevalence of drug resistance like borders of Thailand are certainly valuable. Formulating an appropriate dynamic data-driven model is a powerful predictive tool for exploring the impact of these strategies quantitatively. METHODS: A comprehensive model was constructed incorporating important epidemiological and biological factors of human, mosquito, parasite and treatment. The iterative process of developing the model, identifying data needed, and parameterization has been taken to strongly link the model to the empirical evidence. The model provides quantitative measures of outcomes, such as malaria prevalence/incidence and treatment failure, and illustrates the spread of resistance in low and high transmission settings. The model was used to evaluate different anti-malarial policy options focusing on ACT deployment. RESULTS: The model predicts robustly that in low transmission settings drug resistance spreads faster than in high transmission settings, and treatment failure is the main force driving the spread of drug resistance. In low transmission settings, ACT slows the spread of drug resistance to a partner drug, especially at high coverage rates. This effect decreases exponentially with increasing delay in deploying the ACT and decreasing rates of coverage. In the high transmission settings, however, drug resistance is driven by the proportion of the human population with a residual drug level, which gives resistant parasites some survival advantage. The spread of drug resistance could be slowed down by controlling presumptive drug use and avoiding the use of combination therapies containing drugs with mismatched half-lives, together with reducing malaria transmission through vector control measures. CONCLUSION: This paper has demonstrated the use of a comprehensive mathematical model to describe malaria transmission and the spread of drug resistance. The model is strongly linked to the empirical evidence obtained from extensive data available from various sources. This model can be a useful tool to inform the design of treatment policies, particularly at a time when ACT has been endorsed by WHO as first-line treatment for falciparum malaria worldwide.

Day N, Dondorp AM. 2007. The management of patients with severe malaria. Am J Trop Med Hyg, 77 (6 Suppl), pp. 29-35. | Show Abstract

Severe malaria is a global problem, claiming at least 1 million lives annually. Few adequately powered clinical studies have been directed at improving the management of severe malaria over the years, but this situation is slowly changing. The antimalarial treatment of severe disease is being transformed by the development and deployment of the water-soluble artemisinin derivative artesunate. Parenteral artesunate is now the treatment of choice in low-transmission areas and in the 2nd and 3rd trimesters of pregnancy, and research is underway into whether it should replace quinine as the treatment of choice in African children. Development of good manufacturing practice (GMP) formulations should make parenteral artesunate more widely available in the near future. The development of artesunate suppositories offers another exciting prospect, the ability to treat patients with severe disease in remote rural settings, delaying the evolution of disease and buying them time to reach a health care facility. No adjunctive therapy has been shown to improve the outcome of severe malaria, but most studies have been underpowered. Future trials of interventions shown to be promising in pilot studies should be large and adequately powered. This will require multi-center designs and necessitate close collaboration between groups, as well as agreement on the research agenda. We suggest a list of candidate interventions for debate.

Paris DH, Imwong M, Faiz AM, Hasan M, Yunus EB, Silamut K, Lee SJ, Day NP, Dondorp AM. 2007. Loop-mediated isothermal PCR (LAMP) for the diagnosis of falciparum malaria. Am J Trop Med Hyg, 77 (5), pp. 972-976. | Show Abstract

A recently described loop-mediated isothermal polymerase chain reaction (LAMP) for molecular detection of Plasmodium falciparum was compared with microscopy, PfHRP2-based rapid diagnostic test (RDT), and nested polymerase chain reaction (PCR) as the "gold standard" in 115 Bangladeshi in-patients with fever. DNA extraction for LAMP was conducted by conventional methods or simple heating of the sample; test results were either assessed visually or by gel electrophoresis. Conventional DNA extraction followed by gel electrophoresis had the highest agreement with the reference method (81.7%, kappa = 0.64), with a sensitivity (95% CI) of 76.1% (68.3-83.9%), comparable to RDT and microscopy, but a specificity of 89.6% (84.0-95.2%) compared with 100% for RDT and microscopy. DNA extraction by heat treatment deteriorated specificity to unacceptable levels. LAMP enables molecular diagnosis of falciparum malaria in settings with limited technical resources but will need further optimization. The results are in contrast with a higher accuracy reported in an earlier study comparing LAMP with a non-validated PCR method.

Nuchsongsin F, Chotivanich K, Charunwatthana P, Omodeo-Salè F, Taramelli D, Day NP, White NJ, Dondorp AM. 2007. Effects of malaria heme products on red blood cell deformability. Am J Trop Med Hyg, 77 (4), pp. 617-622. | Show Abstract

In falciparum malaria, the deformability of the entire erythrocyte population is reduced in proportion to disease severity, and this compromises microcirculatory blood flow through vessels partially obstructed by cytoadherent parasitized erythrocytes. The cause of rigidity of uninfected erythrocytes in not known but could be mediated by malaria heme products. In this study, we show that red blood cell deformability (RBC-D), measured by laser-assisted optical rotational cell analyzer, decreased in a dose-dependent manner after incubation with hemin and hydrogen peroxide but not with hemoglobin or beta-hematin. Hemin also reduced mean red cell volume. Albumin decreased and N-acetylcysteine (NAC) both prevented and reversed rigidity induced by hemin. Hemin-induced oxidative damage of the membrane seems to be a more important contributor to pathology than cell shrinkage because the antioxidant NAC restored RBC-D but not red blood cell volume. The findings suggest novel approaches to the treatment of potentially lethal malaria.

Hanson JP, Dondorp AM, Day NP. 2007. Malaria treatment in the United States. JAMA, 298 (12), pp. 1396. | Read more

Lindegårdh N, Dondorp AM, Singhasivanon P, White NJ, Day NP. 2007. Validation and application of a liquid chromatographic-mass spectrometric method for determination of artesunate in pharmaceutical samples. J Pharm Biomed Anal, 45 (1), pp. 149-153. | Show Abstract | Read more

A simple and rapid liquid chromatographic-mass spectrometric assay for the evaluation of artesunate in vials for injection has been developed and validated. The content of each vial was dissolved in 3.0 mL of methanol using a SGE analytical syringe (1.0 mL). Each sample was diluted to a theoretical concentration of 1000 ng/mL and analysed in triplicate. Three replicates of calibration standards at concentrations 500, 1000 and 1500 ng/mL were used to construct a calibration curve. Artesunate was analysed by liquid chromatography with atmospheric pressure chemical ionisation (APCI) mass spectrometric (MS) detection on a Hypersil Gold column (100 mm x 4.6 mm) using a mobile phase containing methanol-ammonium acetate 10 mM pH 5.3 (70:30, v/v) at a flow rate of 1 mL/min. The assay was implemented for the analysis of artesunate for injection purchased from Guilin Pharmaceutical Company in China.

Nguansangiam S, Day NP, Hien TT, Mai NT, Chaisri U, Riganti M, Dondorp AM, Lee SJ et al. 2007. A quantitative ultrastructural study of renal pathology in fatal Plasmodium falciparum malaria. Trop Med Int Health, 12 (9), pp. 1037-1050. | Show Abstract | Read more

OBJECTIVE: To use electron microscopy to examine the role of parasitized red blood cell (PRBC) sequestration in the pathogenesis of acute renal failure in severe falciparum malaria. METHODS: Ultrastructural pathological examination of renal tissues from Southeast Asian adults (n = 63) who died from severe falciparum malaria. Qualitative and quantitative determination of the major pathological features of disease, including PRBC and leukocyte sequestration. Clinico-pathological correlation with the pre-mortem clinical picture and peripheral parasite count. RESULTS: There was a high incidence of malaria-associated renal failure in this population (> 40%) and a correlation between this incidence, severe malarial anaemia and shock. Pathological features included PRBC sequestration in glomerular and tubulo-interstitial vessels, acute tubular damage and mild glomerular hypercellularity resulting from the accumulation of host monocytes within glomerular capillaries. No evidence for an immune complex mediated glomerulonephritis was found. There was a correlation between parasite sequestration in the kidney and pre-mortem renal failure, although overall levels of sequestration were relatively low. Levels of sequestration (Knob+ PRBC) were significantly higher in malaria-associated renal failure than in fatal cases without renal failure (P = 0.005). CONCLUSION: Malaria-associated renal failure is a common and serious complication of severe Plasmodium falciparum malaria in this population, associated with acute tubular injury rather than glomerulonephritis, and linked to localization of host monocytes in the kidney as well as sequestration of PRBCs.

