register interest

Elizabeth Ashley

The Lao-Oxford-Mahosot Hospital-Wellcome Research Unit (LOMWRU) was established in 2002 by Professor Paul Newton, Dr Rattanaphone Phetsouvanh and Associate Professor Mayfong Mayxay. LOMWRU is embedded with the Microbiology Laboratory of Mahosot Hospital in Vientiane, Laos, directed by Dr Manivanh Vongsouvath.

The Unit focuses on conducting clinical and laboratory research and building research capacity in the country. We aim to provide national, regional and global organisations with data that will improve patient care, reduce morbidity and mortality and inform health policy. The main diseases under study include malaria, scrub typhus, murine typhus, melioidosis, typhoid, dengue, leptospirosis and central nervous system infections. We adopt a One Health approach to study the epidemiology of common infectious diseases. Another important focus of our work is on improving access to good quality diagnostics adapted to low and middle income settings. In addition LOMWRU has a well-developed research programme on all aspects of medicine quality.

Looking ahead, LOMWRU plans to continue epidemiological studies to define the burden of the leading infectious diseases in Laos, perform interventional trials to optimize treatments, and support national efforts to monitor and control antimicrobial resistance and to eliminate malaria.

There are no collaborations listed for this principal investigator.

WWARN K13 Genotype-Phenotype Study Group. 2019. Association of mutations in the Plasmodium falciparum Kelch13 gene (Pf3D7_1343700) with parasite clearance rates after artemisinin-based treatments-a WWARN individual patient data meta-analysis. BMC Med, 17 (1), pp. 1. | Show Abstract | Read more

BACKGROUND: Plasmodium falciparum infections with slow parasite clearance following artemisinin-based therapies are widespread in the Greater Mekong Subregion. A molecular marker of the slow clearance phenotype has been identified: single genetic changes within the propeller region of the Kelch13 protein (pfk13; Pf3D7_1343700). Global searches have identified almost 200 different non-synonymous mutant pfk13 genotypes. Most mutations occur at low prevalence and have uncertain functional significance. To characterize the impact of different pfk13 mutations on parasite clearance, we conducted an individual patient data meta-analysis of the associations between parasite clearance half-life (PC1/2) and pfk13 genotype based on a large set of individual patient records from Asia and Africa. METHODS: A systematic literature review following the PRISMA protocol was conducted to identify studies published between 2000 and 2017 which included frequent parasite counts and pfk13 genotyping. Four databases (Ovid Medline, PubMed, Ovid Embase, and Web of Science Core Collection) were searched. Eighteen studies (15 from Asia, 2 from Africa, and one multicenter study with sites on both continents) met inclusion criteria and were shared. Associations between the log transformed PC1/2 values and pfk13 genotype were assessed using multivariable regression models with random effects for study site. RESULTS: Both the pfk13 genotypes and the PC1/2 were available from 3250 (95%) patients (n = 3012 from Asia (93%), n = 238 from Africa (7%)). Among Asian isolates, all pfk13 propeller region mutant alleles observed in five or more specific isolates were associated with a 1.5- to 2.7-fold longer geometric mean PC1/2 compared to the PC1/2 of wild type isolates (all p ≤ 0.002). In addition, mutant allele E252Q located in the P. falciparum region of pfk13 was associated with 1.5-fold (95%CI 1.4-1.6) longer PC1/2. None of the isolates from four countries in Africa showed a significant difference between the PC1/2 of parasites with or without pfk13 propeller region mutations. Previously, the association of six pfk13 propeller mutant alleles with delayed parasite clearance had been confirmed. This analysis demonstrates that 15 additional pfk13 alleles are associated strongly with the slow-clearing phenotype in Southeast Asia. CONCLUSION: Pooled analysis associated 20 pfk13 propeller region mutant alleles with the slow clearance phenotype, including 15 mutations not confirmed previously.

Turner P, Ashley EA. 2019. Standardising the reporting of microbiology and antimicrobial susceptibility data. Lancet Infect Dis, 19 (11), pp. 1163-1164. | Read more

WorldWide Antimalarial Resistance Network Methodology Study Group. 2019. Competing risk events in antimalarial drug trials in uncomplicated Plasmodium falciparum malaria: a WorldWide Antimalarial Resistance Network individual participant data meta-analysis. Malar J, 18 (1), pp. 225. | Show Abstract | Read more

BACKGROUND: Therapeutic efficacy studies in uncomplicated Plasmodium falciparum malaria are confounded by new infections, which constitute competing risk events since they can potentially preclude/pre-empt the detection of subsequent recrudescence of persistent, sub-microscopic primary infections. METHODS: Antimalarial studies typically report the risk of recrudescence derived using the Kaplan-Meier (K-M) method, which considers new infections acquired during the follow-up period as censored. Cumulative Incidence Function (CIF) provides an alternative approach for handling new infections, which accounts for them as a competing risk event. The complement of the estimate derived using the K-M method (1 minus K-M), and the CIF were used to derive the risk of recrudescence at the end of the follow-up period using data from studies collated in the WorldWide Antimalarial Resistance Network data repository. Absolute differences in the failure estimates derived using these two methods were quantified. In comparative studies, the equality of two K-M curves was assessed using the log-rank test, and the equality of CIFs using Gray's k-sample test (both at 5% level of significance). Two different regression modelling strategies for recrudescence were considered: cause-specific Cox model and Fine and Gray's sub-distributional hazard model. RESULTS: Data were available from 92 studies (233 treatment arms, 31,379 patients) conducted between 1996 and 2014. At the end of follow-up, the median absolute overestimation in the estimated risk of cumulative recrudescence by using 1 minus K-M approach was 0.04% (interquartile range (IQR): 0.00-0.27%, Range: 0.00-3.60%). The overestimation was correlated positively with the proportion of patients with recrudescence [Pearson's correlation coefficient (ρ): 0.38, 95% Confidence Interval (CI) 0.30-0.46] or new infection [ρ: 0.43; 95% CI 0.35-0.54]. In three study arms, the point estimates of failure were greater than 10% (the WHO threshold for withdrawing antimalarials) when the K-M method was used, but remained below 10% when using the CIF approach, but the 95% confidence interval included this threshold. CONCLUSIONS: The 1 minus K-M method resulted in a marginal overestimation of recrudescence that became increasingly pronounced as antimalarial efficacy declined, particularly when the observed proportion of new infection was high. The CIF approach provides an alternative approach for derivation of failure estimates in antimalarial trials, particularly in high transmission settings.

van der Pluijm RW, Imwong M, Chau NH, Hoa NT, Thuy-Nhien NT, Thanh NV, Jittamala P, Hanboonkunupakarn B, Chutasmit K, Saelow C et al. 2019. Determinants of dihydroartemisinin-piperaquine treatment failure in Plasmodium falciparum malaria in Cambodia, Thailand, and Vietnam: a prospective clinical, pharmacological, and genetic study. Lancet Infect Dis, 19 (9), pp. 952-961. | Show Abstract | Read more

BACKGROUND: The emergence and spread of resistance in Plasmodium falciparum malaria to artemisinin combination therapies in the Greater Mekong subregion poses a major threat to malaria control and elimination. The current study is part of a multi-country, open-label, randomised clinical trial (TRACII, 2015-18) evaluating the efficacy, safety, and tolerability of triple artemisinin combination therapies. A very high rate of treatment failure after treatment with dihydroartemisinin-piperaquine was observed in Thailand, Cambodia, and Vietnam. The immediate public health importance of our findings prompted us to report the efficacy data on dihydroartemisinin-piperaquine and its determinants ahead of the results of the overall trial, which will be published later this year. METHODS: Patients aged between 2 and 65 years presenting with uncomplicated P falciparum or mixed species malaria at seven sites in Thailand, Cambodia, and Vietnam were randomly assigned to receive dihydroartemisinin-piperaquine with or without mefloquine, as part of the TRACII trial. The primary outcome was the PCR-corrected efficacy at day 42. Next-generation sequencing was used to assess the prevalence of molecular markers associated with artemisinin resistance (kelch13 mutations, in particular Cys580Tyr) and piperaquine resistance (plasmepsin-2 and plasmepsin-3 amplifications and crt mutations). This study is registered with ClinicalTrials.gov, number NCT02453308. FINDINGS: Between Sept 28, 2015, and Jan 18, 2018, 539 patients with acute P falciparum malaria were screened for eligibility, 292 were enrolled, and 140 received dihydroartemisinin-piperaquine. The overall Kaplan-Meier estimate of PCR-corrected efficacy of dihydroartemisinin-piperaquine at day 42 was 50·0% (95% CI 41·1-58·3). PCR-corrected efficacies for individual sites were 12·7% (2·2-33·0) in northeastern Thailand, 38·2% (15·9-60·5) in western Cambodia, 73·4% (57·0-84·3) in Ratanakiri (northeastern Cambodia), and 47·1% (33·5-59·6) in Binh Phuoc (southwestern Vietnam). Treatment failure was associated independently with plasmepsin2/3 amplification status and four mutations in the crt gene (Thr93Ser, His97Tyr, Phe145Ile, and Ile218Phe). Compared with the results of our previous TRACI trial in 2011-13, the prevalence of molecular markers of artemisinin resistance (kelch13 Cys580Tyr mutations) and piperaquine resistance (plasmepsin2/3 amplifications and crt mutations) has increased substantially in the Greater Mekong subregion in the past decade. INTERPRETATION: Dihydroartemisinin-piperaquine is not treating malaria effectively across the eastern Greater Mekong subregion. A highly drug-resistant P falciparum co-lineage is evolving, acquiring new resistance mechanisms, and spreading. Accelerated elimination of P falciparum malaria in this region is needed urgently, to prevent further spread and avoid a potential global health emergency. FUNDING: UK Department for International Development, Wellcome Trust, Bill & Melinda Gates Foundation, Medical Research Council, and National Institutes of Health.

Mansoor R, Dahal P, Humphreys GS, Guerin P, Ashley EA, Stepniewska K. 2019. The effect of dose on the antimalarial efficacy of artesunate-mefloquine against Plasmodium falciparum malaria: a protocol for systematic review and individual patient data (IPD) meta-analysis. BMJ Open, 9 (6), pp. e027738. | Show Abstract | Read more

INTRODUCTION: Antimalarial posology based on weight bands leaves patients at the margins vulnerable to receiving either lower or higher weight-adjusted (mg/kg) dosages. This article aims to describe the protocol for systematic review and individual patient meta-analysis (MA) for a study of the distribution of artesunate and mefloquine dosage administered in patients with uncomplicated Plasmodium falciparum malaria treated with an artesunate-mefloquine (AS-MQ) regimen. Relationship between mg/kg dose and therapeutic outcomes will be assessed through a one-stage individual participant data (IPD) MA. METHODS AND ANALYSIS: Therapeutic efficacy studies with the AS-MQ regimen will be identified by searching the following databases: PUBMED, EMBASE and Web of Science. The corresponding authors of the relevant studies will be requested to share the IPD for the purpose of this MA to a secured repository. All available studies will be standardised using a common data management protocol and pooled into a single database. The relationship between mg/kg dosage and treatment failures will be assessed using a Cox regression model with study sites considered as a shared frailty term. Safety parameters will be explored where available. ETHICS AND DISSEMINATION: This IPD MA met the criteria for waiver of ethical review as defined by the Oxford Tropical Research Ethics Committee as the research consisted of secondary analysis of existing anonymous data. The results of this analysis will be disseminated at conferences, WorldWide Antimalarial Resistance Network website and any peer-reviewed publication arising will be made open source. PROSPERO REGISTRATION NUMBER: CRD42018103776.

Shetty AC, Jacob CG, Huang F, Li Y, Agrawal S, Saunders DL, Lon C, Fukuda MM, Ringwald P, Ashley EA et al. 2019. Genomic structure and diversity of Plasmodium falciparum in Southeast Asia reveal recent parasite migration patterns. Nat Commun, 10 (1), pp. 2665. | Show Abstract | Read more

Estimates of Plasmodium falciparum migration may inform strategies for malaria elimination. Here we elucidate fine-scale parasite population structure and infer recent migration across Southeast Asia using identity-by-descent (IBD) approaches based on genome-wide single nucleotide polymorphisms called in 1722 samples from 54 districts. IBD estimates are consistent with isolation-by-distance. We observe greater sharing of larger IBD segments between artemisinin-resistant parasites versus sensitive parasites, which is consistent with the recent spread of drug resistance. Our IBD analyses reveal actionable patterns, including isolated parasite populations, which may be prioritized for malaria elimination, as well as asymmetrical migration identifying potential sources and sinks of migrating parasites.

O'Flaherty K, Ataíde R, Zaloumis SG, Ashley EA, Powell R, Feng G, Reiling L, Dondorp AM, Day NP, Dhorda M et al. 2019. Contribution of Functional Antimalarial Immunity to Measures of Parasite Clearance in Therapeutic Efficacy Studies of Artemisinin Derivatives. J Infect Dis, 220 (7), pp. 1178-1187. | Show Abstract | Read more

BACKGROUND: Antibodies to the blood stages of malaria parasites enhance parasite clearance and antimalarial efficacy. The antibody subclass and functions that contribute to parasite clearance during antimalarial treatment and their relationship to malaria transmission intensity have not been characterized. METHODS: Levels of immunoglobulin G (IgG) subclasses and C1q fixation in response to Plasmodium falciparum merozoite antigens (erythrocyte-binding antigen [EBA] 175RIII-V, merozoite surface protein 2 [MSP-2], and MSP-142) and opsonic phagocytosis of merozoites were measured in a multinational trial assessing the efficacy of artesunate therapy across 11 Southeast Asian sites. Regression analyses assessed the effects of antibody seropositivity on the parasite clearance half-life (PC½), having a PC½ of ≥5 hours, and having parasitemia 3 days after treatment. RESULTS: IgG3, followed by IgG1, was the predominant IgG subclass detected (seroprevalence range, 5%-35% for IgG1 and 27%-41% for IgG3), varied across study sites, and was lowest in study sites with the lowest transmission intensity and slowest mean PC½. IgG3, C1q fixation, and opsonic-phagocytosis seropositivity were associated with a faster PC½ (range of the mean reduction in PC½, 0.47-1.16 hours; P range, .001-.03) and a reduced odds of having a PC½ of ≥5 hours and having parasitemia 3 days after treatment. CONCLUSIONS: The prevalence of IgG3, complement-fixing antibodies, and merozoite phagocytosis vary according to transmission intensity, are associated with faster parasite clearance, and may be sensitive surrogates of an augmented clearance capacity of infected erythrocytes. Determining the functional immune mechanisms associated with parasite clearance will improve characterization of artemisinin resistance.