Arreesrisom P, Dondorp AM, Looareesuwan S, Udomsangpetch R. 2007. Suppressive effects of the anti-oxidant N-acetylcysteine on the anti-malarial activity of artesunate. Parasitol Int, 56 (3), pp. 221-226. | Show Abstract | Read more

The anti-oxidant drug N-acetylcysteine (NAC) has been proposed as adjunctive treatment in severe falciparum malaria. However, this might inhibit the anti-malarial drug action of the artemisinins, which are thought to exert their parasitocidal action through oxidative damage. We studied the interaction between NAC and artesunate as well as quinine in an in vitro drug sensitivity assay. Combination with NAC reduced the parasitocidal effect of artesunate only within the first 6 h of incubation, whereas no interaction was observed with quinine. Pre-incubation of P. falciparum with NAC resulted in a similar inhibitory effect on the anti-malarial activity of artesunate, whereas no inhibition was observed when NAC was added 2 h after parasite exposure to artesunate. Assessment of parasite maturation inhibition by the standard Giemsa's staining was in accordance with the use of a vital staining. The results herein caution the use of adjunctive treatment for malaria infection. Combination of antagonistic drugs may lead to adverse effects.

Wiersinga WJ, Wieland CW, van der Windt GJ, de Boer A, Florquin S, Dondorp A, Day NP, Peacock SJ, van der Poll T. 2007. Endogenous interleukin-18 improves the early antimicrobial host response in severe melioidosis. Infect Immun, 75 (8), pp. 3739-3746. | Show Abstract | Read more

Melioidosis is caused by the soil saprophyte Burkholderia pseudomallei and is endemic in Southeast Asia. The pathogenesis of melioidosis is still largely unknown, although gamma interferon (IFN-gamma) seems to play an obligatory role in host defense. Previously, we have shown that IFN-gamma production in melioidosis is controlled in part by interleukin-18 (IL-18). The aim of the present study was to determine the role of IL-18 in the immune response to B. pseudomallei. For this the following investigations were performed. (i) Plasma IL-18 and blood monocyte IL-18 mRNA levels were elevated in 34 patients with culture-proven melioidosis compared to the levels in 32 local healthy controls; in addition, IL-18 binding protein levels were markedly elevated in patients, strongly correlating with mortality. (ii) IL-18 gene-deficient (IL-18 knockout [KO]) mice showed accelerated mortality after intranasal infection with a lethal dose of B. pseudomallei, which was accompanied by enhanced bacterial growth in their lungs, livers, spleens, kidneys, and blood at 24 and 48 h postinfection, compared to wild-type mice. In addition, IL-18 KO mice displayed evidence of enhanced hepatocellular injury and renal insufficiency. Together, these data indicate that the enhanced production of IL-18 in melioidosis is an essential part of a protective immune response to this severe infection.

Dondorp AM, Silamut K, Charunwatthana P, Chuasuwanchai S, Ruangveerayut R, Krintratun S, White NJ, Ho M, Day NP. 2007. Levamisole inhibits sequestration of infected red blood cells in patients with falciparum malaria. J Infect Dis, 196 (3), pp. 460-466. | Show Abstract | Read more

BACKGROUND: Sequestration of infected red blood cells (iRBCs) in the microcirculation is central to the pathophysiology of falciparum malaria. It is caused by cytoadhesion of iRBCs to vascular endothelium, mediated through the binding of Plasmodium falciparum erythrocyte membrane protein-1 to several endothelial receptors. Binding to CD36, the major vascular receptor, is stabilized through dephosphorylation of CD36 by an alkaline phosphatase. This is inhibited by the alkaline phosphatase-inhibitor levamisole, resulting in decreased cytoadhesion. METHODS: Patients with uncomplicated falciparum malaria were randomized to receive either quinine treatment alone or treatment with a single 150-mg dose of levamisole as an adjunct to quinine. Peripheral blood parasitemia and parasite stage distribution were monitored closely over time. RESULTS: Compared with those in control subjects, peripheral blood parasitemias of mature P. falciparum parasites increased during the 24 h after levamisole administration (n=21; P=.006). The sequestration ratio (between observed and expected peripheral blood parasitemia) of early trophozoite and midtrophozoite parasites increased after levamisole treatment, with near complete prevention of early trophozoite sequestration and >65% prevention of midtrophozoite sequestration. CONCLUSION: These findings strongly suggest that levamisole decreases iRBC sequestration in falciparum malaria in vivo and should be considered as a potential adjunctive treatment for severe falciparum malaria. TRIAL REGISTRATION: Current Controlled Trials identifier: 15314870.

Dondorp AM, Day NP. 2007. The treatment of severe malaria. Trans R Soc Trop Med Hyg, 101 (7), pp. 633-634. | Show Abstract | Read more

In the SEAQUAMAT trial, parenteral artesunate was shown to be associated with a considerably lower mortality than quinine, and is now the recommended treatment for severe malaria in low-transmission areas and in the second and third trimesters of pregnancy. A trial is underway to establish its role in African children. The development of artesunate suppositories may provide the means to treat patients with severe disease in remote rural settings, potentially buying the time needed to reach a health care facility. The increasing availability of basic intensive care facilities in developing countries also has the potential to further reduce mortality.

Wiersinga WJ, Wieland CW, Dessing MC, Chantratita N, Cheng AC, Limmathurotsakul D, Chierakul W, Leendertse M et al. 2007. Toll-like receptor 2 impairs host defense in gram-negative sepsis caused by Burkholderia pseudomallei (Melioidosis). PLoS Med, 4 (7), pp. e248. | Show Abstract | Read more

BACKGROUND: Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis. METHODS AND FINDINGS: Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury. CONCLUSIONS: Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.

Wiersinga WJ, Dessing MC, Kager PA, Cheng AC, Limmathurotsakul D, Day NP, Dondorp AM, van der Poll T, Peacock SJ. 2007. High-throughput mRNA profiling characterizes the expression of inflammatory molecules in sepsis caused by Burkholderia pseudomallei. Infect Immun, 75 (6), pp. 3074-3079. | Show Abstract | Read more

Sepsis is characterized by an uncontrolled inflammatory response to invading microorganisms. We describe the inflammatory mRNA profiles in whole-blood leukocytes, monocytes, and granulocytes using a multigene system for 35 inflammatory markers that included pro- and anti-inflammatory cytokines, chemokines, and signal transduction molecules in a case-control study with 34 patients with sepsis caused by the gram-negative bacterium Burkholderia pseudomallei (the pathogen causing melioidosis) and 32 healthy volunteers. Relative to healthy controls, patients with sepsis showed increased transcription of a whole array of inflammatory genes in peripheral blood leukocytes, granulocytes, and monocytes. Specific monocyte and granulocyte mRNA profiles were identified. Strong correlations were found between inflammatory mRNA expression levels in monocytes and clinical outcome. These data underline the notion that circulating leukocytes are an important source for inflammatory mediators in patients with gram-negative sepsis. Gene profiling such as was done here provides an excellent tool to obtain insight into the extent of inflammation activation in patients with severe infection.

Paris D, Imwong M, Faiz M, Hasan M, Bin Yunus E, Silamut K, Lee S, Day N, Dondorp A. 2007. Loop-mediated isothermal PCR (LAMP) for the diagnosis of falciparum malaria TROPICAL MEDICINE & INTERNATIONAL HEALTH, 12 pp. 129-129.

Dondorp AM. 2007. The treatment of severe malaria TROPICAL MEDICINE & INTERNATIONAL HEALTH, 12 pp. 21-21.

De Fost M, Chierakul W, Limpaiboon R, Dondorp A, White NJ, van Der Poll T. 2007. Release of granzymes and chemokines in Thai patients with leptospirosis. Clin Microbiol Infect, 13 (4), pp. 433-436. | Show Abstract | Read more

The plasma concentrations of granzymes are considered to reflect the involvement of cytotoxic T-cells and natural killer cells in various disease states. Interferon (IFN)-gamma-inducible protein-10 (IP-10) and monokine induced by IFN-gamma (Mig) are members of the non-ELR CXC chemokine family that act on T-cells and natural killer cells. This study revealed that the plasma concentrations of granzyme B (but not granzyme A), IP-10 and Mig were higher in 44 Thai patients with definite or possible leptospirosis than in healthy blood donors. These data suggest that activation of cell-mediated immunity is part of the early host response to leptospirosis.