Turner P, Fox-Lewis A, Shrestha P, Dance DAB, Wangrangsimakul T, Cusack T-P, Ling CL, Hopkins J, Roberts T, Limmathurotsakul D et al. 2019. Microbiology Investigation Criteria for Reporting Objectively (MICRO): a framework for the reporting and interpretation of clinical microbiology data. BMC Med, 17 (1), pp. 70. | Show Abstract | Read more

BACKGROUND: There is a pressing need to understand better the extent and distribution of antimicrobial resistance on a global scale, to inform development of effective interventions. Collation of datasets for meta-analysis, mathematical modelling and temporo-spatial analysis is hampered by the considerable variability in clinical sampling, variable quality in laboratory practice and inconsistencies in antimicrobial susceptibility testing and reporting. METHODS: The Microbiology Investigation Criteria for Reporting Objectively (MICRO) checklist was developed by an international working group of clinical and laboratory microbiologists, infectious disease physicians, epidemiologists and mathematical modellers. RESULTS: In keeping with the STROBE checklist, but applicable to all study designs, MICRO defines items to be included in reports of studies involving human clinical microbiology data. It provides a concise and comprehensive reference for clinicians, researchers, reviewers and journals working on, critically appraising, and publishing clinical microbiology datasets. CONCLUSIONS: Implementation of the MICRO checklist will enhance the quality and scientific reporting of clinical microbiology data, increasing data utility and comparability to improve surveillance, grade data quality, facilitate meta-analyses and inform policy and interventions from local to global levels.

Cusack TP, Ashley EA, Ling CL, Roberts T, Turner P, Wangrangsimakul T, Dance DAB. 2019. Time to switch from CLSI to EUCAST? A Southeast Asian perspective. Clin Microbiol Infect, 25 (7), pp. 782-785. | Read more

Cusack TP, Ashley EA, Ling CL, Rattanavong S, Roberts T, Turner P, Wangrangsimakul T, Dance DAB. 2019. Impact of CLSI and EUCAST breakpoint discrepancies on reporting of antimicrobial susceptibility and AMR surveillance. Clin Microbiol Infect, 25 (7), pp. 910-911. | Read more

Ashley EA, Phyo AP, Carrara VI, Tun KM, Nosten F, Smithuis F, White NJ. 2019. Plasmodium vivax Relapse Rates Following Plasmodium falciparum Malaria Reflect Previous Transmission Intensity. J Infect Dis, 220 (1), pp. 100-104. | Show Abstract | Read more

From 2003 through 2009, 687 of 2885 patients (23.8%) treated for Plasmodium falciparum malaria in clinical studies in Myanmar or on the Thailand-Myanmar border had recurrent Plasmodium vivax malaria within 63 days, compared with 18 of 429 patients (4.2%) from 2010 onward (risk ratio [RR], 0.176; 95% confidence interval, .112-.278; P < .0001). Corresponding data from 42 days of follow-up revealed that 820 of 3883 patients (21.1%) had recurrent P. vivax malaria before 2010, compared with 22 of 886 (2.5%) from 2010 onward (RR, 0.117; 95% CI, .077-.177; P < .0001). This 6-fold reduction suggests a recent decline in P. vivax transmission intensity and, thus, a substantial reduction in the proportion of individuals harboring hypnozoites.

Intharabut B, Kingston HW, Srinamon K, Ashley EA, Imwong M, Dhorda M, Woodrow C, Stepniewska K, Silamut K, Day NPJ et al. 2019. Artemisinin Resistance and Stage Dependency of Parasite Clearance in Falciparum Malaria. J Infect Dis, 219 (9), pp. 1483-1489. | Show Abstract | Read more

BACKGROUND: Artemisinin resistance in falciparum malaria is associated with kelch13 propeller mutations, reduced ring stage parasite killing, and, consequently, slow parasite clearance. We assessed how parasite age affects parasite clearance in artemisinin resistance. METHODS: Developmental stages of Plasmodium falciparum parasites on blood films performed at hospital admission and their kelch13 genotypes were assessed for 816 patients enrolled in a multinational clinical trial of artemisinin combination therapy. RESULTS: Early changes in parasitemia level (ie, 0-6 hours after admission) were determined mainly by modal stage of asexual parasite development, whereas the subsequent log-linear decline was determined mainly by kelch13 propeller mutations. Older circulating parasites on admission were associated with more-rapid parasite clearance, particularly in kelch13 mutant infections. The geometric mean parasite clearance half-life decreased by 11.6% (95% CI 3.4%-19.1%) in kelch13 wild-type infections and by 30% (95% CI 17.8%-40.4%) in kelch13 mutant infections as the mean age of circulating parasites rose from 3 to 21 hours. CONCLUSION: Following the start of antimalarial treatment, ongoing parasite sequestration and schizogony both affect initial changes in parasitemia. The greater dependency of parasite clearance half-life on parasite age in artemisinin resistant infections is consistent with ring stage resistance and consequent parasite clearance by sequestration. The stage of parasite development should be incorporated in individual assessments of artemisinin resistance.

Dailey PJ, Osborn J, Ashley EA, Baron EJ, Dance DAB, Fusco D, Fanello C, Manabe YC, Mokomane M, Newton PN et al. 2019. Defining System Requirements for Simplified Blood Culture to Enable Widespread Use in Resource-Limited Settings. Diagnostics (Basel), 9 (1), pp. 10-10. | Show Abstract | Read more

Bacterial blood stream infections (BSI) are a common cause of mortality and morbidity globally. As the causative agents and the resulting treatment decisions vary, near-patient testing and surveillance tools are necessary to monitor bacterial causes and resistance to antimicrobial agents. The gold standard to identify BSIs is blood culture (BC), a methodology not widely available in resource-limited settings. The aim of the study was to map out a target product profile of a simplified BC system (SBCS) to inform product development efforts. To identify the desired characteristics of a SBCS, we enlisted a small group of specialists working in Africa and Asia. Questions were used to understand challenges and how these constraints inform system requirements. The specialists were infectious disease physicians, public health/clinical microbiologists, clinical researchers, and technology experts with different geographical backgrounds. All suggested that BC should ideally be available at the district hospital level. Many of the same operational challenges, such as limited availability of culture bottles, electricity and internet connectivity, profuse dust, the lack of ambient temperature control, and human capacity constraints were identified across the different regions. BCs, although the accepted gold standard for diagnosis of BSIs, are not widely available outside of reference/research centers in Africa and Asia. To extend the reach of this important tool, it is crucial to engage product developers and academic research partners to develop accessible alternatives.

Win TT, Su KK, Than AM, Htut ZM, Pyar KP, Ashley EA, Dance DAB, Tun KM. 2019. Presence of Burkholderia pseudomallei in the 'Granary of Myanmar'. Trop Med Infect Dis, 4 (1), pp. 8-8. | Show Abstract | Read more

Melioidosis is a frequently fatal infectious disease caused by the Gram negative bacillus Burkholderia pseudomallei. Although it was originally discovered in Myanmar, the disease disappeared from sight for many decades. This study focuses on detection of B. pseudomallei in soil in selected sampling sites in an attempt to start to fill the gaps in the current status of our knowledge of the geographical distribution of B. pseudomallei in soil in Myanmar. This cross-sectional study consists of 400 soil samples from 10 selected study townships from two major paddy growing regions. Bacterial isolation was done using a simplified method for the isolation of Burkholderia pseudomallei from soil. In this study, only 1% (4/400) of soil samples were found to be positive; two of four were found at 90 cm depth and another two positive samples were found at 30 cm and 60 cm. This survey has confirmed the presence of environmental B. pseudomallei in Myanmar indicating that the conditions are in place for melioidosis acquisition.

Ashley EA, Shetty N, Patel J, van Doorn R, Limmathurotsakul D, Feasey NA, Okeke IN, Peacock SJ. 2019. Harnessing alternative sources of antimicrobial resistance data to support surveillance in low-resource settings. J Antimicrob Chemother, 74 (3), pp. 541-546. | Show Abstract | Read more

One of the most pressing challenges facing the global surveillance of antimicrobial resistance (AMR) is the generation, sharing, systematic analysis and dissemination of data in low-resource settings. Numerous agencies and initiatives are working to support the development of globally distributed microbiology capacity, but the routine generation of a sustainable flow of reliable data will take time to establish before it can deliver a clinical and public health impact. By contrast, there are a large number of pharma- and academia-led initiatives that have generated a wealth of data on AMR and drug-resistant infections in low-resource settings, together with high-volume data generation by private laboratories. Here, we explore how untapped sources of data could provide a short-term solution that bridges the gap between now and the time when routine surveillance capacity will have been established and how this could continue to support surveillance efforts in the future. We discuss the benefits and limitations of data generated by these sources, the mechanisms and barriers to making this accessible and how academia and pharma might support the development of laboratory and analytical capacity. We provide key actions that will be required to harness these data, including: a mapping exercise; creating mechanisms for data sharing; use of data to support national action plans; facilitating access to and use of data by the WHO Global Antimicrobial Resistance Surveillance System; and innovation in data capture, analysis and sharing.

Zhu L, Tripathi J, Rocamora FM, Miotto O, van der Pluijm R, Voss TS, Mok S, Kwiatkowski DP, Nosten F, Day NPJ et al. 2018. The origins of malaria artemisinin resistance defined by a genetic and transcriptomic background. Nat Commun, 9 (1), pp. 5158. | Show Abstract | Read more

The predisposition of parasites acquiring artemisinin resistance still remains unclear beyond the mutations in Pfk13 gene and modulation of the unfolded protein response pathway. To explore the chain of casualty underlying artemisinin resistance, we reanalyze 773 P. falciparum isolates from TRACI-study integrating TWAS, GWAS, and eQTL analyses. We find the majority of P. falciparum parasites are transcriptomically converged within each geographic site with two broader physiological profiles across the Greater Mekong Subregion (GMS). We report 8720 SNP-expression linkages in the eastern GMS parasites and 4537 in the western. The minimal overlap between them suggests differential gene regulatory networks facilitating parasite adaptations to their unique host environments. Finally, we identify two genetic and physiological backgrounds associating with artemisinin resistance in the GMS, together with a farnesyltransferase protein and a thioredoxin-like protein which may act as vital intermediators linking the Pfk13 C580Y mutation to the prolonged parasite clearance time.

McLean ARD, Wai HP, Thu AM, Khant ZS, Indrasuta C, Ashley EA, Kyaw TT, Day NPJ, Dondorp A, White NJ, Smithuis FM. 2018. Malaria elimination in remote communities requires integration of malaria control activities into general health care: an observational study and interrupted time series analysis in Myanmar. BMC Med, 16 (1), pp. 183. | Show Abstract | Read more

BACKGROUND: Community health workers (CHWs) can provide diagnosis and treatment of malaria in remote rural areas and are therefore key to the elimination of malaria. However, as incidence declines, uptake of their services could be compromised if they only treat malaria. METHODS: We conducted a retrospective analysis of 571,286 malaria rapid diagnostic tests conducted between 2011 and 2016 by 1335 CHWs supported by Medical Action Myanmar. We assessed rates of decline in Plasmodium falciparum and Plasmodium vivax incidence and rapid diagnostic test (RDT) positivity rates using negative binomial mixed effects models. We investigated whether broadening the CHW remit to provide a basic health care (BHC) package was associated with a change in malaria blood examination rates. RESULTS: Communities with CHWs providing malaria diagnosis and treatment experienced declines in P. falciparum and P. vivax malaria incidence of 70% (95% CI 66-73%) and 64% (59-68%) respectively each year of operation. RDT positivity rates declined similarly with declines of 70% (95% CI 66-73%) for P. falciparum and 65% (95% CI 61-69%) for P. vivax with each year of CHW operation. In four cohorts studied, adding a BHC package was associated with an immediate and sustained increase in blood examination rates (step-change rate ratios 2.3 (95% CI 2.0-2.6), 5.4 (95% CI 4.0-7.3), 1.7 (95% CI 1.4-2.1), and 1.1 (95% CI 1.0.1.3)). CONCLUSIONS: CHWs have overseen dramatic declines in P. falciparum and P. vivax malaria in rural Myanmar. Expanding their remit to general health care has sustained community uptake of malaria services. In similar settings, expanding health services offered by CHWs beyond malaria testing and treatment can improve rural health care while ensuring continued progress towards the elimination of malaria.

Perez-Guzman PN, Carlos Junior Alcantara L, Obolski U, de Lima MM, Ashley EA, Smithuis F, Horby P, Maude RJ, Lin Z, Kyaw AMM, Lourenço J. 2018. Measuring Mosquito-borne Viral Suitability in Myanmar and Implications for Local Zika Virus Transmission. PLoS Curr, 10 | Show Abstract | Read more

Introduction: In South East Asia, mosquito-borne viruses (MBVs) have long been a cause of high disease burden and significant economic costs. While in some SEA countries the epidemiology of MBVs is spatio-temporally well characterised and understood, in others such as Myanmar our understanding is largely incomplete. Materials and Methods: Here, we use a simple mathematical approach to estimate a climate-driven suitability index aiming to better characterise the intrinsic, spatio-temporal potential of MBVs in Myanmar. Results: Results show that the timing and amplitude of the natural oscillations of our suitability index are highly informative for the temporal patterns of DENV case counts at the country level, and a mosquito-abundance measure at a city level. When projected at fine spatial scales, the suitability index suggests that the time period of highest MBV transmission potential is between June and October independently of geographical location. Higher potential is nonetheless found along the middle axis of the country and in particular in the southern corridor of international borders with Thailand. Discussion: This research complements and expands our current understanding of MBV transmission potential in Myanmar, by identifying key spatial heterogeneities and temporal windows of importance for surveillance and control. We discuss our findings in the context of Zika virus given its recent worldwide emergence, public health impact, and current lack of information on its epidemiology and transmission potential in Myanmar. The proposed suitability index here demonstrated is applicable to other regions of the world for which surveillance data is missing, either due to lack of resources or absence of an MBV of interest.