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Scopus

de Veij M, Vandenabeele P, Hall KA, Fernandez FM, Green MD, White NJ, Dondorp AM, Newton PN, Moens L. 2007. Fast detection and identification of counterfeit antimalarial tablets by Raman spectroscopy JOURNAL OF RAMAN SPECTROSCOPY, 38 (2), pp. 181-187. | Show Abstract | Read more

During the last decade there has been an apparent increase in the prevalence of counterfeit medicines in developing as well as developed countries. The pivotal antimalarial artesunate has been counterfeited on a large scale in SE Asia. In this work, the possibilities of Raman spectroscopy are explored as a fast and reliable screening method for the detection of counterfeit artesunate tablets. In this study, 50 'artesunate tablets', purchased in SE Asia, were examined. This spectroscopic method was able to distinguish between genuine and counterfeit artesunate and to identify the composition of the counterfeit tablets. These contained no detectable levels of artesunate, but consisted mostly of starch, calcite (CaCO3), and paracetamol (4-acetamidophenol). In one particular case an admixture of rutile (TiO2) and artesunate was detected. The results of the investigation by Raman spectroscopy were in agreement with those of colorimetric tests and of liquid chromatography-mass spectrometry on the artesunate. Moreover, principal components analysis (PCA) was combined with hierarchical cluster analysis to establish an automated approach for the discrimination between different groups of counterfeits and genuine artesunate tablets. These results demonstrate that Raman spectroscopy combined with multivariate analysis is a promising and reliable methodology for the fast characterization of genuine and counterfeit artesunate antimalarial tablets. Copyright © 2006 John Wiley & Sons, Ltd.

White NJ, Day NP, Dondorp A, Anstey N. 2007. UK recommendations for severe malaria are worrying. BMJ, 334 (7592), pp. 490. | Read more

Hall KA, Newton PN, Green MD, De Veij M, Vandenabeele P, Pizzanelli D, Mayxay M, Dondorp A, Fernandez FM. 2006. Characterization of counterfeit artesunate antimalarial tablets from southeast Asia. Am J Trop Med Hyg, 75 (5), pp. 804-811. | Show Abstract

In southeast Asia, the widespread high prevalence of counterfeits tablets of the vital antimalarial artesunate is of great public health concern. To assess the seriousness of this problem, we quantified the amount of active ingredient present in artesunate tablets by liquid chromatography coupled to mass spectrometry. This method, in conjunction with analysis of the packaging, classified tablets as genuine, substandard, or fake and validated results of the colorimetric Fast Red TR test. Eight (35%) of 23 fake artesunate samples contained the wrong active ingredients, which were identified as different erythromycins and paracetamol. Raman spectroscopy identified calcium carbonate as an excipient in 9 (39%) of 23 fake samples. Multivariate unsupervised pattern recognition results indicated two major clusters of artesunate counterfeits, those with counterfeit foil stickers and containing calcium carbonate, erythromycin, and paracetamol, and those with counterfeit holograms and containing starch but without evidence of erythromycin or paracetamol.

Blacksell SD, Doust JA, Newton PN, Peacock SJ, Day NP, Dondorp AM. 2006. A systematic review and meta-analysis of the diagnostic accuracy of rapid immunochromatographic assays for the detection of dengue virus IgM antibodies during acute infection. Trans R Soc Trop Med Hyg, 100 (8), pp. 775-784. | Show Abstract | Read more

A meta-analysis of rapid (</=60 min) dengue diagnostic assays was conducted to determine accuracy and identify causes of between-study heterogeneity. A systematic review identified 302 potentially suitable studies, of which 11 were selected for meta-analysis. All selected studies evaluated the immunochromatographic test (ICT) manufactured by Panbio Pty Ltd. Individual study results for sensitivity ranged from 0.45 to 1.0, specificity 0.57-1.0, diagnostic odds ratio 4.5-1287, and positive:negative likelihood ratios 2.3-59 and 0.01-0.56, respectively. Results indicated that the ICT evaluated in the selected studies can both rule in and rule out disease but is more accurate when samples are collected later in the acute phase of infection. Limitations of this meta-analysis were significant between-study heterogeneity caused by inconsistencies in evaluation methodologies, and the evaluation of only the Panbio ICT. It is recommended that additional, standardized evaluations are required for other dengue ICTs.

Newton PN, Ward S, Angus BJ, Chierakul W, Dondorp A, Ruangveerayuth R, Silamut K, Teerapong P, Suputtamongkol Y, Looareesuwan S, White NJ. 2006. Early treatment failure in severe malaria resulting from abnormally low plasma quinine concentrations. Trans R Soc Trop Med Hyg, 100 (2), pp. 184-186. | Show Abstract | Read more

A patient admitted with severe Plasmodium falciparum malaria in western Thailand had an early treatment failure with quinine, despite full dosing. Plasma quinine concentrations were subtherapeutic. Abnormal quinine pharmacokinetics may explain sporadic reports of quinine treatment failures in severe malaria.

White NJ, Dondorp AM, Nosten F, Day NPJ, Grp SEAQUAMATS. 2006. Artesunate versus quinine for severe falciparum malaria - Reply LANCET, 367 (9505), pp. 111-112. | Read more

White NJ, Dondorp AM, Nosten F, Day NPJ. 2006. Authors' reply [9] Lancet, 367 (9505), pp. 111-112. | Read more

Dondorp A, White N, Day N. 2006. Authors' Reply: Response to Ian Clark PLoS Medicine, 3 (1), pp. e69-e69. | Read more

Dondorp A, White N, Day N. 2006. PfHRP2 measures schizogony, not mechanical blockage - Authors' reply: Response to Ian Clark PLOS MEDICINE, 3 (1), pp. 145-145. | Read more

Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, Chotivanich K, Newton PN, Pitisuttithum P, Smithyman AM, White NJ, Day NPJ. 2005. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2 (vol 2, art. no. e204, 2005) PLOS MEDICINE, 2 (10), pp. 1047-1047. | Read more

Dondorp A, Nosten F, Stepniewska K, Day N, White N, South East Asian Quinine Artesunate Malaria Trial (SEAQUAMAT) group. 2005. Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial. Lancet, 366 (9487), pp. 717-725. | Show Abstract | Read more

BACKGROUND: In the treatment of severe malaria, intravenous artesunate is more rapidly acting than intravenous quinine in terms of parasite clearance, is safer, and is simpler to administer, but whether it can reduce mortality is uncertain. METHODS: We did an open-label randomised controlled trial in patients admitted to hospital with severe falciparum malaria in Bangladesh, India, Indonesia, and Myanmar. We assigned individuals intravenous artesunate 2.4 mg/kg bodyweight given as a bolus (n=730) at 0, 12, and 24 h, and then daily, or intravenous quinine (20 mg salt per kg loading dose infused over 4 h then 10 mg/kg infused over 2-8 h three times a day; n=731). Oral medication was substituted when possible to complete treatment. Our primary endpoint was death from severe malaria, and analysis was by intention to treat. FINDINGS: We assessed all patients randomised for the primary endpoint. Mortality in artesunate recipients was 15% (107 of 730) compared with 22% (164 of 731) in quinine recipients; an absolute reduction of 34.7% (95% CI 18.5-47.6%; p=0.0002). Treatment with artesunate was well tolerated, whereas quinine was associated with hypoglycaemia (relative risk 3.2, 1.3-7.8; p=0.009). INTERPRETATION: Artesunate should become the treatment of choice for severe falciparum malaria in adults.

Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, Chotivanich K, Newton PN, Pitisuttithum P, Smithyman AM, White NJ, Day NP. 2005. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2. PLoS Med, 2 (8), pp. e204. | Show Abstract | Read more

BACKGROUND: In falciparum malaria sequestration of erythrocytes containing mature forms of Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria. METHODS AND FINDINGS: We measured plasma concentrations of PfHRP2, using a quantitative antigen-capture enzyme-linked immunosorbent assay, in 337 adult patients with falciparum malaria of varying severity hospitalised on the Thai-Burmese border. Based on in vitro production rates, we constructed a model to link this measure to the total parasite burden in the patient. The estimated geometric mean parasite burden was 7 x 10(11) (95% confidence interval [CI] 5.8 x 10(11) to 8.5 x 10(11)) parasites per body, and was over six times higher in severe malaria (geometric mean 1.7 x 10(12), 95% CI 1.3 x 10(12) to 2.3 x 10(12)) than in patients hospitalised without signs of severity (geometric mean 2.8 x 10(11), 95% CI 2.3 x 10(11) to 3.5 x 10(11); p < 0.001). Parasite burden was highest in patients who died (geometric mean 3.4 x 10(12), 95% CI 1.9 x 10(12) to 6.3 x 10(12); p = 0.03). The calculated number of sequestered parasites increased with disease severity and was higher in patients with late developmental stages of P. falciparum present on peripheral blood smears. Comparing model and laboratory estimates of the time of sequestration suggested that admission to hospital with uncomplicated malaria often follows schizogony-but in severe malaria is unrelated to stage of parasite development. CONCLUSION: Plasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes.

Desakorn V, Dondorp AM, Silamut K, Pongtavornpinyo W, Sahassananda D, Chotivanich K, Pitisuttithum P, Smithyman AM, Day NP, White NJ. 2005. Stage-dependent production and release of histidine-rich protein 2 by Plasmodium falciparum. Trans R Soc Trop Med Hyg, 99 (7), pp. 517-524. | Show Abstract | Read more

Because of their sequestration in the microcirculation, the pathogenic late stages of Plasmodium falciparum are under-represented in peripheral blood samples from patients with falciparum malaria. Excreted products of the parasite might help to estimate this sequestered biomass. We quantified the stage-dependent production and release per parasite of P. falciparum histidine-rich protein 2 (PfHRP2) with the objective of measuring the sequestered biomass. A simple method to relate parasite stage to parasite age was developed to facilitate this. In four isolates of P. falciparum, the median (range) PfHRP2 content was 2.0fg (0.5-4.3fg) for a young ring stage infected erythrocyte, and 5.4fg (2.1-10.2fg) for the schizont stage. The amount of PfHRP2 in the parasitized erythrocyte increased most during development to the mature trophozoite stage. The median (range) amount of PfHRP2 secreted per parasite per entire erythrocytic cycle was 5.2fg (1.1-13.0fg). A median of 89% of the total PfHRP2 was excreted at the moment of schizont rupture. This assessment of the stage-dependent release of PfHRP2 is an essential prerequisite for future studies aimed at estimating the total patient parasite mass from the peripheral blood PfHRP2 concentration.

de Fost M, Chierakul W, Pimda K, Dondorp AM, White NJ, Van der Poll T. 2005. Activation of cytotoxic lymphocytes in patients with scrub typhus. Am J Trop Med Hyg, 72 (4), pp. 465-467. | Show Abstract

Thai patients with scrub typhus caused by the intracellular pathogen Orientia tsutsugamushi displayed elevated plasma concentrations of granzymes A and B, interferon-gamma (IFN)-gamma-inducible protein 10, and monokine induced by IFN-gamma. These data suggest that activation of cytotoxic lymphocytes is part of the early host response to scrub typhus.

Omodeo-Salè F, Motti A, Dondorp A, White NJ, Taramelli D. 2005. Destabilisation and subsequent lysis of human erythrocytes induced by Plasmodium falciparum haem products. Eur J Haematol, 74 (4), pp. 324-332. | Show Abstract | Read more

In falciparum malaria, both infected and uninfected red cells have structural and functional alterations. To investigate the mechanisms of these modifications, we studied the effects of two Plasmodium falciparum haem products (haematin and malaria pigment in the synthetic form beta-haematin) on isolated human red blood cells (RBCs) and purified RBC ghosts. A dose- and time-dependent incorporation of haematin into RBC ghosts and intact cells was observed, which was in proportion to the extent of haematin- induced haemolysis. RBCs pre-incubated with haematin were more sensitive to haemolysis induced by hypotonic shock, low pH, H2O2 or haematin itself. Haemolysis was not related to membrane lipid peroxidation and only partially to oxidation of protein sulphydryl groups and it could not be prevented by scavengers of lipid peroxidation or hydroperoxide groups. N-acetylcysteine partly protected the oxidation of SH groups and significantly reduced haemolysis. In contrast, beta-haematin was neither haemolytic nor oxidative towards protein sulphydryl groups. Beta-haematin did destabilise the RBC membrane, but to a lesser extent than haematin, inducing increased susceptibility to lysis caused by hypotonic medium, H2O2 or haematin. This study suggests that the destabilising effect of haematin and, to a much less extent, beta-haematin on the RBC membrane does not result from oxidative damage of membrane lipids but from direct binding or incorporation which may affect the reciprocal interactions between the membrane and cytoskeleton proteins. These changes could contribute to the reduced red cell deformability associated with severe malaria.

Newton PN, Chaulet JF, Brockman A, Chierakul W, Dondorp A, Ruangveerayuth R, Looareesuwan S, Mounier C, White NJ. 2005. Pharmacokinetics of oral doxycycline during combination treatment of severe falciparum malaria. Antimicrob Agents Chemother, 49 (4), pp. 1622-1625. | Show Abstract | Read more

The pharmacokinetics of oral doxycycline administered at 200 mg every 24 h were investigated in 17 patients recovering from severe Plasmodium falciparum malaria. The data suggest that the doses of doxycycline currently recommended (circa 3.5 mg/kg of body weight daily) may not be optimal.

Eziefula A, Mwakisha N, Macharia A, Williams T, Maitland K, Lowe B, Dondorp A, Newton C. 2005. Correlates of red blood cell deformability (RCD) with adverse outcome in severe falciparum malaria: The effect of sequestered parasitized red cells [MIM-AE-70550] ACTA TROPICA, 95 pp. S384-S385.

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Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, Chotivanich K, Newton PN, Pitisuttithum P, Smithyman AM, White NJ, Day NPJ. 2005. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2 PLoS Medicine, 2 (8), pp. 0788-0797. | Show Abstract | Read more

Background: In falciparum malaria sequestration of erythrocytes containing mature forms of Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria. Methods and Findings: We measured plasma concentrations of PfHRP2, using a quantitative antigen-capture enzyme-linked immunosorbent assay, in 337 adult patients with falciparum malaria of varying severity hospitalised on the Thai-Burmese border. Based on in vitro production rates, we constructed a model to link this measure to the total parasite burden in the patient. The estimated geometric mean parasite burden was 7 × 1011 (95% confidence interval [CI] 5.8 × 1011 to 8.5 × 1011) parasites per body, and was over six times higher in severe malaria (geometric mean 1.7 × 1012, 95% CI 1.3 × 1012 to 2.3 × 1012) than in patients hospitalised without signs of severity (geometric mean 2.8 × 1011, 95% CI 2.3 × 1011 to 3.5 × 1011; p < 0.001). Parasite burden was highest in patients who died (geometric mean 3.4 × 1012, 95% CI 1.9 × 1012 to 6.3 × 10 12; p = 0.03). The calculated number of sequestered parasites increased with disease severity and was higher in patients with late developmental stages of P. falciparum present on peripheral blood smears. Comparing model and laboratory estimates of the time of sequestration suggested that admission to hospital with uncomplicated malaria often follows schizogony - but in severe malaria is unrelated to stage of parasite development. Conclusion: Plasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes. © 2005 Dondorp et al.

Dondorp AM, Newton PN, Mayxay M, Van Damme W, Smithuis FM, Yeung S, Petit A, Lynam AJ et al. 2004. Fake antimalarials in Southeast Asia are a major impediment to malaria control: multinational cross-sectional survey on the prevalence of fake antimalarials. Trop Med Int Health, 9 (12), pp. 1241-1246. | Show Abstract | Read more

OBJECTIVE: To assess the prevalence of counterfeit antimalarial drugs in Southeast (SE) Asia. DESIGN: Cross-sectional survey. SETTING: Pharmacies and shops selling antimalarial drugs in Myanmar (Burma), Lao PDR, Vietnam, Cambodia and Thailand. MAIN OUTCOME MEASURES: Proportion of artemisinin derivatives or mefloquine containing drugs of substandard quality. RESULTS: Of the 188 tablet packs purchased which were labelled as 'artesunate' 53% did not contain any artesunate. All counterfeit artesunate tablets were labelled as manufactured by 'Guilin Pharma', and refinements of the fake blisterpacks made them often hard to distinguish from their genuine counterparts. No other artemisinin derivatives were found to be counterfeited. Of the 44 mefloquine samples, 9% contained <10% of the expected amount of active ingredient. CONCLUSIONS: An alarmingly high proportion of antimalarial drugs bought in pharmacies and shops in mainland SE Asia are counterfeit, and the problem has increased significantly compared with our previous survey in 1999-2000. This is a serious threat to public health in the region.