Tun KM, Jeeyapant A, Myint AH, Kyaw ZT, Dhorda M, Mukaka M, Cheah PY, Imwong M, Hlaing T, Kyaw TH et al. 2018. Effectiveness and safety of 3 and 5 day courses of artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in an area of emerging artemisinin resistance in Myanmar. Malar J, 17 (1), pp. 258. | Show Abstract | Read more

BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged and spread in Southeast Asia. In areas where resistance is established longer courses of artemisinin-based combination therapy have improved cure rates. METHODS: The standard 3-day course of artemether-lumefantrine (AL) was compared with an extended 5-day regimen for the treatment of uncomplicated falciparum malaria in Kayin state in South-East Myanmar, an area of emerging artemisinin resistance. Late parasite clearance dynamics were described by microscopy and quantitative ultra-sensitive PCR. Patients were followed up for 42 days. RESULTS: Of 154 patients recruited (105 adults and 49 children < 14 years) 78 were randomized to 3 days and 76 to 5 days AL. Mutations in the P. falciparum kelch13 propeller gene (k13) were found in 46% (70/152) of infections, with F446I the most prevalent propeller mutation (29%; 20/70). Both regimens were well-tolerated. Parasite clearance profiles were biphasic with a slower submicroscopic phase which was similar in k13 wild-type and mutant infections. The cure rates were 100% (70/70) and 97% (68/70) in the 3- and 5-day arms respectively. Genotyping of the two recurrences was unsuccessful. CONCLUSION: Despite a high prevalence of k13 mutations, the current first-line treatment, AL, was still highly effective in this area of South-East Myanmar. The extended 5 day regimen was very well tolerated, and would be an option to prolong the useful therapeutic life of AL. Trial registration NCT02020330. Registered 24 December 2013, https://clinicaltrials.gov/NCT02020330.

Thriemer K, Bobogare A, Ley B, Gudo CS, Alam MS, Anstey NM, Ashley E, Baird JK, Gryseels C, Jambert E et al. 2018. Quantifying primaquine effectiveness and improving adherence: a round table discussion of the APMEN Vivax Working Group. Malar J, 17 (1), pp. 241. | Show Abstract | Read more

The goal to eliminate malaria from the Asia-Pacific by 2030 will require the safe and widespread delivery of effective radical cure of malaria. In October 2017, the Asia Pacific Malaria Elimination Network Vivax Working Group met to discuss the impediments to primaquine (PQ) radical cure, how these can be overcome and the methodological difficulties in assessing clinical effectiveness of radical cure. The salient discussions of this meeting which involved 110 representatives from 18 partner countries and 21 institutional partner organizations are reported. Context specific strategies to improve adherence are needed to increase understanding and awareness of PQ within affected communities; these must include education and health promotion programs. Lessons learned from other disease programs highlight that a package of approaches has the greatest potential to change patient and prescriber habits, however optimizing the components of this approach and quantifying their effectiveness is challenging. In a trial setting, the reactivity of participants results in patients altering their behaviour and creates inherent bias. Although bias can be reduced by integrating data collection into the routine health care and surveillance systems, this comes at a cost of decreasing the detection of clinical outcomes. Measuring adherence and the factors that relate to it, also requires an in-depth understanding of the context and the underlying sociocultural logic that supports it. Reaching the elimination goal will require innovative approaches to improve radical cure for vivax malaria, as well as the methods to evaluate its effectiveness.

Kloprogge F, Workman L, Borrmann S, Tékété M, Lefèvre G, Hamed K, Piola P, Ursing J, Kofoed PE, Mårtensson A et al. 2018. Artemether-lumefantrine dosing for malaria treatment in young children and pregnant women: A pharmacokinetic-pharmacodynamic meta-analysis. PLoS Med, 15 (6), pp. e1002579. | Show Abstract | Read more

BACKGROUND: The fixed dose combination of artemether-lumefantrine (AL) is the most widely used treatment for uncomplicated Plasmodium falciparum malaria. Relatively lower cure rates and lumefantrine levels have been reported in young children and in pregnant women during their second and third trimester. The aim of this study was to investigate the pharmacokinetic and pharmacodynamic properties of lumefantrine and the pharmacokinetic properties of its metabolite, desbutyl-lumefantrine, in order to inform optimal dosing regimens in all patient populations. METHODS AND FINDINGS: A search in PubMed, Embase, ClinicalTrials.gov, Google Scholar, conference proceedings, and the WorldWide Antimalarial Resistance Network (WWARN) pharmacology database identified 31 relevant clinical studies published between 1 January 1990 and 31 December 2012, with 4,546 patients in whom lumefantrine concentrations were measured. Under the auspices of WWARN, relevant individual concentration-time data, clinical covariates, and outcome data from 4,122 patients were made available and pooled for the meta-analysis. The developed lumefantrine population pharmacokinetic model was used for dose optimisation through in silico simulations. Venous plasma lumefantrine concentrations 7 days after starting standard AL treatment were 24.2% and 13.4% lower in children weighing <15 kg and 15-25 kg, respectively, and 20.2% lower in pregnant women compared with non-pregnant adults. Lumefantrine exposure decreased with increasing pre-treatment parasitaemia, and the dose limitation on absorption of lumefantrine was substantial. Simulations using the lumefantrine pharmacokinetic model suggest that, in young children and pregnant women beyond the first trimester, lengthening the dose regimen (twice daily for 5 days) and, to a lesser extent, intensifying the frequency of dosing (3 times daily for 3 days) would be more efficacious than using higher individual doses in the current standard treatment regimen (twice daily for 3 days). The model was developed using venous plasma data from patients receiving intact tablets with fat, and evaluations of alternative dosing regimens were consequently only representative for venous plasma after administration of intact tablets with fat. The absence of artemether-dihydroartemisinin data limited the prediction of parasite killing rates and recrudescent infections. Thus, the suggested optimised dosing schedule was based on the pharmacokinetic endpoint of lumefantrine plasma exposure at day 7. CONCLUSIONS: Our findings suggest that revised AL dosing regimens for young children and pregnant women would improve drug exposure but would require longer or more complex schedules. These dosing regimens should be evaluated in prospective clinical studies to determine whether they would improve cure rates, demonstrate adequate safety, and thereby prolong the useful therapeutic life of this valuable antimalarial treatment.

Ashley EA, Dance DAB, Turner P. 2018. Grading antimicrobial susceptibility data quality: room for improvement. Lancet Infect Dis, 18 (6), pp. 603-604. | Read more

Ashley EA, Phyo AP. 2018. Drugs in Development for Malaria. Drugs, 78 (9), pp. 861-879. | Show Abstract | Read more

The last two decades have seen a surge in antimalarial drug development with product development partnerships taking a leading role. Resistance of Plasmodium falciparum to the artemisinin derivatives, piperaquine and mefloquine in Southeast Asia means new antimalarials are needed with some urgency. There are at least 13 agents in clinical development. Most of these are blood schizonticides for the treatment of uncomplicated falciparum malaria, under evaluation either singly or as part of two-drug combinations. Leading candidates progressing through the pipeline are artefenomel-ferroquine and lumefantrine-KAF156, both in Phase 2b. Treatment of severe malaria continues to rely on two parenteral drugs with ancient forebears: artesunate and quinine, with sevuparin being evaluated as an adjuvant therapy. Tafenoquine is under review by stringent regulatory authorities for approval as a single-dose treatment for Plasmodium vivax relapse prevention. This represents an advance over standard 14-day primaquine regimens; however, the risk of acute haemolytic anaemia in patients with glucose-6-phosphate dehydrogenase deficiency remains. For disease prevention, several of the newer agents show potential but are unlikely to be recommended for use in the main target groups of pregnant women and young children for some years. Latest predictions are that the malaria burden will continue to be high in the coming decades. This fact, coupled with the repeated loss of antimalarials to resistance, indicates that new antimalarials will be needed for years to come. Failure of the artemisinin-based combinations in Southeast Asia has stimulated a reappraisal of current approaches to combination therapy for malaria with incorporation of three or more drugs in a single treatment under consideration.

Bopp S, Magistrado P, Wong W, Schaffner SF, Mukherjee A, Lim P, Dhorda M, Amaratunga C, Woodrow CJ, Ashley EA et al. 2018. Plasmepsin II-III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum. Nat Commun, 9 (1), pp. 1769. | Show Abstract | Read more

Multidrug resistant Plasmodium falciparum in Southeast Asia endangers regional malaria elimination and threatens to spread to other malaria endemic areas. Understanding mechanisms of piperaquine (PPQ) resistance is crucial for tracking its emergence and spread, and to develop effective strategies for overcoming it. Here we analyze a mechanism of PPQ resistance in Cambodian parasites. Isolates exhibit a bimodal dose-response curve when exposed to PPQ, with the area under the curve quantifying their survival in vitro. Increased copy number for plasmepsin II and plasmepsin III appears to explain enhanced survival when exposed to PPQ in most, but not all cases. A panel of isogenic subclones reinforces the importance of plasmepsin II-III copy number to enhanced PPQ survival. We conjecture that factors producing increased parasite survival under PPQ exposure in vitro may drive clinical PPQ failures in the field.

Vachot-Ganée L, Khim N, Iannello A, Legrand E, Kim S, Eam R, Khean C, Ken M, Ashley E, Tun KM et al. 2018. A novel field-based molecular assay to detect validated artemisinin-resistant k13 mutants. Malar J, 17 (1), pp. 175. | Show Abstract | Read more

BACKGROUND: Given the risk of artemisinin resistance spreading from the Greater Mekong sub-region, prospective monitoring in sub-Saharan Africa should be expedited. Molecular biology techniques used for monitoring rely on the detection of k13 validated mutants by using PCR and Sanger sequencing approach, usually not available in malaria endemic areas. METHODS: A semi-automated workflow based on the easyMAG® platform and the Argene Solution® (bioMérieux, Marcy l'Etoile, France) as a field-based surveillance tool operable at national level was developed in four steps. Clinical and analytical performances of this tool detecting five of the most frequent and validated k13 mutants (Y493H, I543T, R539T, F446I and C580Y) from dried blood spots (DBS) were compared to the gold standard approach (PCR and Sanger sequencing). RESULTS: By using the ARMS (amplification-refractory mutation system) strategy, the best multiplexing options were found in 3 separate real-time PCR duplexes (IC as internal control/I543T, C580Y/Y493H and F446I/R539T) with limits of detection ranging from 50 (C580Y) to 6.25 parasites/µL (Y493H). In field conditions, using 642 clinical DBS (from symptomatic patients and asymptomatic individuals) collected from Cambodia, Myanmar and Africa (Chad), the overall sensitivity and specificity of the K13 bMx prototype assay developed by bioMérieux were ≥ 90%. Areas under the ROC curves were estimated to be > 0.90 for all k13 mutants in samples from symptomatic patients. CONCLUSION: The K13 ready-to-use bMx prototype assay, considered by the end-users as a user-friendly assay to perform (in shorter time than the K13 reference assay) and easy to interpret, was found to require less budget planning and had fewer logistical constraints. Its excellent performance qualifies the prototype as a reliable screening tool usable in malaria endemic countries recognized to be at risk of emergence or spread of validated k13 mutants. Additional multi-site studies are needed to evaluate the performances of the K13 bMx prototype assay in different epidemiological contexts such as Africa, India, or South America.

Recht J, Ashley EA, White NJ. 2018. Use of primaquine and glucose-6-phosphate dehydrogenase deficiency testing: Divergent policies and practices in malaria endemic countries. PLoS Negl Trop Dis, 12 (4), pp. e0006230. | Show Abstract | Read more

Primaquine is the only available antimalarial drug that kills dormant liver stages of Plasmodium vivax and Plasmodium ovale malarias and therefore prevents their relapse ('radical cure'). It is also the only generally available antimalarial that rapidly sterilises mature P. falciparum gametocytes. Radical cure requires extended courses of primaquine (usually 14 days; total dose 3.5-7 mg/kg), whereas transmissibility reduction in falciparum malaria requires a single dose (formerly 0.75 mg/kg, now a single low dose [SLD] of 0.25 mg/kg is recommended). The main adverse effect of primaquine is dose-dependent haemolysis in glucose 6-phosphate dehydrogenase (G6PD) deficiency, the most common human enzymopathy. X-linked mutations conferring varying degrees of G6PD deficiency are prevalent throughout malaria-endemic regions. Phenotypic screening tests usually detect <30% of normal G6PD activity, identifying nearly all male hemizygotes and female homozygotes and some heterozygotes. Unfortunately, G6PD deficiency screening is usually unavailable at point of care, and, as a consequence, radical cure is greatly underused. Both haemolytic risk (determined by the prevalence and severity of G6PD deficiency polymorphisms) and relapse rates vary, so there has been considerable uncertainty in both policies and practices related to G6PD deficiency testing and use of primaquine for radical cure. Review of available information on the prevalence and severity of G6PD variants together with countries' policies for the use of primaquine and G6PD deficiency testing confirms a wide range of practices. There remains lack of consensus on the requirement for G6PD deficiency testing before prescribing primaquine radical cure regimens. Despite substantially lower haemolytic risks, implementation of SLD primaquine as a P. falciparum gametocytocide also varies. In Africa, a few countries have recently adopted SLD primaquine, yet many with areas of low seasonal transmission do not use primaquine as an antimalarial at all. Most countries that recommended the higher 0.75 mg/kg single primaquine dose for falciparum malaria (e.g., most countries in the Americas) have not changed their recommendation. Some vivax malaria-endemic countries where G6PD deficiency testing is generally unavailable have adopted the once-weekly radical cure regimen (0.75 mg/kg/week for 8 weeks), known to be safer in less severe G6PD deficiency variants. There is substantial room for improvement in radical cure policies and practices.

Ashley EA, Pyae Phyo A, Woodrow CJ. 2018. Malaria. Lancet, 391 (10130), pp. 1608-1621. | Show Abstract | Read more

Following unsuccessful eradication attempts there was a resurgence of malaria towards the end of the 20th century. Renewed control efforts using a range of improved tools, such as long-lasting insecticide-treated bednets and artemisinin-based combination therapies, have more than halved the global burden of disease, but it remains high with 445 000 deaths and more than 200 million cases in 2016. Pitfalls in individual patient management are delayed diagnosis and overzealous fluid resuscitation in severe malaria. Even in the absence of drug resistance, parasite recurrence can occur, owing to high parasite densities, low host immunity, or suboptimal drug concentrations. Malaria elimination is firmly back as a mainstream policy but resistance to the artemisinin derivatives, their partner drugs, and insecticides present major challenges. Vaccine development continues on several fronts but none of the candidates developed to date have been shown to provide long-lasting benefits at a population level. Increased resources and unprecedented levels of regional cooperation and societal commitment will be needed if further substantial inroads into the malaria burden are to be made.