Chierakul W, de Fost M, Suputtamongkol Y, Limpaiboon R, Dondorp A, White NJ, van der Poll T. 2004. Differential expression of interferon-gamma and interferon-gamma-inducing cytokines in Thai patients with scrub typhus or leptospirosis. Clin Immunol, 113 (2), pp. 140-144. | Show Abstract | Read more

Interferon (IFN)-gamma plays an important role in the induction of a type 1 immune response against intracellular pathogens. We compared the plasma levels of IFN-gamma and IFN-gamma-inducing cytokines in adult Thai patients with scrub typhus, caused by the obligate intracellular bacterium Orientia tsutsugamushi, and leptospirosis, caused by extracellular Leptospira interrogans. IFN-gamma, interleukin (IL)-18, and IL-15 levels were elevated only in patients with scrub typhus, whereas IL-12p40 and tumor necrosis factor-alpha concentrations were elevated in both patient groups, although more so in scrub typhus. These data suggest a role for a cell-mediated immune response in host defense against O. tsutsugamushi.

Dondorp AM, Chau TT, Phu NH, Mai NT, Loc PP, Chuong LV, Sinh DX, Taylor A, Hien TT, White NJ, Day NP. 2004. Unidentified acids of strong prognostic significance in severe malaria. Crit Care Med, 32 (8), pp. 1683-1688. | Show Abstract | Read more

OBJECTIVE: To calculate, using the Stewart approach to acid-base disorders, the strong anion gap as an estimate for the contribution of unmeasured plasma anions other than lactate to the metabolic acidosis that characterizes severe falciparum malaria and to assess its relative prognostic significance. DESIGN: Cohort study. SETTING: The intensive care unit of an infectious diseases hospital in southern Vietnam. PATIENTS: Consecutive adult patients (n = 268) with severe falciparum malaria. INTERVENTIONS: The intervention was clinical management in a dedicated unit. We measured baseline venous lactate, electrolytes, biochemical variables, admission arterial blood pH, and gas tensions for calculation of the strong anion gap. MEASUREMENTS AND MAIN RESULTS: The mean (95% confidence interval) admission strong anion gap was 11.1 (10.4-11.9) mEq/L, compared with lactate (geometric mean, 95% confidence interval) at 2.9 (2.7-3.2) mmol/L. Strong anion gap had a high predictive value for mortality (area under the receiver operating characteristic curve 0.73 (95% confidence interval, 0.65-0.82), which was independent of plasma lactate and creatinine concentrations. Renal failure and hepatic dysfunction were both associated with, but were not the sole determinants of, high levels of strong anion gap. CONCLUSIONS: In severe malaria, unidentified anions other than lactate are the most important contributors to metabolic acidosis, a major cause of death. The strong anion gap is a powerful prognostic indicator in patients with severe malaria.

Dondorp AM, Pongponratn E, White NJ. 2004. Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology. Acta Trop, 89 (3), pp. 309-317. | Show Abstract | Read more

The pathophysiology of severe falciparum malaria is complex, but evidence is mounting that its central feature is the old concept of a mechanical microcirculatory obstruction. Autopsy studies, but also in vivo observations of the microcirculation, demonstrate variable obstruction of the microcirculation in severe malaria. The principal cause of this is cytoadherence to the vascular endothelium of erythrocytes containing the mature forms of the parasite, leading to sequestration and obstruction of small vessels. Besides, parasitized red cells become rigid, compromising their flow through capillaries whose lumen has been reduced by sequestered erythrocytes. Adhesive forces between infected red cells (auto-agglutination), between infected and uninfected red cells (rosetting) and between uninfected erythrocytes (aggregation) could further slow down microcirculatory flow. A more recent finding is that uninfected erythrocytes also become rigid in severe malaria. Reduction in the overall red cell deformability has a strong predictive value for a fatal outcome. Rigidity may be caused by oxidative damage to the red blood cell membrane by malaria pigment released at the moment of schizont rupture. Anti-oxidants, such as N-acetylcysteine can reverse this effect and are promising as adjunctive treatment in severe malaria.

Suwanarusk R, Cooke BM, Dondorp AM, Silamut K, Sattabongkot J, White NJ, Udomsangpetch R. 2004. The deformability of red blood cells parasitized by Plasmodium falciparum and P. vivax. J Infect Dis, 189 (2), pp. 190-194. | Show Abstract | Read more

Red blood cells (RBCs) must deform considerably during their multiple passages through the microvasculature and the sinusoids of the spleen. RBCs infected with Plasmodium falciparum (Pf-IRBCs) become increasingly rigid as they mature but avoid splenic clearance by sequestering in venules and capillaries. In contrast, RBCs infected with P. vivax (Pv-IRBCs) do not sequester. We compared the effects of P. vivax and P. falciparum infection on RBC deformability in a laminar shear flow system. Pf-IRBCs became more rigid as the parasite matured, but equivalent maturation of Pv-IRBCs resulted in a doubling of flexibility. Coincidentally, the IRBC surface area increased from 56.7+/-1.3 microm2 to 74.7+/-0.6 microm2 to 90.9+/-1.1 microm2 in ring-, trophozoite-, and schizont-stage Pv-IRBCs, respectively, whereas Pf-IRBCs did not increase in size. P. vivax increases the deformability of IRBCs and thereby avoids splenic entrapment.

Newton PN, Dondorp A, Green M, Mayxay M, White NJ. 2003. Counterfeit artesunate antimalarials in southeast Asia. Lancet, 362 (9378), pp. 169. | Read more

Newton PN, Angus BJ, Chierakul W, Dondorp A, Ruangveerayuth R, Silamut K, Teerapong P, Suputtamongkol Y, Looareesuwan S, White NJ. 2003. Randomized comparison of artesunate and quinine in the treatment of severe falciparum malaria. Clin Infect Dis, 37 (1), pp. 7-16. | Show Abstract | Read more

A randomized, open-label comparison of artesunate and quinine was conducted in 113 adults with clinically severe falciparum malaria in western Thailand. Mortality was 12% with artesunate and 22% with quinine treatment (relative risk, 0.53; 95% confidence interval, 0.23-1.26; P=.22). Multiple logistic regression analysis found admission plasma lactate level, Glasgow Coma Scale score, and total serum bilirubin level to be independent risk factors for death. Coma recovery and times to normalize plasma lactate levels were similar, but the parasite clearance time was much shorter among artesunate-treated patients (P=.019). Fewer patients became hypoglycemic during artesunate therapy (10%) than during quinine therapy (28%) (P=.03). Artesunate is at least as effective as quinine in the treatment of adults with severe malaria. Larger trials are required to determine whether mortality is reduced among patients treated with artesunate.

Dondorp AM, Omodeo-Salè F, Chotivanich K, Taramelli D, White NJ. 2003. Oxidative stress and rheology in severe malaria. Redox Rep, 8 (5), pp. 292-294. | Show Abstract | Read more

There is mounting evidence that the release of haemozoin (beta-haematin), which is produced in large amounts during malaria infection and is released into the circulation during schizont rupture, is associated with damage to cell membranes through an oxidative mechanism. The red blood cell membrane is thus oxidised, causing rigidity of the cell. This can contribute to the pathophysiology of severe malaria, since red blood cells will have to deform considerably in order to squeeze through the microcirculation, the patency of which is disturbed by sequestered red blood cells containing the mature forms of the parasite. Rigidity of red blood cells forms a new target for intervention. Since this seems to be caused by oxidative damage to the red blood cell membrane, the anti-oxidant N-acetylcysteine is a promising candidate for adjunctive treatment in severe malaria, which still has a mortality rate as high as 20%.