Ashley EA, Recht J, Chua A, Dance D, Dhorda M, Thomas NV, Ranganathan N, Turner P, Guerin PJ, White NJ, Day NP. 2018. An inventory of supranational antimicrobial resistance surveillance networks involving low- and middle-income countries since 2000. J Antimicrob Chemother, 73 (7), pp. 1737-1749. | Show Abstract | Read more

Low- and middle-income countries (LMICs) shoulder the bulk of the global burden of infectious diseases and drug resistance. We searched for supranational networks performing antimicrobial resistance (AMR) surveillance in LMICs and assessed their organization, methodology, impacts and challenges. Since 2000, 72 supranational networks for AMR surveillance in bacteria, fungi, HIV, TB and malaria have been created that have involved LMICs, of which 34 are ongoing. The median (range) duration of the networks was 6 years (1-70) and the number of LMICs included was 8 (1-67). Networks were categorized as WHO/governmental (n = 26), academic (n = 24) or pharma initiated (n = 22). Funding sources varied, with 30 networks receiving public or WHO funding, 25 corporate, 13 trust or foundation, and 4 funded from more than one source. The leading global programmes for drug resistance surveillance in TB, malaria and HIV gather data in LMICs through periodic active surveillance efforts or combined active and passive approaches. The biggest challenges faced by these networks has been achieving high coverage across LMICs and complying with the recommended frequency of reporting. Obtaining high quality, representative surveillance data in LMICs is challenging. Antibiotic resistance surveillance requires a level of laboratory infrastructure and training that is not widely available in LMICs. The nascent Global Antimicrobial Resistance Surveillance System (GLASS) aims to build up passive surveillance in all member states. Past experience suggests complementary active approaches may be needed in many LMICs if representative, clinically relevant, meaningful data are to be obtained. Maintaining an up-to-date registry of networks would promote a more coordinated approach to surveillance.

Win MM, Ashley EA, Zin KN, Aung MT, Swe MMM, Ling CL, Nosten F, Thein WM, Zaw NN, Aung MY et al. 2018. Melioidosis in Myanmar. Trop Med Infect Dis, 3 (1), pp. 28-28. | Show Abstract | Read more

Sporadic cases of melioidosis have been diagnosed in Myanmar since the disease was first described in Yangon in 1911. Published and unpublished cases are summarized here, along with results from environmental and serosurveys. A total of 298 cases have been reported from seven states or regions between 1911 and 2018, with the majority of these occurring before 1949. Findings from soil surveys confirm the presence of Burkholderia pseudomallei in the environment in all three regions examined. The true epidemiology of the disease in Myanmar is unknown. Important factors contributing to the current gaps in knowledge are lack of awareness among clinicians and insufficient laboratory diagnostic capacity in many parts of the country. This is likely to have led to substantial under-reporting.

Phyo AP, Win KK, Thu AM, Swe LL, Htike H, Beau C, Sriprawat K, Winterberg M, Proux S, Imwong M et al. 2018. Poor response to artesunate treatment in two patients with severe malaria on the Thai-Myanmar border. Malar J, 17 (1), pp. 30. | Show Abstract | Read more

BACKGROUND: Malaria has declined dramatically along the Thai-Myanmar border in recent years due to malaria control and elimination programmes. However, at the same time, artemisinin resistance has spread, raising concerns about the efficacy of parenteral artesunate for the treatment of severe malaria. CASE PRESENTATION: In November 2015 and April 2017, two patients were treated for severe malaria with parenteral artesunate. Quinine was added within 24 h due to an initial poor response to treatment. The first patient died within 24 h of starting treatment and the second did not clear his peripheral parasitaemia until 11 days later. Genotyping revealed artemisinin resistance Kelch-13 markers. CONCLUSIONS: Reliable efficacy of artesunate for the treatment of severe malaria may no longer be assured in areas where artemisinin resistance has emerged. Empirical addition of parenteral quinine to artesunate for treatment is recommended as a precautionary measure.

Bonnington CA, Phyo AP, Ashley EA, Imwong M, Sriprawat K, Parker DM, Proux S, White NJ, Nosten F. 2017. Plasmodium falciparum Kelch 13 mutations and treatment response in patients in Hpa-Pun District, Northern Kayin State, Myanmar. Malar J, 16 (1), pp. 480. | Show Abstract | Read more

BACKGROUND: Artemisinin resistance, linked to polymorphisms in the Kelch gene on chromosome 13 of Plasmodium falciparum (k13), has outpaced containment efforts in South East Asia. For national malaria control programmes in the region, it is important to establish a surveillance system which includes monitoring for k13 polymorphisms associated with the clinical phenotype. METHODS: Between February and December 2013, parasite clearance was assessed in 35 patients with uncomplicated P. falciparum treated with artesunate monotherapy followed by 3-day ACT in an isolated area on the Myanmar-Thai border with relatively low artemisinin drug pressure. Molecular testing for k13 mutations was performed on dry blood spots collected on admission. RESULTS: The proportion of k13 mutations in these patients was 41.7%, and only 5 alleles were detected: C580Y, I205T, M476I, R561H, and F446I. Of these, F446I was the most common, and was associated with a longer parasite clearance half-life (median) 4.1 (min-max 2.3-6.7) hours compared to 2.5 (min-max 1.6-8.7) in wildtype (p = 0·01). The prevalence of k13 mutant parasites was much lower than the proportion of k13 mutants detected 200 km south in a much less remote setting where the prevalence of k13 mutants was 84% with 15 distinct alleles in 2013 of which C580Y predominated. CONCLUSIONS: This study provides evidence of artemisinin resistance in a remote part of eastern Myanmar. The prevalence of k13 mutations as well as allele diversity varies considerably across short distances, presumably because of historical patterns of artemisinin use and population movements.

Zellweger RM, Carrique-Mas J, Limmathurotsakul D, Day NPJ, Thwaites GE, Baker S, Southeast Asia Antimicrobial Resistance Network. 2017. A current perspective on antimicrobial resistance in Southeast Asia. J Antimicrob Chemother, 72 (11), pp. 2963-2972. | Show Abstract | Read more

Southeast Asia, a vibrant region that has recently undergone unprecedented economic development, is regarded as a global hotspot for the emergence and spread of antimicrobial resistance (AMR). Understanding AMR in Southeast Asia is crucial for assessing how to control AMR on an international scale. Here we (i) describe the current AMR situation in Southeast Asia, (ii) explore the mechanisms that make Southeast Asia a focal region for the emergence of AMR, and (iii) propose ways in which Southeast Asia could contribute to a global solution.

White NJ, Watson J, Ashley EA. 2017. Split dosing of artemisinins does not improve antimalarial therapeutic efficacy. Sci Rep, 7 (1), pp. 12132. | Show Abstract | Read more

It has been suggested recently, based on pharmacokinetic-pharmacodynamic modelling exercises, that twice daily dosing of artemisinins increases malaria parasite killing and so could "dramatically enhance and restore drug effectiveness" in artemisinin resistant P. falciparum malaria infections. It was recommended that split dosing should be incorporated into all artemisinin combination regimen designs. To explain why parasite clearance rates were not faster with split dose regimens it was concluded that splenic malaria parasite clearance capacity was readily exceeded, resulting in the accumulation of dead parasites in the circulation, that parasite clearance was therefore an unreliable measure of drug efficacy, and instead that human immunity is the primary determinant of clearance rates. To test these various hypotheses we performed a logistic meta-regression analysis of cure rates from all falciparum malaria treatment trials (n = 40) with monotherapy arms containing artemisinin or a derivative (76 arms). There was no evidence that split dosing enhanced cure rates.

Tadesse BT, Ashley EA, Ongarello S, Havumaki J, Wijegoonewardena M, González IJ, Dittrich S. 2017. Antimicrobial resistance in Africa: a systematic review. BMC Infect Dis, 17 (1), pp. 616. | Show Abstract | Read more

BACKGROUND: Antimicrobial resistance (AMR) is widely acknowledged as a global problem, yet in many parts of the world its magnitude is still not well understood. This review, using a public health focused approach, aimed to understand and describe the current status of AMR in Africa in relation to common causes of infections and drugs recommended in WHO treatment guidelines. METHODS: PubMed, EMBASE and other relevant databases were searched for recent articles (2013-2016) in accordance with the PRISMA guidelines. Article retrieval and screening were done using a structured search string and strict inclusion/exclusion criteria. Median and interquartile ranges of percent resistance were calculated for each antibiotic-bacterium combination. RESULTS: AMR data was not available for 42.6% of the countries in the African continent. A total of 144 articles were included in the final analysis. 13 Gram negative and 5 Gram positive bacteria were tested against 37 different antibiotics. Penicillin resistance in Streptococcus pneumoniae was reported in 14/144studies (median resistance (MR): 26.7%). Further 18/53 (34.0%) of Haemophilus influenza isolates were resistant to amoxicillin. MR of Escherichia coli to amoxicillin, trimethoprim and gentamicin was 88.1%, 80.7% and 29.8% respectively. Ciprofloxacin resistance in Salmonella Typhi was rare. No documented ceftriaxone resistance in Neisseria gonorrhoeae was reported, while the MR for quinolone was 37.5%. Carbapenem resistance was common in Acinetobacter spp. and Pseudomonas aeruginosa but uncommon in Enterobacteriaceae. CONCLUSION: Our review highlights three important findings. First, recent AMR data is not available for more than 40% of the countries. Second, the level of resistance to commonly prescribed antibiotics was significant. Third, the quality of microbiological data is of serious concern. Our findings underline that to conserve our current arsenal of antibiotics it is imperative to address the gaps in AMR diagnostic standardization and reporting and use available information to optimize treatment guidelines.

Ashley E. 2017. Investment in antimalarial drug development is bearing fruit The Lancet Infectious Diseases, 17 (6), pp. 568-570. | Read more

Mukherjee A, Bopp S, Magistrado P, Wong W, Daniels R, Demas A, Schaffner S, Amaratunga C, Lim P, Dhorda M et al. 2017. Artemisinin resistance without pfkelch13 mutations in Plasmodium falciparum isolates from Cambodia. Malar J, 16 (1), pp. 195. | Show Abstract | Read more

BACKGROUND: Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin-piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance. METHODS: Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA0-3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates. RESULTS: Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA0-3h = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance. CONCLUSIONS: This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine resistance and development of strategies to prevent or overcome anti-malarial resistance.

Cerqueira GC, Cheeseman IH, Schaffner SF, Nair S, McDew-White M, Phyo AP, Ashley EA, Melnikov A, Rogov P, Birren BW et al. 2017. Longitudinal genomic surveillance of Plasmodium falciparum malaria parasites reveals complex genomic architecture of emerging artemisinin resistance. Genome Biol, 18 (1), pp. 78. | Show Abstract | Read more

BACKGROUND: Artemisinin-based combination therapies are the first line of treatment for Plasmodium falciparum infections worldwide, but artemisinin resistance has risen rapidly in Southeast Asia over the past decade. Mutations in the kelch13 gene have been implicated in this resistance. We used longitudinal genomic surveillance to detect signals in kelch13 and other loci that contribute to artemisinin or partner drug resistance. We retrospectively sequenced the genomes of 194 P. falciparum isolates from five sites in Northwest Thailand, over the period of a rapid increase in the emergence of artemisinin resistance (2001-2014). RESULTS: We evaluate statistical metrics for temporal change in the frequency of individual SNPs, assuming that SNPs associated with resistance increase in frequency over this period. After Kelch13-C580Y, the strongest temporal change is seen at a SNP in phosphatidylinositol 4-kinase, which is involved in a pathway recently implicated in artemisinin resistance. Furthermore, other loci exhibit strong temporal signatures which warrant further investigation for involvement in artemisinin resistance evolution. Through genome-wide association analysis we identify a variant in a kelch domain-containing gene on chromosome 10 that may epistatically modulate artemisinin resistance. CONCLUSIONS: This analysis demonstrates the potential of a longitudinal genomic surveillance approach to detect resistance-associated gene loci to improve our mechanistic understanding of how resistance develops. Evidence for additional genomic regions outside of the kelch13 locus associated with artemisinin-resistant parasites may yield new molecular markers for resistance surveillance, which may be useful in efforts to reduce the emergence or spread of artemisinin resistance in African parasite populations.

Dondorp AM, Limmathurotsakul D, Ashley EA. 2018. What's wrong in the control of antimicrobial resistance in critically ill patients from low- and middle-income countries? Intensive Care Med, 44 (1), pp. 79-82. | Read more

Ataide R, Ashley EA, Powell R, Chan J-A, Malloy MJ, O'Flaherty K, Takashima E, Langer C, Tsuboi T, Dondorp AM et al. 2017. Host immunity to Plasmodium falciparum and the assessment of emerging artemisinin resistance in a multinational cohort. Proc Natl Acad Sci U S A, 114 (13), pp. 3515-3520. | Show Abstract | Read more

Artemisinin-resistant falciparum malaria, defined by a slow-clearance phenotype and the presence of kelch13 mutants, has emerged in the Greater Mekong Subregion. Naturally acquired immunity to malaria clears parasites independent of antimalarial drugs. We hypothesized that between- and within-population variations in host immunity influence parasite clearance after artemisinin treatment and the interpretation of emerging artemisinin resistance. Antibodies specific to 12 Plasmodium falciparum sporozoite and blood-stage antigens were determined in 959 patients (from 11 sites in Southeast Asia) participating in a multinational cohort study assessing parasite clearance half-life (PCt1/2) after artesunate treatment and kelch13 mutations. Linear mixed-effects modeling of pooled individual patient data assessed the association between antibody responses and PCt1/2.P. falciparum antibodies were lowest in areas where the prevalence of kelch13 mutations and slow PCt1/2 were highest [Spearman ρ = -0.90 (95% confidence interval, -0.97, -0.65), and Spearman ρ = -0.94 (95% confidence interval, -0.98, -0.77), respectively]. P. falciparum antibodies were associated with faster PCt1/2 (mean difference in PCt1/2 according to seropositivity, -0.16 to -0.65 h, depending on antigen); antibodies have a greater effect on the clearance of kelch13 mutant compared with wild-type parasites (mean difference in PCt1/2 according to seropositivity, -0.22 to -0.61 h faster in kelch13 mutants compared with wild-type parasites). Naturally acquired immunity accelerates the clearance of artemisinin-resistant parasites in patients with falciparum malaria and may confound the current working definition of artemisinin resistance. Immunity may also play an important role in the emergence and transmission potential of artemisinin-resistant parasites.