Nontprasert A, Pukrittayakamee S, Dondorp AM, Clemens R, Looareesuwan S, White NJ. 2002. Neuropathologic toxicity of artemisinin derivatives in a mouse model. Am J Trop Med Hyg, 67 (4), pp. 423-429. | Show Abstract

Intramuscular administration of high doses of artemether and arteether to experimental mammals produces selective damage to brain stem centers involved predominantly in auditory processing and vestibular reflexes. The relationship between clinical signs of neurotoxicity and neuropathologic toxicity was studied in the mouse. Intramuscular artemether (50-100 mg/kg/day for 28 days) caused dose-dependent neuropathologic damage to the brain stem. There was no pathologic evidence of neuronal death in mice receiving either oral artemether, or oral or intramuscular artesunate, in doses up to 300 mg/kg/day. The neurons in the lower brain stem trapezoid nucleus, the gigantocellular reticular nucleus, and the inferior cerebellar peduncle were the most sensitive to the toxic effects of artemether. All mice with neuropathologic changes also showed behavioral changes, whereas in some mice with gait disturbance, no corresponding histopathologic damage could be detected. Thus clinical assessment was a sensitive measure of neurotoxicity. There may be a reversible component to artemether neurotoxicity.

Dondorp AM, Nyanoti M, Kager PA, Mithwani S, Vreeken J, Marsh K. 2002. The role of reduced red cell deformability in the pathogenesis of severe falciparum malaria and its restoration by blood transfusion. Trans R Soc Trop Med Hyg, 96 (3), pp. 282-286. | Show Abstract | Read more

As reduced red cell deformability (RCD) can contribute to derangement of the microcirculation, a central feature in the pathogenesis of severe malaria, RCD was measured with a laser diffraction technique in 232 consecutive patients with falciparum malaria on the Kenyan coast, of whom 99 had severe disease. RCD on admission (measured as elongation index [EI] at shear stress = 1.7 Pa) was reduced in proportion with severity of disease (fatal outcome: EI = 0.182 (SD = 0.048), survivors from severe disease: EI = 0.217 (SD = 0.043), uncomplicated malaria: EI = 0.249 (SD = 0.030), healthy controls: EI = 0.268 (SD = 0.022). All but 2 survivors with severe malaria and rigid erythrocytes received a blood transfusion restoring RCD. Reduced RCD may contribute to impaired microcirculatory flow and a fatal outcome in falciparum malaria. RCD can be improved by blood transfusion. Since severely reduced RCD has a strong predictive value for mortality, blood transfusion possibly improves disease outcome not only through its beneficial effect on anaemia but also on RCD.

Kager PA, Dondorp AM. 2001. [Fever and vesiculopapular exanthema due to infection with Rickettsia africae after a sojourn in South Africa]. Ned Tijdschr Geneeskd, 145 (3), pp. 138-141. | Show Abstract

A 26-year-old woman, who had visited the Krugerpark in South Africa 5 days before, presented with fever, a skin lesion with a black crust (eschar), lymphadenopathy and a vesiculo papular rash. The clinical diagnosis 'Rickettsia africae infection' was confirmed by specific serological tests. A second patient aged 43 years, whose vesicular rash did not respond to flucloxacillin had been in the Krugerpark one week before and on examination was found with 2 eschars. Based on epidemiological and clinical grounds African tick fever can be distinguished from Mediterranean spotted fever (fièvre boutonneuse). In the Netherlands specific diagnostic tests are not available. For treatment the distinction is not necessary; treatment is with tetracycline or doxycycline. Both patients recovered upon this treatment.

van der Vorm ER, Dondorp AM, van Ketel RJ, Dankert J. 2000. Apparent culture-negative prosthetic valve endocarditis caused by Peptostreptococcus magnus. J Clin Microbiol, 38 (12), pp. 4640-4642. | Show Abstract

In two patients with prosthetic valve endocarditis due to Peptostreptococcus magnus, blood cultures in the BacT/Alert and BACTEC 9240 systems were signal negative. The capability of the BacT/Alert system to detect various Peptostreptococcus species was assessed. P. magnus and P. anaerobius could not be detected, and subcultures remained negative. The growth in conventional media of these two species and other Peptostreptococcus species was similar.

Dondorp AM, Kager PA, Vreeken J, White NJ. 2000. Abnormal blood flow and red blood cell deformability in severe malaria. (vol 16, pg 228, 2000) PARASITOLOGY TODAY, 16 (7), pp. 272-272. | Read more

Dondorp AM, Kager PA, Vreeken J, White NJ. 2000. Abnormal blood flow and red blood cell deformability in severe malaria. Parasitol Today, 16 (6), pp. 228-232. | Show Abstract | Read more

Obstruction of the microcirculation plays a central role in the pathophysiology of severe malaria. Here, Arjen Dondorp and colleagues describe the various contributors to impaired microcirculatory flow in falciparum malaria: sequestration, rosetting and recent findings regarding impaired red blood cell deformability. The correlation with clinical findings and possible therapeutic consequences are discussed.

Chotivanich KT, Dondorp AM, White NJ, Peters K, Vreeken J, Kager PA, Udomsangpetch R. 2000. The resistance to physiological shear stresses of the erythrocytic rosettes formed by cells infected with Plasmodium falciparum. Ann Trop Med Parasitol, 94 (3), pp. 219-226. | Show Abstract | Read more

Rosetting forces are believed to be an important contributor to the microcirculatory obstruction that occurs in malaria caused by Plasmodium falciparum. In this study, rosettes of erythrocytes from cultures of this parasite were suspended in different media and exposed to shear stresses corresponding to those encountered on the arterial and venous sides of the human circulation. The rosettes formed by infected erythrocytes in malaria culture medium containing 10% AB serum were disrupted easily (approximately 50% being broken) when exposed to very low shear stresses of < 0.5 Pa. However, use of higher concentrations of serum strengthened the rosetting binding forces considerably. Suspension of rosettes in a viscous colloid (e.g. dextran) increased the adherence forces between infected and uninfected red cells. The results indicate that rosettes do resist the physiological shear forces that are encountered in the venular side of the circulation and could thus contribute to microvascular obstruction in falciparum malaria.

Dondorp AM, Angus BJ, Chotivanich K, Silamut K, Ruangveerayuth R, Hardeman MR, Kager PA, Vreeken J, White NJ. 1999. Red blood cell deformability as a predictor of anemia in severe falciparum malaria. Am J Trop Med Hyg, 60 (5), pp. 733-737. | Show Abstract

Decreased erythropoiesis and increased clearance of both parasitized and noninfected erythrocytes both contribute to the pathogenesis of anemia in falciparum malaria. Erythrocytes with reduced deformability are more likely to be cleared from the circulation by the spleen, a process that is augmented in acute malaria. Using a laser diffraction technique, we measured red blood cell (RBC) deformability over a range of shear stresses and related this to the severity of anemia in 36 adults with severe falciparum malaria. The RBC deformability at a high shear stress of 30 Pa, similar to that encountered in the splenic sinusoids, showed a significant positive correlation with the nadir in hemoglobin concentration during hospitalization (r = 0.49, P < 0.002). Exclusion of five patients with microcytic anemia strengthened this relationship (r = 0.64, P < 0.001). Reduction in RBC deformability resulted mainly from changes in unparasitized erythrocytes. Reduced deformability of uninfected erythrocytes at high shear stresses and subsequent splenic removal of these cells may be an important contributor to the anemia of severe malaria.

Dondorp AM, Chotivanich KT, Fucharoen S, Silamut K, Vreeken J, Kager PA, White NJ. 1999. Red cell deformability, splenic function and anaemia in thalassaemia. Br J Haematol, 105 (2), pp. 505-508. | Show Abstract | Read more

Red cell deformability (RCD) was measured in 38 patients with alpha-thalassaemia and 48 patients with beta-thalassaemia, of whom 13 had undergone splenectomy. All splenectomized patients, but none of those with intact spleens, had very rigid erythrocytes with an elongation index <0.45 at a high shear stress of 30 Pa suggesting a splenic recognition threshold for removal of rigid red cells. At this shear stress RCD correlated strongly with the degree of anaemia in both the splenectomized (r = 0.81, P < 0.001) and non-splenectomized beta-thalassaemic patients (all patients r = 0.81, P < 0.001; homozygous beta-thalassaemic patients r = 0.51, P = 0. 01). These data suggest that reduced RCD is a major determinant of anaemia in thalassaemia.