Ashley EA. 2017. Post-malaria neurological syndromes. Clin Med (Lond), 17 (1), pp. 95. | Read more

Hoglund RM, Workman L, Edstein MD, Thanh NX, Quang NN, Zongo I, Ouedraogo JB, Borrmann S, Mwai L, Nsanzabana C et al. 2017. Population Pharmacokinetic Properties of Piperaquine in Falciparum Malaria: An Individual Participant Data Meta-Analysis. PLoS Med, 14 (1), pp. e1002212. | Show Abstract | Read more

BACKGROUND: Artemisinin-based combination therapies (ACTs) are the mainstay of the current treatment of uncomplicated Plasmodium falciparum malaria, but ACT resistance is spreading across Southeast Asia. Dihydroartemisinin-piperaquine is one of the five ACTs currently recommended by the World Health Organization. Previous studies suggest that young children (<5 y) with malaria are under-dosed. This study utilised a population-based pharmacokinetic approach to optimise the antimalarial treatment regimen for piperaquine. METHODS AND FINDINGS: Published pharmacokinetic studies on piperaquine were identified through a systematic literature review of articles published between 1 January 1960 and 15 February 2013. Individual plasma piperaquine concentration-time data from 11 clinical studies (8,776 samples from 728 individuals) in adults and children with uncomplicated malaria and healthy volunteers were collated and standardised by the WorldWide Antimalarial Resistance Network. Data were pooled and analysed using nonlinear mixed-effects modelling. Piperaquine pharmacokinetics were described successfully by a three-compartment disposition model with flexible absorption. Body weight influenced clearance and volume parameters significantly, resulting in lower piperaquine exposures in small children (<25 kg) compared to larger children and adults (≥25 kg) after administration of the manufacturers' currently recommended dose regimens. Simulated median (interquartile range) day 7 plasma concentration was 29.4 (19.3-44.3) ng/ml in small children compared to 38.1 (25.8-56.3) ng/ml in larger children and adults, with the recommended dose regimen. The final model identified a mean (95% confidence interval) increase of 23.7% (15.8%-32.5%) in piperaquine bioavailability between each piperaquine dose occasion. The model also described an enzyme maturation function in very young children, resulting in 50% maturation at 0.575 (0.413-0.711) y of age. An evidence-based optimised dose regimen was constructed that would provide piperaquine exposures across all ages comparable to the exposure currently seen in a typical adult with standard treatment, without exceeding the concentration range observed with the manufacturers' recommended regimen. Limited data were available in infants and pregnant women with malaria as well as in healthy individuals. CONCLUSIONS: The derived population pharmacokinetic model was used to develop a revised dose regimen of dihydroartemisinin-piperaquine that is expected to provide equivalent piperaquine exposures safely in all patients, including in small children with malaria. Use of this dose regimen is expected to prolong the useful therapeutic life of dihydroartemisinin-piperaquine by increasing cure rates and thereby slowing resistance development. This work was part of the evidence that informed the World Health Organization technical guidelines development group in the development of the recently published treatment guidelines (2015).

Anderson TJC, Nair S, McDew-White M, Cheeseman IH, Nkhoma S, Bilgic F, McGready R, Ashley E, Pyae Phyo A, White NJ, Nosten F. 2017. Population Parameters Underlying an Ongoing Soft Sweep in Southeast Asian Malaria Parasites. Mol Biol Evol, 34 (1), pp. 131-144. | Show Abstract | Read more

Multiple kelch13 alleles conferring artemisinin resistance (ART-R) are currently spreading through Southeast Asian malaria parasite populations, providing a unique opportunity to observe an ongoing soft selective sweep, investigate why resistance alleles have evolved multiple times and determine fundamental population genetic parameters for Plasmodium We sequenced kelch13 (n = 1,876), genotyped 75 flanking SNPs, and measured clearance rate (n = 3,552) in parasite infections from Western Thailand (2001-2014). We describe 32 independent coding mutations including common mutations outside the kelch13 propeller associated with significant reductions in clearance rate. Mutations were first observed in 2003 and rose to 90% by 2014, consistent with a selection coefficient of ∼0.079. ART-R allele diversity rose until 2012 and then dropped as one allele (C580Y) spread to high frequency. The frequency with which adaptive alleles arise is determined by the rate of mutation and the population size. Two factors drive this soft sweep: (1) multiple kelch13 amino-acid mutations confer resistance providing a large mutational target-we estimate the target is 87-163 bp. (2) The population mutation parameter (Θ = 2Neμ) can be estimated from the frequency distribution of ART-R alleles and is ∼5.69, suggesting that short term effective population size is 88 thousand to 1.2 million. This is 52-705 times greater than Ne estimated from fluctuation in allele frequencies, suggesting that we have previously underestimated the capacity for adaptive evolution in Plasmodium Our central conclusions are that retrospective studies may underestimate the complexity of selective events and the Ne relevant for adaptation for malaria is considerably higher than previously estimated.

Phyo AP, Ashley EA, Anderson TJC, Carrara VI, Woodrow CJ, White NJ, Nosten F. 2016. Reply to Meshnick and Hastings et al. Clin Infect Dis, 63 (11), pp. 1528-1529. | Read more

Grist EPM, Flegg JA, Humphreys G, Mas IS, Anderson TJC, Ashley EA, Day NPJ, Dhorda M, Dondorp AM, Faiz MA et al. 2016. Optimal health and disease management using spatial uncertainty: a geographic characterization of emergent artemisinin-resistant Plasmodium falciparum distributions in Southeast Asia. Int J Health Geogr, 15 (1), pp. 37. | Show Abstract | Read more

BACKGROUND: Artemisinin-resistant Plasmodium falciparum malaria parasites are now present across much of mainland Southeast Asia, where ongoing surveys are measuring and mapping their spatial distribution. These efforts require substantial resources. Here we propose a generic 'smart surveillance' methodology to identify optimal candidate sites for future sampling and thus map the distribution of artemisinin resistance most efficiently. METHODS: The approach uses the 'uncertainty' map generated iteratively by a geostatistical model to determine optimal locations for subsequent sampling. RESULTS: The methodology is illustrated using recent data on the prevalence of the K13-propeller polymorphism (a genetic marker of artemisinin resistance) in the Greater Mekong Subregion. CONCLUSION: This methodology, which has broader application to geostatistical mapping in general, could improve the quality and efficiency of drug resistance mapping and thereby guide practical operations to eliminate malaria in affected areas.

Otienoburu SD, Maïga-Ascofaré O, Schramm B, Jullien V, Jones JJ, Zolia YM, Houzé P, Ashley EA, Kiechel J-R, Guérin PJ et al. 2016. Selection of Plasmodium falciparum pfcrt and pfmdr1 polymorphisms after treatment with artesunate-amodiaquine fixed dose combination or artemether-lumefantrine in Liberia. Malar J, 15 (1), pp. 452. | Show Abstract | Read more

BACKGROUND: Plasmodium falciparum uncomplicated malaria can successfully be treated with an artemisinin-based combination therapy (ACT). However resistance is spreading to the different ACT compounds; the artemisinin derivative and the partner drug. Studies of P. falciparum polymorphisms associated with drug resistance can provide a useful tool to track resistance and guide treatment policy as well as an in-depth understanding of the development and spread of resistance. METHODS: The role of P. falciparum molecular markers in selection of reinfections was assessed in an efficacy trial comparing artesunate-amodiaquine fixed-dose combination with artemether-lumefantrine to treat malaria in Nimba County, Liberia 2008-2009. P. falciparum polymorphisms in pfcrt 76, pfmdr1 86, 184 and 1246, and pfmrp1 876 and 1466 were analysed by PCR-RFLP and pyrosequencing. RESULTS: High baseline prevalence of pfmdr1 1246Y was found in Nimba county (38 %). Pfmdr1 1246Y and pfmdr1 86+184+1246 haplotypes NYY and YYY were selected in reinfections in the artesunate-amodiaquine arm and pfcrt K76, pfmdr1 N86 and pfmdr1 haplotype NFD were selected in artemether-lumefantrine reinfections. Parasites harbouring pfmdr1 1246Y could reinfect earlier after treatment with artesunate-amodiaquine and parasites carrying pfmdr1 N86 could reinfect at higher lumefantrine concentrations in patients treated with artemether-lumefantrine. CONCLUSIONS: Although treatment is highly efficacious, selection of molecular markers in reinfections could indicate a decreased sensitivity or tolerance of parasites to the current treatments and the baseline prevalence of molecular markers should be closely monitored. Since individual drug levels and the day of reinfection were demonstrated to be key determinants for selection of reinfections, this data needs to be collected and taken into account for accurate evaluation of molecular markers for anti-malarial treatments. The protocols for the clinical trial was registered with Current Controlled Trials, under the Identifier Number ISRCTN51688713 on 9 October 2008.

Phyo AP, Ashley EA, Anderson TJC, Bozdech Z, Carrara VI, Sriprawat K, Nair S, White MM, Dziekan J, Ling C et al. 2016. Declining Efficacy of Artemisinin Combination Therapy Against P. Falciparum Malaria on the Thai-Myanmar Border (2003-2013): The Role of Parasite Genetic Factors. Clin Infect Dis, 63 (6), pp. 784-791. | Show Abstract | Read more

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

WWARN Gametocyte Study Group. 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 Med, 14 (1), pp. 79. | Show Abstract | Read more

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.

Tun KM, Jeeyapant A, Imwong M, Thein M, Aung SSM, Hlaing TM, Yuentrakul P, Promnarate C, Dhorda M, Woodrow CJ 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.

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.

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

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

WorldWide Antimalarial Resistance Network (WWARN) Lumefantrine PK/PD Study Group. 2015. Artemether-lumefantrine treatment of uncomplicated Plasmodium falciparum malaria: a systematic review and meta-analysis of day 7 lumefantrine concentrations and therapeutic response using individual patient data. BMC Med, 13 (1), pp. 227. | Show Abstract | Read more

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

WWARN Artemisinin based Combination Therapy (ACT) Africa Baseline Study Group, Dahal P, d'Alessandro U, Dorsey G, Guerin PJ, Nsanzabana C, Price RN, Sibley CH, Stepniewska K, Talisuna AO. 2015. Clinical determinants of early parasitological response to ACTs in African patients with uncomplicated falciparum malaria: a literature review and meta-analysis of individual patient data. BMC Med, 13 (1), pp. 212. | Show Abstract | Read more

BACKGROUND: Artemisinin-resistant Plasmodium falciparum has emerged in the Greater Mekong sub-region and poses a major global public health threat. Slow parasite clearance is a key clinical manifestation of reduced susceptibility to artemisinin. This study was designed to establish the baseline values for clearance in patients from Sub-Saharan African countries with uncomplicated malaria treated with artemisinin-based combination therapies (ACTs). METHODS: A literature review in PubMed was conducted in March 2013 to identify all prospective clinical trials (uncontrolled trials, controlled trials and randomized controlled trials), including ACTs conducted in Sub-Saharan Africa, between 1960 and 2012. Individual patient data from these studies were shared with the WorldWide Antimalarial Resistance Network (WWARN) and pooled using an a priori statistical analytical plan. Factors affecting early parasitological response were investigated using logistic regression with study sites fitted as a random effect. The risk of bias in included studies was evaluated based on study design, methodology and missing data. RESULTS: In total, 29,493 patients from 84 clinical trials were included in the analysis, treated with artemether-lumefantrine (n = 13,664), artesunate-amodiaquine (n = 11,337) and dihydroartemisinin-piperaquine (n = 4,492). The overall parasite clearance rate was rapid. The parasite positivity rate (PPR) decreased from 59.7 % (95 % CI: 54.5-64.9) on day 1 to 6.7 % (95 % CI: 4.8-8.7) on day 2 and 0.9 % (95 % CI: 0.5-1.2) on day 3. The 95th percentile of observed day 3 PPR was 5.3 %. Independent risk factors predictive of day 3 positivity were: high baseline parasitaemia (adjusted odds ratio (AOR) = 1.16 (95 % CI: 1.08-1.25); per 2-fold increase in parasite density, P <0.001); fever (>37.5 °C) (AOR = 1.50 (95 % CI: 1.06-2.13), P = 0.022); severe anaemia (AOR = 2.04 (95 % CI: 1.21-3.44), P = 0.008); areas of low/moderate transmission setting (AOR = 2.71 (95 % CI: 1.38-5.36), P = 0.004); and treatment with the loose formulation of artesunate-amodiaquine (AOR = 2.27 (95 % CI: 1.14-4.51), P = 0.020, compared to dihydroartemisinin-piperaquine). CONCLUSIONS: The three ACTs assessed in this analysis continue to achieve rapid early parasitological clearance across the sites assessed in Sub-Saharan Africa. A threshold of 5 % day 3 parasite positivity from a minimum sample size of 50 patients provides a more sensitive benchmark in Sub-Saharan Africa compared to the current recommended threshold of 10 % to trigger further investigation of artemisinin susceptibility.

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.

White LJ, Flegg JA, Phyo AP, Wiladpai-ngern JH, Bethell D, Plowe C, Anderson T, Nkhoma S, Nair S, Tripura R 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.

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

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

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

Tun KM, Imwong M, Lwin KM, Win AA, Hlaing TM, Hlaing T, Lin K, Kyaw MP, Plewes K, Faiz MA 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, Oyola SO, Dhorda M 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.

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

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

Mok S, Ashley EA, Ferreira PE, Zhu L, Lin Z, Yeo T, Chotivanich K, Imwong M, Pukrittayakamee S, Dhorda 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, Drakeley C, Chotivanich K 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.

Lubell Y, Dondorp A, Guérin PJ, Drake T, Meek S, Ashley E, Day NPJ, 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.

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

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

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

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

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

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

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

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

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

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

Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, Sreng S, Anderson JM, Mao S, Sam B 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.).

Das D, Cheah PY, Akter F, Paul D, Islam A, Sayeed AA, Samad R, Rahman R, Hossain A, Dondorp A 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.

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

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

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

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

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

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

Chantler T, Cheah PY, Miiro G, Hantrakum V, Nanvubya A, Ayuo E, Kivaya E, Kidola J, Kaleebu P, Parker M et al. 2014. International health research monitoring: exploring a scientific and a cooperative approach using participatory action research. BMJ Open, 4 (2), pp. e004104. | Show Abstract | Read more

OBJECTIVES: To evaluate and determine the value of monitoring models developed by the Mahidol Oxford Tropical Research Unit and the East African Consortium for Clinical Research, consider how this can be measured and explore monitors' and investigators' experiences of and views about the nature, purpose and practice of monitoring. RESEARCH DESIGN: A case study approach was used within the context of participatory action research because one of the aims was to guide and improve practice. 34 interviews, five focus groups and observations of monitoring practice were conducted. SETTING AND PARTICIPANTS: Fieldwork occurred in the places where the monitoring models are coordinated and applied in Thailand, Cambodia, Uganda and Kenya. Participants included those coordinating the monitoring schemes, monitors, senior investigators and research staff. ANALYSIS: Transcribed textual data from field notes, interviews and focus groups was imported into a qualitative data software program (NVIVO V. 10) and analysed inductively and thematically by a qualitative researcher. The initial coding framework was reviewed internally and two main categories emerged from the subsequent interrogation of the data. RESULTS: The categories that were identified related to the conceptual framing and nature of monitoring, and the practice of monitoring, including relational factors. Particular emphasis was given to the value of a scientific and cooperative style of monitoring as a means of enhancing data quality, trust and transparency. In terms of practice the primary purpose of monitoring was defined as improving the conduct of health research and increasing the capacity of researchers and trial sites. CONCLUSIONS: The models studied utilise internal and network wide expertise to improve the ethics and quality of clinical research. They demonstrate how monitoring can be a scientific and constructive exercise rather than a threatening process. The value of cooperative relations needs to be given more emphasis in monitoring activities, which seek to ensure that research protects human rights and produces reliable data.

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

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

WorldWide Antimalarial Resistance Network (WWARN) DP Study Group. 2013. The effect of dosing regimens on the antimalarial efficacy of dihydroartemisinin-piperaquine: a pooled analysis of individual patient data. PLoS Med, 10 (12), pp. 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.

Flegg JA, Guérin PJ, Nosten F, Ashley EA, Phyo AP, Dondorp AM, Fairhurst RM, Socheat D, Borrmann S, Björkman A 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.

Lim P, Dek D, Try V, Eastman RT, Chy S, Sreng S, Suon S, Mao S, Sopha C, Sam B 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.

Schramm B, Valeh P, Baudin E, Mazinda CS, Smith R, Pinoges L, Sundaygar T, Zolia YM, Jones JJ, Comte E et al. 2013. Tolerability and safety of artesunate-amodiaquine and artemether-lumefantrine fixed dose combinations for the treatment of uncomplicated Plasmodium falciparum malaria: two open-label, randomized trials in Nimba County, Liberia. Malar J, 12 (1), pp. 250. | Show Abstract | Read more

BACKGROUND: Safety surveillance of widely used artemisinin-based combination therapy (ACT) is essential, but tolerability data in the over five years age group are largely anecdotal. METHODS: Two open-label, randomized trials were conducted in Nimba County, Liberia: i) the main tolerability trial with 1,000 Plasmodium falciparum malaria patients aged over five years (Study-T), and, ii) an efficacy trial with a secondary objective of collecting tolerability data among 300 children age six to 59 months (Study-E). In both studies patients were randomized to fixed-dose artesunate-amodiaquine (ASAQ Winthrop®) or artemether-lumefantrine (AL, Coartem®), respectively. Clinical- and laboratory-adverse events (AEs) were recorded until day 28. RESULTS: Study-T: most patients experienced at least one AE. Severe AEs were few, primarily asymptomatic blood system disorders or increased liver enzyme values. No treatment or study discontinuation occurred. Mild or moderate fatigue (39.8% vs 16.3%, p < 0.001), vomiting (7.1% vs 1.6%, p < 0.001), nausea (3.2% vs 1.0%, p = 0.01), and anaemia (14.9% vs 9.8%, p = 0.01) were more frequently recorded in the ASAQ versus AL arm. Study-E: mild or moderate AEs were common, including anaemia, fatigue, vomiting or diarrhoea. The few severe events were asymptomatic blood system disorders and four clinical events (pneumonia, malaria, vomiting and stomatitis). CONCLUSION: Both ASAQ and AL were well tolerated in patients of all age groups. No unexpected AEs occurred. Certain mild or moderate AEs were more frequent in the ASAQ arm. Standardised safety surveillance should continue for all forms of ACT. TRIAL REGISTRATION: The protocols were registered with Current Controlled Trials, under the identifier numbers ISRCTN40020296, ISRCTN51688713, (http://www.controlled-trials.com).

Schramm B, Valeh P, Baudin E, Mazinda CS, Smith R, Pinoges L, Dhorda M, Boum Y, Sundaygar T, Zolia YM et al. 2013. Efficacy of artesunate-amodiaquine and artemether-lumefantrine fixed-dose combinations for the treatment of uncomplicated Plasmodium falciparum malaria among children aged six to 59 months in Nimba County, Liberia: an open-label randomized non-inferiority trial. Malar J, 12 (1), pp. 251. | Show Abstract | Read more

BACKGROUND: Prospective efficacy monitoring of anti-malarial treatments is imperative for timely detection of resistance development. The in vivo efficacy of artesunate-amodiaquine (ASAQ) fixed-dose combination (FDC) was compared to that of artemether-lumefantrine (AL) among children aged six to 59 months in Nimba County, Liberia, where Plasmodium falciparum malaria is endemic and efficacy data are scarce. METHODS: An open-label, randomized controlled non-inferiority trial compared the genotyping adjusted day 42 cure rates of ASAQ FDC (ASAQ Winthrop®) to AL (Coartem®) in 300 children aged six to 59 months with uncomplicated falciparum malaria. Inclusion was between December 2008 and May 2009. Randomization (1:1) was to a three-day observed oral regimen (ASAQ: once a day; AL: twice a day, given with fatty food). Day 7 desethylamodiaquine and lumefantrine blood-concentrations were also measured. RESULTS: The day 42 genotyping-adjusted cure rate estimates were 97.3% [95% CI: 91.6-99.1] for ASAQ and 94.2% [88.1-97.2] for AL (Kaplan-Meier survival estimates). The difference in day 42 cure rates was -3.1% [upper limit 95% CI: 1.2%]. These results were confirmed by observed proportion of patients cured at day 42 on the per-protocol population. Parasite clearance was 100% (ASAQ) and 99.3% (AL) on day 3. The probability to remain free of re-infection was 0.55 [95% CI: 0.46-0.63] (ASAQ) and 0.66 [0.57-0.73] (AL) (p = 0.017). CONCLUSIONS: Both ASAQ and AL were highly efficacious and ASAQ was non-inferior to AL. The proportion of patients with re-infection was high in both arms in this highly endemic setting. In 2010, ASAQ FDC was adopted as the first-line national treatment in Liberia. Continuous efficacy monitoring is recommended. TRIAL REGISTRATION: The protocols were registered with Current Controlled Trials, under the identifier numbers ISRCTN51688713, ISRCTN40020296.

Carrara VI, Lwin KM, Phyo AP, Ashley E, Wiladphaingern J, Sriprawat K, Rijken M, Boel M, McGready R, Proux S et al. 2013. Malaria burden and artemisinin resistance in the mobile and migrant population on the Thai-Myanmar border, 1999-2011: an observational study. PLoS Med, 10 (3), pp. e1001398. | Show Abstract | Read more

BACKGROUND: The Shoklo Malaria Research Unit has been working on the Thai-Myanmar border for 25 y providing early diagnosis and treatment (EDT) of malaria. Transmission of Plasmodium falciparum has declined, but resistance to artesunate has emerged. We expanded malaria activities through EDT and evaluated the impact over a 12-y period. METHODS AND FINDINGS: Between 1 October 1999 and 30 September 2011, the Shoklo Malaria Research Unit increased the number of cross-border (Myanmar side) health facilities from two to 11 and recorded the number of malaria consultations. Changes in malaria incidence were estimated from a cohort of pregnant women, and prevalence from cross-sectional surveys. In vivo and in vitro antimalarial drug efficacy were monitored. Over this period, the number of malaria cases detected increased initially, but then declined rapidly. In children under 5 y, the percentage of consultations due to malaria declined from 78% (95% CI 76-80) (1,048/1,344 consultations) to 7% (95% CI 6.2-7.1) (767/11,542 consultations), p<0.001. The ratio of P. falciparum/P. vivax declined from 1.4 (95% CI 1.3-1.4) to 0.7 (95% CI 0.7-0.8). The case fatality rate was low (39/75,126; 0.05% [95% CI 0.04-0.07]). The incidence of malaria declined from 1.1 to 0.1 episodes per pregnant women-year. The cumulative proportion of P. falciparum decreased significantly from 24.3% (95% CI 21.0-28.0) (143/588 pregnant women) to 3.4% (95% CI 2.8-4.3) (76/2,207 pregnant women), p<0.001. The in vivo efficacy of mefloquine-artesunate declined steadily, with a sharp drop in 2011 (day-42 PCR-adjusted cure rate 42% [95% CI 20-62]). The proportion of patients still slide positive for malaria at day 3 rose from 0% in 2000 to reach 28% (95% CI 13-45) (8/29 patients) in 2011. CONCLUSIONS: Despite the emergence of resistance to artesunate in P. falciparum, the strategy of EDT with artemisinin-based combination treatments has been associated with a reduction in malaria in the migrant population living on the Thai-Myanmar border. Although limited by its observational nature, this study provides useful data on malaria burden in a strategically crucial geographical area. Alternative fixed combination treatments are needed urgently to replace the failing first-line regimen of mefloquine and artesunate. Please see later in the article for the Editors' Summary.

Nkhoma SC, Nair S, Al-Saai S, Ashley E, McGready R, Phyo AP, Nosten F, Anderson TJC. 2013. Population genetic correlates of declining transmission in a human pathogen. Mol Ecol, 22 (2), pp. 273-285. | Show Abstract | Read more

Pathogen control programs provide a valuable, but rarely exploited, opportunity to directly examine the relationship between population decline and population genetics. We investigated the impact of an ~12-fold decline in transmission on the population genetics of Plasmodium falciparum infections (n = 1731) sampled from four clinics on the Thai-Burma border over 10 years and genotyped using 96 genome-wide SNPs. The most striking associated genetic change was a reduction in the frequency of infections containing multiple parasite genotypes from 63% in 2001 to 14% in 2010 (P = 3 × 10(-15)). Two measures of the clonal composition of populations (genotypic richness and the β-parameter of the Pareto distribution) declined over time as more people were infected by parasites with identical multilocus genotypes, consistent with increased selfing and a reduction in the rate at which multilocus genotypes are broken apart by recombination. We predicted that the reduction in transmission, multiple clone carriage and outbreeding would be mirrored by an increased influence of genetic drift. However, geographical differentiation and expected heterozygosity remained stable across the sampling period. Furthermore, N(e) estimates derived from allele frequencies fluctuation between years remained high (582 to ∞) and showed no downward trend. These results demonstrate how genetic data can compliment epidemiological assessments of infectious disease control programs. The temporal changes in a single declining population parallel to those seen in comparisons of parasite genetics in regions of differing endemicity, strongly supporting the notion that reduced opportunity for outbreeding is the key driver of these patterns.

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

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

Chue AL, Moore RL, Cavey A, Ashley EA, Stepniewska K, Nosten F, McGready R. 2012. Comparability of tympanic and oral mercury thermometers at high ambient temperatures. BMC Res Notes, 5 (1), pp. 356. | Show Abstract | Read more

BACKGROUND: Body temperature can be measured in seconds with tympanic thermometers as opposed to minutes with mercury ones. The aim of this study was to compare tympanic and oral mercury thermometer measurements under high ambient field temperatures. RESULTS: Tympanic temperature (measured thrice by 3 operators) was compared to oral temperature measured once with a mercury-in-glass thermometer in 201 patients (aged ≥5 years), on the Thai-Myanmar border. Ambient temperature was measured with an electronic thermo-hygrometer. Participants had a mean [min-max] age of 27 [5-60] years and 42% (84) were febrile by oral thermometer. The mean difference in the mercury and tympanic temperature measurement for all observers/devices was 0.09 (95%CI 0.07-0.12)°C and intra-class correlation for repeat tympanic measurements was high (≥0.97) for each observer. Deviations in tympanic temperatures were not related to ambient temperature. CONCLUSION: Clinically significant differences were not observed between oral and tympanic temperature measurements at high ambient temperatures in a rural tropical setting.

Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, McGready R, ler Moo C, Al-Saai S, Dondorp AM, Lwin KM 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.

Cheeseman IH, Miller BA, Nair S, Nkhoma S, Tan A, Tan JC, Al Saai S, Phyo AP, Moo CL, Lwin KM 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.

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

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

Dhorda M, Piola P, Nyehangane D, Tumwebaze B, Nalusaji A, Nabasumba C, Turyakira E, McGready R, Ashley E, Guerin PJ, Snounou G. 2012. Performance of a histidine-rich protein 2 rapid diagnostic test, Paracheck Pf®, for detection of malaria infections in Ugandan pregnant women. Am J Trop Med Hyg, 86 (1), pp. 93-95. | Show Abstract | Read more

Improved laboratory diagnosis is critical to reduce the burden of malaria in pregnancy. Peripheral blood smears appear less sensitive than Plasmodium falciparum histidine-rich protein 2-based rapid diagnostic tests (RDTs) for placental malaria infections in studies conducted at delivery. In this study, 81 women in Uganda in the second or third trimester of pregnancy were followed-up until delivery. At each visit, peripheral blood was tested by blood smear, RDT, and nested species-specific polymerase chain reaction (PCR). Sensitivity and specificity of the tests was calculated with PCR, which detected 22 infections of P. falciparum, as the gold standard. The sensitivity and specificity of blood smears were 36.4% (95% confidence interval [CI] = 18.0-59.2%) and 99.6% (95% CI = 97.7-100%), respectively. The corresponding values for RDT were 31.8% (95% CI = 14.7-54.9%) and 100% (95% CI = 98.3-100%). The RDTs could replace blood smears for diagnosis of malaria in pregnancy by virtue of their relative ease of use. Field-based sensitive tests for malaria in pregnancy are urgently needed.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Piola P, Nabasumba C, Turyakira E, Dhorda M, Lindegardh N, Nyehangane D, Snounou G, Ashley EA, McGready R, Nosten F, Guerin PJ. 2010. Efficacy and safety of artemether-lumefantrine compared with quinine in pregnant women with uncomplicated Plasmodium falciparum malaria: an open-label, randomised, non-inferiority trial. Lancet Infect Dis, 10 (11), pp. 762-769. | Show Abstract | Read more

BACKGROUND: Malaria in pregnancy is associated with maternal and fetal morbidity and mortality. In 2006, WHO recommended use of artemisinin-based combination treatments during the second or third trimesters, but data on efficacy and safety in Africa were scarce. We aimed to assess whether artemether-lumefantrine was at least as efficacious as oral quinine for the treatment of uncomplicated falciparum malaria during the second and third trimesters of pregnancy in Mbarara, Uganda. METHODS: We did an open-label, randomised, non-inferiority trial between October, 2006, and May, 2009, at the antenatal clinics of the Mbarara University of Science and Technology Hospital in Uganda. Pregnant women were randomly assigned (1:1) by computer generated sequence to receive either quinine hydrochloride or artemether-lumefantrine, and were followed up weekly until delivery. Our primary endpoint was cure rate at day 42, confirmed by PCR. The non-inferiority margin was a difference in cure rate of 5%. Analysis of efficacy was for all randomised patients without study deviations that could have affected the efficacy outcome. This study was registered with ClinicalTrials.gov, number NCT00495508. FINDINGS: 304 women were randomly assigned, 152 to each treatment group. By day 42, 16 patients were lost to follow-up and 25 were excluded from the analysis. At day 42, 137 (99.3%) of 138 patients taking artemether-lumefantrine and 122 (97.6%) of 125 taking quinine were cured-difference 1.7% (lower limit of 95% CI -0.9). There were 290 adverse events in the quinine group and 141 in the artemether-lumefantrine group. INTERPRETATION: Artemisinin derivatives are not inferior to oral quinine for the treatment of uncomplicated malaria in pregnancy and might be preferable on the basis of safety and efficacy. FUNDING: Médecins Sans Frontières and the European Commission.