Dondorp AM, Planche T, de Bel EE, Angus BJ, Chotivanich KT, Silamut K, Romijn JA, Ruangveerayuth R et al. 1998. Nitric oxides in plasma, urine, and cerebrospinal fluid in patients with severe falciparum malaria. Am J Trop Med Hyg, 59 (3), pp. 497-502. | Show Abstract

It has been suggested that nitric oxide (NO) plays an important role in the pathogenesis of severe falciparum malaria. Since NO has a very short half-life, nitrate and nitrite (NOx) levels, stable metabolites of NO, are used as measures of NO production. We measured plasma NOx levels in 24 adults with severe falciparum malaria on the Thai-Burmese border. After correction for renal function, there was no correlation between plasma NOx levels, or the total amount of NOx excreted in the urine, and disease severity. Plasma NOx levels decreased after the first 48 hr in all patients (P = 0.007), suggesting decreased NO production. The NOx levels in cerebrospinal fluid (CSF) correlated well with plasma NOx levels, but these did not show a correlation with coma depth, and were not significantly different from those in a healthy control group. These findings do not support the hypothesis that excessive NO production contributes to the pathogenesis of severe falciparum malaria. However, local changes in NO production, e.g., in the central nervous system, might not be reflected in the total NOx production or NOx levels in the CSF.

Dondorp AM, de Groot GH. 1998. Onset of coeliac disease after a spontaneous miscarriage during a holiday in Australia: coincidence or causal relationship? Neth J Med, 52 (4), pp. 147-149. | Show Abstract | Read more

Diarrhoea contracted whilst travelling in a (sub)tropical country often has an infectious cause. However, dietary changes can also be of importance. We describe the case of a 28-year-old woman, who developed severe coeliac disease during a trip in the Australian outback. The nutritional history revealed that the patient's diet contained more wheat products during her trip than she was used to in Holland. Moreover, the onset of symptoms coincided with a spontaneous miscarriage of an 8-week-old embryo. This correlation has been described in several case reports in the literature. However, the pathophysiologic mechanism behind this correlation is unknown. Some speculative mechanisms are proposed here. Further investigations into this relationship could increase our understanding of the immunopathogenesis of coeliac disease.

Dondorp AM, Angus BJ, Hardeman MR, Chotivanich KT, Silamut K, Ruangveerayuth R, Kager PA, White NJ, Vreeken J. 1997. Prognostic significance of reduced red blood cell deformability in severe falciparum malaria. Am J Trop Med Hyg, 57 (5), pp. 507-511. | Show Abstract

Severe falciparum malaria is associated with microvascular obstruction resulting from sequestration of erythrocytes containing mature stages of the parasite. Since reduced red blood cell deformability (RBC-D) can contribute to impaired microcirculatory flow, RBC-D was measured in 23 patients with severe falciparum malaria (seven of whom subsequently died), 30 patients with uncomplicated malaria, and 17 healthy controls. The RBC-D, measured by ektacytometry, was significantly reduced in severe malaria and was particularly low in all fatal cases. At a low shear stress of 1.7 Pascal (Pa), a red blood cell elongation index less than 0.21 on admission to the hospital predicted fatal outcome with a sensitivity of 100% (confidence interval [CI] = 59-100%) and a specificity of 88% (CI = 61-98%). The reduction in the RBC-D appeared to result mainly from changes in unparasitized erythrocytes. Reduced deformability of unparasitized red blood cells in severe malaria may contribute to impaired microcirculatory flow and a fatal outcome in severe falciparum malaria.

Dondorp AM, Veenstra J, van der Poll T, Mulder JW, Reiss P. 1994. Activation of the cytokine network in a patient with AIDS and the recalcitrant erythematous desquamating disorder. Clin Infect Dis, 18 (6), pp. 942-945. | Show Abstract | Read more

An AIDS patient with the recalcitrant erythematous desquamating (RED) disorder, a presumed variant of the Staphylococcus aureus toxic shock syndrome that is characterized by relapses, skin involvement, and variable multiple-organ involvement, is described. A strain of S. aureus producing toxic shock syndrome toxin 1 was isolated from the patient, but no antibodies to this exotoxin were detectable before and during the most severe episode of the RED disorder. Levels of cytokines were measured during episodes and at other times. Tumor necrosis factor and interleukin 6 were detectable only during the most severe exacerbation of the disorder; this finding suggests a pathogenetic role for these cytokines.

Imwong M, Stepniewska K, Tripura R, Peto TJ, Lwin KM, Vihokhern B, Wongsaen K, von Seidlein L et al. 2016. Numerical Distributions of Parasite Densities During Asymptomatic Malaria. J Infect Dis, 213 (8), pp. 1322-1329. | Show Abstract | Read more

BACKGROUND: Asymptomatic parasitemia is common even in areas of low seasonal malaria transmission, but the true proportion of the population infected has not been estimated previously because of the limited sensitivity of available detection methods. METHODS: Cross-sectional malaria surveys were conducted in areas of low seasonal transmission along the border between eastern Myanmar and northwestern Thailand and in western Cambodia. DNA was quantitated by an ultrasensitive polymerase chain reaction (uPCR) assay (limit of accurate detection, 22 parasites/mL) to characterize parasite density distributions for Plasmodium falciparum and Plasmodium vivax, and the proportions of undetected infections were imputed. RESULTS: The prevalence of asymptomatic malaria as determined by uPCR was 27.5% (1303 of 4740 people tested). Both P. vivax and P. falciparum density distributions were unimodal and log normal, with modal values well within the quantifiable range. The estimated proportions of all parasitemic individuals identified by uPCR were >70% among individuals infected with P. falciparum and >85% among those infected with P. vivax. Overall, 83% of infections were predicted to be P. vivax infections, 13% were predicted to be P. falciparum infections, and 4% were predicted to be mixed infections. Geometric mean parasite densities were similar; 5601 P. vivax parasites/mL and 5158 P. falciparum parasites/mL. CONCLUSIONS: This uPCR method identified most infected individuals in malaria-endemic areas. Malaria parasitemia persists in humans at levels that optimize the probability of generating transmissible gametocyte densities without causing illness.

Slater HC, Ross A, Ouédraogo AL, White LJ, Nguon C, Walker PG, Ngor P, Aguas R et al. 2015. Assessing the impact of next-generation rapid diagnostic tests on Plasmodium falciparum malaria elimination strategies. Nature, 528 (7580), pp. S94-101. | Show Abstract | Read more

Mass-screen-and-treat and targeted mass-drug-administration strategies are being considered as a means to interrupt transmission of Plasmodium falciparum malaria. However, the effectiveness of such strategies will depend on the extent to which current and future diagnostics are able to detect those individuals who are infectious to mosquitoes. We estimate the relationship between parasite density and onward infectivity using sensitive quantitative parasite diagnostics and mosquito feeding assays from Burkina Faso. We find that a diagnostic with a lower detection limit of 200 parasites per microlitre would detect 55% of the infectious reservoir (the combined infectivity to mosquitoes of the whole population weighted by how often each individual is bitten) whereas a test with a limit of 20 parasites per microlitre would detect 83% and 2 parasites per microlitre would detect 95% of the infectious reservoir. Using mathematical models, we show that increasing the diagnostic sensitivity from 200 parasites per microlitre (equivalent to microscopy or current rapid diagnostic tests) to 2 parasites per microlitre would increase the number of regions where transmission could be interrupted with a mass-screen-and-treat programme from an entomological inoculation rate below 1 to one of up to 4. The higher sensitivity diagnostic could reduce the number of treatment rounds required to interrupt transmission in areas of lower prevalence. We predict that mass-screen-and-treat with a highly sensitive diagnostic is less effective than mass drug administration owing to the prophylactic protection provided to uninfected individuals by the latter approach. In low-transmission settings such as those in Southeast Asia, we find that a diagnostic tool with a sensitivity of 20 parasites per microlitre may be sufficient for targeted mass drug administration because this diagnostic is predicted to identify a similar village population prevalence compared with that currently detected using polymerase chain reaction if treatment levels are high and screening is conducted during the dry season. Along with other factors, such as coverage, choice of drug, timing of the intervention, importation of infections, and seasonality, the sensitivity of the diagnostic can play a part in increasing the chance of interrupting transmission.

Guyant P, Corbel V, Guérin PJ, Lautissier A, Nosten F, Boyer S, Coosemans M, Dondorp AM, Sinou V, Yeung S, White N. 2015. Past and new challenges for malaria control and elimination: the role of operational research for innovation in designing interventions. Malar J, 14 pp. 279. | Show Abstract | Read more

This meeting report presents the outcomes of a workshop held in Bangkok on December 1st 2014, where the following challenges were discussed: the threat of resistance to artemisinin and artemisinin-based combination therapy in the Greater Mekong Sub-region (GMS) and in Africa; access to treatment for most at risk and hard to reach population; insecticide resistance, residual and outdoors transmission. The role of operational research and the interactions between research institutions, National Malaria Control Programmes, Civil Society Organizations, and of financial and technical partners to address those challenges and to accelerate translation of research into policies and programmes were debated. The threat and the emergency of the artemisinin resistance spread and independent emergence in the GMS was intensely debated as it is now close to the border of India. The need for key messages, based on scientific evidence and information available and disseminated without delay, was highlighted as crucial for an effective and urgent response.