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

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

Inthavilay S, Franchard T, Meimei Y, Ashley EA, Barennes H. 2010. Knowledge and acceptability of the rectal treatment route in Laos and its application for pre-referral emergency malaria treatment. Malar J, 9 (1), pp. 342. | Show Abstract | Read more

BACKGROUND: Rectal artesunate has been shown to reduce death and disability from severe malaria caused by delays in reaching facilities capable of providing appropriate treatment. Acceptability of this mode of drug delivery in Laos is not known. In 2009 the acceptability of rectal treatments was evaluated among the general Lao population and Lao doctors in a national survey. METHODS: A cross sectional survey was performed of 985 households selected through a multi-stage random sampling process from 85 villages in 12/18 provinces and of 315 health staff randomly selected at each administrative level. RESULTS: Out of 985 families, 9% had used the rectal route to treat children (the main indication was seizures or constipation). The population considered it less effective than other routes. Other concerns raised included pain (28%), discomfort for children (40%) and the possibility of other side effects (20%). Of 300 health staff surveyed (nurses 44%, doctors 66%), only 51% had already used the rectal route with a suppository, mostly to treat fever (76%). Health staff working in provincial hospitals had more experience of using the rectal route than those in urban areas. The majority (92%) were keen to use the rectal route to treat malaria although oral and intramuscular routes were preferred and considered to be more efficacious. DISCUSSION AND CONCLUSION: Use of rectal treatments is uncommon in Laos and generally not considered to be very effective. This view is shared by the population and health care workers. More information and training are needed to convince the population and health staff of the efficacy and advantages of the rectal route for malaria treatment.

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

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

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

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

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

Ashley EA, Touabi M, Ahrer M, Hutagalung R, Htun K, Luchavez J, Dureza C, Proux S, Leimanis M, Lwin MM et al. 2009. Evaluation of three parasite lactate dehydrogenase-based rapid diagnostic tests for the diagnosis of falciparum and vivax malaria. Malar J, 8 (1), pp. 241. | Show Abstract | Read more

BACKGROUND: In areas where non-falciparum malaria is common rapid diagnostic tests (RDTs) capable of distinguishing malaria species reliably are needed. Such tests are often based on the detection of parasite lactate dehydrogenase (pLDH). METHODS: In Dawei, southern Myanmar, three pLDH based RDTs (CareStart Malaria pLDH (Pan), CareStart Malaria pLDH (Pan, Pf) and OptiMAL-IT)were evaluated in patients presenting with clinically suspected malaria. Each RDT was read independently by two readers. A subset of patients with microscopically confirmed malaria had their RDTs repeated on days 2, 7 and then weekly until negative. At the end of the study, samples of study batches were sent for heat stability testing. RESULTS: Between August and November 2007, 1004 patients aged between 1 and 93 years were enrolled in the study. Slide microscopy (the reference standard) diagnosed 213 Plasmodium vivax (Pv) monoinfections, 98 Plasmodium falciparum (Pf) mono-infections and no malaria in 650 cases. The sensitivities (sens) and specificities (spec), of the RDTs for the detection of malaria were- CareStart Malaria pLDH (Pan) test: sens 89.1% [CI95 84.2-92.6], spec 97.6% [CI95 96.5-98.4]. OptiMal-IT: Pf+/- other species detection: sens 95.2% [CI95 87.5-98.2], spec 94.7% [CI95 93.3-95.8]; non-Pf detection alone: sens 89.6% [CI95 83.6-93.6], spec 96.5% [CI95 94.8-97.7]. CareStart Malaria pLDH (Pan, Pf): Pf+/- other species: sens 93.5% [CI95 85.4-97.3], spec 97.4% [95.9-98.3]; non-Pf: sens 78.5% [CI95 71.1-84.4], spec 97.8% [CI95 96.3-98.7]. Inter-observer agreement was excellent for all tests (kappa > 0.9). The median time for the RDTs to become negative was two days for the CareStart Malaria tests and seven days for OptiMAL-IT. Tests were heat stable up to 90 days except for OptiMAL-IT (Pf specific pLDH stable to day 20 at 35 degrees C). CONCLUSION: None of the pLDH-based RDTs evaluated was able to detect non-falciparum malaria with high sensitivity, particularly at low parasitaemias. OptiMAL-IT performed best overall and would perform best in an area of high malaria prevalence among screened fever cases. However, heat stability was unacceptable and the number of steps to perform this test is a significant drawback in the field. A reliable, heat-stable, highly sensitive RDT, capable of diagnosing all Plasmodium species has yet to be identified.

Zwang J, Ashley EA, Karema C, D'Alessandro U, Smithuis F, Dorsey G, Janssens B, Mayxay M, Newton P, Singhasivanon P et al. 2009. Safety and efficacy of dihydroartemisinin-piperaquine in falciparum malaria: a prospective multi-centre individual patient data analysis. PLoS One, 4 (7), pp. e6358. | Show Abstract | Read more

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

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

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

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

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

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

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

Lwin KM, Ashley EA, Proux S, Silamut K, Nosten F, McGready R. 2008. Clinically uncomplicated Plasmodium falciparum malaria with high schizontaemia: a case report. Malar J, 7 (1), pp. 57. | Show Abstract | Read more

BACKGROUND: The treatment options for acute Plasmodium falciparum malaria are based on the clinician classifying the patient as uncomplicated or severe according to the clinical and parasitological findings. This process is not always straightforward. CASE PRESENTATION: An adult male presented to a clinic on the western border of Thailand with a physical examination and P. falciparum trophozoite count (1.2% of infected red blood cells, IRBC) from malaria blood smear, consistent with a diagnosis of uncomplicated P. falciparum infection. However, the physician on duty treated the patient for severe malaria based on the reported P. falciparum schizont count, which was very high (0.3% IRBC), noticeably in relation to the trophozoite count and schizont:trophozoite ratio 0.25:1. On intravenous artesunate, the patient deteriorated clinically in the first 24 hours. The trophozoite count increased from 1.2% IRBC at baseline to 20.5% IRBC 18 hours following the start of treatment. By day three, the patient recovered and was discharged on day seven having completed a seven-day treatment with artesunate and mefloquine. CONCLUSION: The malaria blood smear provides only a guide to the overall parasite biomass in the body, due to the ability of P. falciparum to sequester in the microvasculature. In severe malaria, high schizont counts are associated with worse prognosis. In low transmission areas or in non-immune travelers the presence of schizonts in the peripheral circulation is an indication for close patient supervision. In this case, an unusually high schizont count in a clinically uncomplicated patient was indicative of potential deterioration. Prompt treatment with intravenous artesunate is likely to have been responsible for the good clinical outcome in this case.

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

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

Lee SJ, Stepniewska K, Anstey N, Ashley E, Barnes K, Binh TQ, D'Alessandro U, Day NPJ, de Vries PJ, Dorsey G et al. 2008. The relationship between the haemoglobin concentration and the haematocrit in Plasmodium falciparum malaria. Malar J, 7 (1), pp. 149. | Show Abstract | Read more

BACKGROUND: Malaria is a very important cause of anaemia in tropical countries. Anaemia is assessed either by measurement of the haematocrit or the haemoglobin concentration. For comparisons across studies, it is often necessary to derive one measure from the other. METHODS: Data on patients with slide-confirmed uncomplicated falciparum malaria were pooled from 85 antimalarial drug trials conducted in 25 different countries, to assess the haemoglobin/haematocrit relationship at different time points in malaria. Using a linear random effects model, a conversion equation for haematocrit was derived based on 3,254 measurements from various time points (ranging from day 0 to day 63) from 1,810 patients with simultaneous measurements of both parameters. Haemoglobin was also estimated from haematocrit with the commonly used threefold conversion. RESULTS: A good fit was obtained using Haematocrit = 5.62 + 2.60 * Haemoglobin. On average, haematocrit/3 levels were slightly higher than haemoglobin measurements with a mean difference (+/- SD) of -0.69 (+/- 1.3) for children under the age of 5 (n = 1,440 measurements from 449 patients). CONCLUSION: Based on this large data set, an accurate and robust conversion factor both in acute malaria and in convalescence was obtained. The commonly used threefold conversion is also valid.

Ashley EA, Pinoges L, Turyakira E, Dorsey G, Checchi F, Bukirwa H, van den Broek I, Zongo I, Urruta PPP, van Herp M et al. 2008. Different methodological approaches to the assessment of in vivo efficacy of three artemisinin-based combination antimalarial treatments for the treatment of uncomplicated falciparum malaria in African children. Malar J, 7 (1), pp. 154. | Show Abstract | Read more

BACKGROUND: Use of different methods for assessing the efficacy of artemisinin-based combination antimalarial treatments (ACTs) will result in different estimates being reported, with implications for changes in treatment policy. METHODS: Data from different in vivo studies of ACT treatment of uncomplicated falciparum malaria were combined in a single database. Efficacy at day 28 corrected by PCR genotyping was estimated using four methods. In the first two methods, failure rates were calculated as proportions with either (1a) reinfections excluded from the analysis (standard WHO per-protocol analysis) or (1b) reinfections considered as treatment successes. In the second two methods, failure rates were estimated using the Kaplan-Meier product limit formula using either (2a) WHO (2001) definitions of failure, or (2b) failure defined using parasitological criteria only. RESULTS: Data analysed represented 2926 patients from 17 studies in nine African countries. Three ACTs were studied: artesunate-amodiaquine (AS+AQ, N = 1702), artesunate-sulphadoxine-pyrimethamine (AS+SP, N = 706) and artemether-lumefantrine (AL, N = 518).Using method (1a), the day 28 failure rates ranged from 0% to 39.3% for AS+AQ treatment, from 1.0% to 33.3% for AS+SP treatment and from 0% to 3.3% for AL treatment. The median [range] difference in point estimates between method 1a (reference) and the others were: (i) method 1b = 1.3% [0 to 24.8], (ii) method 2a = 1.1% [0 to 21.5], and (iii) method 2b = 0% [-38 to 19.3].The standard per-protocol method (1a) tended to overestimate the risk of failure when compared to alternative methods using the same endpoint definitions (methods 1b and 2a). It either overestimated or underestimated the risk when endpoints based on parasitological rather than clinical criteria were applied. The standard method was also associated with a 34% reduction in the number of patients evaluated compared to the number of patients enrolled. Only 2% of the sample size was lost when failures were classified on the first day of parasite recurrence and survival analytical methods were used. CONCLUSION: The primary purpose of an in vivo study should be to provide a precise estimate of the risk of antimalarial treatment failure due to drug resistance. Use of survival analysis is the most appropriate way to estimate failure rates with parasitological recurrence classified as treatment failure on the day it occurs.

Price RN, Dorsey G, Ashley EA, Barnes KI, Baird JK, d'Alessandro U, Guerin PJ, Laufer MK, Naidoo I, Nosten F et al. 2007. World Antimalarial Resistance Network I: clinical efficacy of antimalarial drugs. Malar J, 6 (1), pp. 119. | Show Abstract | Read more

The proliferation of antimalarial drug trials in the last ten years provides the opportunity to launch a concerted global surveillance effort to monitor antimalarial drug efficacy. The diversity of clinical study designs and analytical methods undermines the current ability to achieve this. The proposed World Antimalarial Resistance Network (WARN) aims to establish a comprehensive clinical database from which standardised estimates of antimalarial efficacy can be derived and monitored over time from diverse geographical and endemic regions. The emphasis of this initiative is on five key variables which define the therapeutic response. Ensuring that these data are collected at the individual patient level in a consistent format will facilitate better data management and analytical practices, and ensure that clinical data can be readily collated and made amenable for pooled analyses. Such an approach, if widely adopted will permit accurate and timely recognition of trends in drug efficacy. This will guide not only appropriate interventions to deal with established multidrug resistant strains of malaria, but also facilitate prompt action when new strains of drug resistant plasmodia first emerge. A comprehensive global database incorporating the key determinants of the clinical response with in vitro, molecular and pharmacokinetic parameters will bring together relevant data on host, drug and parasite factors that are fundamental contributors to treatment efficacy. This resource will help guide rational drug policies that optimize antimalarial drug use, in the hope that the emergence and spread of resistance to new drugs can be, if not prevented, at least delayed.

Mytton OT, Ashley EA, Peto L, Price RN, La Y, Hae R, Singhasivanon P, White NJ, Nosten F. 2007. Electrocardiographic safety evaluation of dihydroartemisinin piperaquine in the treatment of uncomplicated falciparum malaria. Am J Trop Med Hyg, 77 (3), pp. 447-450. | Show Abstract | Read more

Dihydroartemisinin-piperaquine (DP) could become a leading fixed combination malaria treatment worldwide. Although there is accumulating evidence of efficacy and safety from clinical trials, data on cardiotoxicity are limited. In two randomized controlled trials in Thailand, 56 patients had ECGs performed before treatment, 4 hours after the first dose, and 4 hours after the last dose. The mean (95% CI) changes in QTc interval (Bazett's correction) were 2 (-6 to 9) ms and 14 (7 to 21) ms, respectively. These small changes on the third day of treatment are similar to those observed elsewhere in the convalescent phase following antimalarial treatment with drugs known to have no cardiac effects and are therefore likely to result from recovery from acute malaria and not the treatment given. At therapeutic doses, DP does not have clinically significant effects on the electrocardiogram.