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.)

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.

Dondorp AM, Ince C, Charunwatthana P, Hanson J, van Kuijen A, Faiz MA, Rahman MR, Hasan M et al. 2008. Direct in vivo assessment of microcirculatory dysfunction in severe falciparum malaria. J Infect Dis, 197 (1), pp. 79-84. | Show Abstract | Read more

BACKGROUND: This study sought to describe and quantify microcirculatory changes in the mucosal surfaces of patients with severe malaria, by direct in vivo observation using orthogonal polarization spectral (OPS) imaging. METHODS: The microcirculation in the rectal mucosa of adult patients with severe malaria was assessed by use of OPS imaging, at admission and then daily. Comparison groups comprised patients with uncomplicated falciparum malaria, patients with bacterial sepsis, and healthy individuals. RESULTS: Erythrocyte velocities were measured directly in 43 adult patients with severe falciparum malaria, of whom 20 died. Microcirculatory blood flow was markedly disturbed, with heterogeneous obstruction that was proportional to severity of disease. Blocked capillaries were found in 29 patients (67%) and were associated with concurrent hyperdynamic blood flow (erythrocyte velocity, >750 mm/s) in adjacent vessels in 27 patients (93%). The proportion of blocked capillaries correlated with the base deficit in plasma and with the concentration of lactate. Abnormalities disappeared when the patients recovered. In healthy individuals and in patients with uncomplicated malaria or sepsis, no stagnant erythrocytes were detected, and, in patients with sepsis, hyperdynamic blood flow was prominent. CONCLUSION: Patients with severe falciparum malaria show extensive microvascular obstruction that is proportional to the severity of the disease. This finding underscores the prominent role that microvascular obstruction plays in the pathophysiology of severe malaria and illustrates the fundamental difference between the microvascular pathophysiology of malaria and that of bacterial sepsis.

Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, Chotivanich K, Newton PN, Pitisuttithum P, Smithyman AM, White NJ, Day NP. 2005. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2. PLoS Med, 2 (8), pp. e204. | Show Abstract | Read more

BACKGROUND: In falciparum malaria sequestration of erythrocytes containing mature forms of Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria. METHODS AND FINDINGS: We measured plasma concentrations of PfHRP2, using a quantitative antigen-capture enzyme-linked immunosorbent assay, in 337 adult patients with falciparum malaria of varying severity hospitalised on the Thai-Burmese border. Based on in vitro production rates, we constructed a model to link this measure to the total parasite burden in the patient. The estimated geometric mean parasite burden was 7 x 10(11) (95% confidence interval [CI] 5.8 x 10(11) to 8.5 x 10(11)) parasites per body, and was over six times higher in severe malaria (geometric mean 1.7 x 10(12), 95% CI 1.3 x 10(12) to 2.3 x 10(12)) than in patients hospitalised without signs of severity (geometric mean 2.8 x 10(11), 95% CI 2.3 x 10(11) to 3.5 x 10(11); p < 0.001). Parasite burden was highest in patients who died (geometric mean 3.4 x 10(12), 95% CI 1.9 x 10(12) to 6.3 x 10(12); p = 0.03). The calculated number of sequestered parasites increased with disease severity and was higher in patients with late developmental stages of P. falciparum present on peripheral blood smears. Comparing model and laboratory estimates of the time of sequestration suggested that admission to hospital with uncomplicated malaria often follows schizogony-but in severe malaria is unrelated to stage of parasite development. CONCLUSION: Plasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes.

Dondorp AM, Newton PN, Mayxay M, Van Damme W, Smithuis FM, Yeung S, Petit A, Lynam AJ et al. 2004. Fake antimalarials in Southeast Asia are a major impediment to malaria control: multinational cross-sectional survey on the prevalence of fake antimalarials. Trop Med Int Health, 9 (12), pp. 1241-1246. | Show Abstract | Read more

OBJECTIVE: To assess the prevalence of counterfeit antimalarial drugs in Southeast (SE) Asia. DESIGN: Cross-sectional survey. SETTING: Pharmacies and shops selling antimalarial drugs in Myanmar (Burma), Lao PDR, Vietnam, Cambodia and Thailand. MAIN OUTCOME MEASURES: Proportion of artemisinin derivatives or mefloquine containing drugs of substandard quality. RESULTS: Of the 188 tablet packs purchased which were labelled as 'artesunate' 53% did not contain any artesunate. All counterfeit artesunate tablets were labelled as manufactured by 'Guilin Pharma', and refinements of the fake blisterpacks made them often hard to distinguish from their genuine counterparts. No other artemisinin derivatives were found to be counterfeited. Of the 44 mefloquine samples, 9% contained <10% of the expected amount of active ingredient. CONCLUSIONS: An alarmingly high proportion of antimalarial drugs bought in pharmacies and shops in mainland SE Asia are counterfeit, and the problem has increased significantly compared with our previous survey in 1999-2000. This is a serious threat to public health in the region.

Dondorp AM, Chau TT, Phu NH, Mai NT, Loc PP, Chuong LV, Sinh DX, Taylor A, Hien TT, White NJ, Day NP. 2004. Unidentified acids of strong prognostic significance in severe malaria. Crit Care Med, 32 (8), pp. 1683-1688. | Show Abstract | Read more

OBJECTIVE: To calculate, using the Stewart approach to acid-base disorders, the strong anion gap as an estimate for the contribution of unmeasured plasma anions other than lactate to the metabolic acidosis that characterizes severe falciparum malaria and to assess its relative prognostic significance. DESIGN: Cohort study. SETTING: The intensive care unit of an infectious diseases hospital in southern Vietnam. PATIENTS: Consecutive adult patients (n = 268) with severe falciparum malaria. INTERVENTIONS: The intervention was clinical management in a dedicated unit. We measured baseline venous lactate, electrolytes, biochemical variables, admission arterial blood pH, and gas tensions for calculation of the strong anion gap. MEASUREMENTS AND MAIN RESULTS: The mean (95% confidence interval) admission strong anion gap was 11.1 (10.4-11.9) mEq/L, compared with lactate (geometric mean, 95% confidence interval) at 2.9 (2.7-3.2) mmol/L. Strong anion gap had a high predictive value for mortality (area under the receiver operating characteristic curve 0.73 (95% confidence interval, 0.65-0.82), which was independent of plasma lactate and creatinine concentrations. Renal failure and hepatic dysfunction were both associated with, but were not the sole determinants of, high levels of strong anion gap. CONCLUSIONS: In severe malaria, unidentified anions other than lactate are the most important contributors to metabolic acidosis, a major cause of death. The strong anion gap is a powerful prognostic indicator in patients with severe malaria.

Dondorp AM, Pongponratn E, White NJ. 2004. Reduced microcirculatory flow in severe falciparum malaria: pathophysiology and electron-microscopic pathology. Acta Trop, 89 (3), pp. 309-317. | Show Abstract | Read more

The pathophysiology of severe falciparum malaria is complex, but evidence is mounting that its central feature is the old concept of a mechanical microcirculatory obstruction. Autopsy studies, but also in vivo observations of the microcirculation, demonstrate variable obstruction of the microcirculation in severe malaria. The principal cause of this is cytoadherence to the vascular endothelium of erythrocytes containing the mature forms of the parasite, leading to sequestration and obstruction of small vessels. Besides, parasitized red cells become rigid, compromising their flow through capillaries whose lumen has been reduced by sequestered erythrocytes. Adhesive forces between infected red cells (auto-agglutination), between infected and uninfected red cells (rosetting) and between uninfected erythrocytes (aggregation) could further slow down microcirculatory flow. A more recent finding is that uninfected erythrocytes also become rigid in severe malaria. Reduction in the overall red cell deformability has a strong predictive value for a fatal outcome. Rigidity may be caused by oxidative damage to the red blood cell membrane by malaria pigment released at the moment of schizont rupture. Anti-oxidants, such as N-acetylcysteine can reverse this effect and are promising as adjunctive treatment in severe malaria.

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