Myint HY, Ashley EA, Day NPJ, Nosten F, White NJ. 2007. Efficacy and safety of dihydroartemisinin-piperaquine. Trans R Soc Trop Med Hyg, 101 (9), pp. 858-866. | Show Abstract | Read more

Dihydroartemisinin-piperaquine, a fixed-dose combination antimalarial, is an inexpensive, safe and highly effective treatment for uncomplicated falciparum or vivax malaria. Efficacy assessed over 28-63 days has consistently exceeded 95% in the treatment of multidrug-resistant falciparum malaria. More than 2600 patients have been treated with this combination in prospective studies, mainly in Southeast Asia. Tolerability was uniformly good, and no serious adverse effects have been identified. The dosing regimen has been simplified from four doses to once daily over 3 days. More information on efficacy in Africa, and more pharmacokinetic and efficacy data in children are needed.

Mytton OT, McGready R, Lee SJ, Roberts CH, Ashley EA, Carrara VI, Thwai KL, Jay MP, Wiangambun T, Singhasivanon P, Nosten F. 2007. Safety of benzyl benzoate lotion and permethrin in pregnancy: a retrospective matched cohort study. BJOG, 114 (5), pp. 582-587. | Show Abstract | Read more

OBJECTIVE: To assess the safety of benzyl benzoate lotion (BBL) and permethrin, topical treatments for scabies, during pregnancy. DESIGN: A retrospective controlled cohort study. POPULATION: Refugee and migrant women attending antenatal clinics (ANC) on the Thai-Burmese border between August 1993 and April 2006. METHODS: Women treated with either BBL (25%) or permethrin (4%) were identified from a manual search of antenatal records. Each case of scabies was matched with four scabies-free controls for gravidity, age, smoking status, malaria, period of treatment and gestational age at treatment. Conditional Poisson regression was used to estimate risk ratios for outcomes of pregnancy (proportion of abortions, congenital abnormalities, neonatal deaths, stillbirths and premature babies), mean birthweight and estimated median gestational age, for scabies and scabies-free women, independently for BBL and permethrin. RESULTS: There were no statistically significant differences in pregnancy outcomes between women who were treated with either BBL (n = 444) compared with their matched controls (n = 1,776) or permethrin (n = 196) treated women and their matched controls (n = 784). Overall, only 10.9% (n = 66) of treatments were in the first trimester. Retreatment rates were higher with BBL 16.4%, than permethrin 9.7%, P = 0.038. Scabies was more common during cooler periods. CONCLUSION: We found no evidence of adverse effects on pregnancy outcome due to topical 25% BBL or 4% permethrin.

Ashley EA, Stepniewska K, Lindegårdh N, McGready R, Annerberg A, Hutagalung R, Singtoroj T, Hla G, Brockman A, Proux S et al. 2007. Pharmacokinetic study of artemether-lumefantrine given once daily for the treatment of uncomplicated multidrug-resistant falciparum malaria. Trop Med Int Health, 12 (2), pp. 201-208. | Show Abstract | Read more

BACKGROUND: Adherence to antimalarial drug regimens is improved by simple dosing. If the fixed antimalarial drug combination artemether-lumefantrine (AL) could be given once daily, this should improve adherence and thus effectiveness and lower the risk of selecting for resistance. METHODS: In an open randomized study, 43 patients with uncomplicated falciparum malaria were given equivalent doses of AL with 200 ml flavoured milk either as the conventional twice-daily regimen or as a single daily dose for 3 days. The primary end point was a comparison of the areas under the plasma lumefantrine concentration-time curves (AUC). Secondary end points were the day 42 polymerase chain reaction (PCR)-adjusted cure rates and the tolerability profiles. RESULTS: Lumefantrine pharmacokinetic profiles were obtained for 36 patients. The AUC((0-->infinity)) of the once-daily regimen was 30% lower than that in the conventional regimen (P = 0.011) with a median (range) value of 306 (114-5781) microg/ml h, compared with 432 (308-992) microg/ml h. There was no significant difference in the peak plasma concentrations reached. PCR-adjusted cure rate estimates at day 42 of follow-up were 94% (95% CI: 84-100) in the six-dose arm and 85% (70-100) in the three-dose arm (P = 0.3). CONCLUSION: Artemether-lumefantrine efficacy is reduced by once-daily dosing, because absorption of lumefantrine is dose limited. At currently recommended doses, this antimalarial should be given twice daily in a 3-day regimen, with food containing fat.

Ashley EA, Stepniewska K, Lindegårdh N, Annerberg A, Kham A, Brockman A, Singhasivanon P, White NJ, Nosten F. 2007. How much fat is necessary to optimize lumefantrine oral bioavailability? Trop Med Int Health, 12 (2), pp. 195-200. | Show Abstract | Read more

BACKGROUND: Artemether-lumefantrine (AL) is the only fixed, artemisinin-based combination antimalarial drug which is registered internationally and deployed on a large scale. Absorption of the hydrophobic lipophilic lumefantrine component varies widely between individuals and is greatly increased by fat coadministration; but patients with acute malaria are frequently nauseated and anorexic, making dietary advice difficult to comply with. The aim of this study was to describe the dose-response relationship between coadministration of fat and relative lumefantrine bioavailability, in order to determine the minimum amount of fat necessary to optimize absorption. METHOD: We conducted a multiple crossover pharmacokinetic study in 12 healthy volunteers. This compared the area under the plasma concentration-time curve (AUC) for lumefantrine after administration of a single dose of AL in the fasting state given with 0, 10, 40, 150 and 500 ml of soya milk corresponding to 0, 0.32, 1.28, 4.8 and 16 g of fat. All volumes of milk supplements were tested in all subjects with a 3- to 4-week washout period in-between. RESULTS: A dose-response relationship was demonstrated between the volume of soya milk administered and lumefantrine bioavailability. AL administration with soya milk increased the lumefantrine AUC more than five fold. The population mean estimated volume of soya milk required to obtain 90% of maximum effect (in terms of lumefantrine AUC) was 36 ml (corresponding to 1.2 g of fat). CONCLUSIONS: Coadministration of artemether-lumefantrine with a relatively small amount of fat (as soya milk) was required to ensure maximum absorption of lumefantrine in healthy adult volunteers.

Uhlemann A-C, McGready R, Ashley EA, Brockman A, Singhasivanon P, Krishna S, White NJ, Nosten F, Price RN. 2007. Intrahost selection of Plasmodium falciparum pfmdr1 alleles after antimalarial treatment on the northwestern border of Thailand. J Infect Dis, 195 (1), pp. 134-141. | Show Abstract | Read more

BACKGROUND: Increased pfmdr1 copy number is associated with reduced susceptibility to structurally unrelated antimalarial drugs. We assessed how administration of different antimalarial drugs altered pfmdr1 polymorphism in parasites from patients who experienced treatment failure. METHODS: In studies conducted on the northwestern border of Thailand, amplifications and single-nucleotide polymorphisms in pfmdr1 were compared before and after antimalarial drug treatment. RESULTS: Intrahost changes in pfmdr1 copy number were observed in 20% (26/132) of patients with recurrent infections. Among infections that recrudesced after mefloquine-containing regimens, increases in pfmdr1 copy number occurred in 68% (95% confidence interval [CI], 46%-85%), and decreases occurred in 2% (95% CI, 0.4%-11%) of isolates; corresponding proportions after artemether-lumefantrine were 25% (2/8) and 11% (2/19); after quinine, 50% (1/2) and 40% (4/10); and after artemisinins alone, 0% (0/10) and 19% (3/16) of isolates (overall P<.001). CONCLUSIONS: Intrahost selection based on pfmdr1 copy number occurs frequently in parasite populations within individual patients. Amplification confers multidrug resistance but probably imposes a significant fitness cost to the parasites.

McGready R, Stepniewska K, Lindegardh N, Ashley EA, La Y, Singhasivanon P, White NJ, Nosten F. 2006. The pharmacokinetics of artemether and lumefantrine in pregnant women with uncomplicated falciparum malaria. Eur J Clin Pharmacol, 62 (12), pp. 1021-1031. | Show Abstract | Read more

OBJECTIVE: To determine the pharmacokinetic properties of artemether and lumefantrine (AL) in pregnant women with recrudescent uncomplicated multi-drug resistant falciparum malaria. METHODS: Pregnant women who had recurrence of parasitaemia following 7 days supervised quinine treatment were treated with AL. Serial blood samples were taken over a 7-day period, and pharmacokinetic parameters were estimated. For lumefantrine, these data were compared in a population pharmacokinetic model with data from non-pregnant, mainly male adults with acute malaria. RESULTS: The pregnant women (five in the second trimester and eight in the third trimester) had lower concentrations of artemether, dihydroartemisinin and lumefantrine, and the elimination of lumefantrine in pregnant women was more rapid than reported previously in non-pregnant adults. CONCLUSION: Pregnancy is associated with reduced plasma concentrations of both artemether and lumefantrine. This is likely to be of therapeutic significance as plasma concentrations of lumefantrine, after elimination of artemether, are an important determinant of cure. Further studies are needed to determine the optimum dose regimen of artemether-lumefantrine in pregnancy.

Ashley EA, Lwin KM, McGready R, Simon WH, Phaiphun L, Proux S, Wangseang N, Taylor W, Stepniewska K, Nawamaneerat W et al. 2006. An open label randomized comparison of mefloquine-artesunate as separate tablets vs. a new co-formulated combination for the treatment of uncomplicated multidrug-resistant falciparum malaria in Thailand. Trop Med Int Health, 11 (11), pp. 1653-1660. | Show Abstract | Read more

BACKGROUND: Delivering drugs in a fixed combination is essential to the success of the strategy of artemisinin-based combination therapy. This prevents one drug being taken without the protection of the other, reducing the chance of emergence and spread of drug resistant strains of Plasmodium falciparum. A lower tablet burden should also facilitate adherence to treatment. A new fixed combination of mefloquine plus artesunate has been developed. This was compared with the conventional regimen of separate tablets for the treatment of uncomplicated multidrug-resistant falciparum malaria. METHODS: On the north-western border of Thailand 500 adults and children with uncomplicated falciparum malaria were randomized to receive either the new fixed combination or separate tablets. They were followed up weekly for 63 days. RESULTS: The day 63 polymerase chain reaction-adjusted cure rates were 91.9% (95% CI 88.2-95.6) in the fixed combination group and 89.2% (85.0-93.4) in the loose tablets group (P=0.3). There was a lower incidence of early vomiting in the group receiving the fixed combination. CONCLUSION: This new fixed combination of mefloquine and artesunate was efficacious, well tolerated and convenient to administer.

Nosten F, Ashley E, McGready R, Price R. 2006. We still need artesunate monotherapy. BMJ, 333 (7557), pp. 45. | Read more

Ashley EA, Stepniewska K, Lindegårdh N, McGready R, Hutagalung R, Hae R, Singhasivanon P, White NJ, Nosten F. 2006. Population pharmacokinetic assessment of a new regimen of mefloquine used in combination treatment of uncomplicated falciparum malaria. Antimicrob Agents Chemother, 50 (7), pp. 2281-2285. | Show Abstract | Read more

A fixed artesunate-mefloquine combination, comprising three daily doses of 8 mg of mefloquine/kg of body weight and 4 mg of artesunate/kg, has been developed recently. This study was designed to construct a population pharmacokinetic model describing this new dosage regimen of mefloquine given as loose tablets together with artesunate. In two randomized trials in Thailand which evaluated the efficacy, safety, and tolerability of this new regimen, the members of a subgroup of 50 patients were randomized to have capillary blood sampling before treatment and at five randomly assigned time points during the 63-day follow-up period. Mefloquine levels in capillary whole blood were assayed by liquid chromatography with UV detection. A pharmacokinetic model for mefloquine was constructed using mixed-effects modeling. A one-compartment model with first-order absorption and elimination was selected to describe the kinetic properties of mefloquine. For capillary whole-blood mefloquine, the area under the concentration curve (AUC) was 40% higher than previous estimates for patients given the equivalent conventional-dose regimen (mefloquine given as 15 mg/kg and then 10 mg/kg on the second and third days of treatment). The half-life (t1/2) of the carboxylic acid metabolite was estimated as 26 days, and the metabolite was eliminated more slowly than the parent drug (population t1/2 estimate, 10.5 days). Splitting the 25 mg/kg dose of mefloquine into three doses of 8 mg/kg each resulted in improved oral bioavailability compared to the conventional split-dose regimen results. This new regimen is well tolerated and results in an equivalent therapeutic response.

Smithuis F, Kyaw MK, Phe O, Aye KZ, Htet L, Barends M, Lindegardh N, Singtoroj T, Ashley E, Lwin S et al. 2006. Efficacy and effectiveness of dihydroartemisinin-piperaquine versus artesunate-mefloquine in falciparum malaria: an open-label randomised comparison. Lancet, 367 (9528), pp. 2075-2085. | Show Abstract | Read more

BACKGROUND: Artemisinin-based combinations are judged the best treatments for multidrug-resistant Plasmodium falciparum malaria. Artesunate-mefloquine is widely recommended in southeast Asia, but its high cost and tolerability profile remain obstacles to widespread deployment. To assess whether dihydroartemisinin-piperaquine is a suitable alternative to artesunate-mefloquine, we compared the safety, tolerability, efficacy, and effectiveness of the two regimens for the treatment of uncomplicated falciparum in western Myanmar (Burma). METHODS: We did an open randomised comparison of 3-day regimens of artesunate-mefloquine (12/25 mg/kg) versus dihydroartemisinin-piperaquine (6.3/50 mg/kg) for the treatment of children aged 1 year or older and in adults with uncomplicated falciparum malaria in Rakhine State, western Myanmar. Within each group, patients were randomly assigned supervised or non-supervised treatment. The primary endpoint was the PCR-confirmed parasitological failure rate by day 42. Failure rates at day 42 were estimated by Kaplan-Meier survival analysis. This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN27914471. FINDINGS: Of 652 patients enrolled, 327 were assigned dihydroartemisinin-piperaquine (156 supervised and 171 not supervised), and 325 artesunate-mefloquine (162 and 163, respectively). 16 patients were lost to follow-up, and one patient died 22 days after receiving dihydroartemisinin-piperaquine. Recrudescent parasitaemias were confirmed in only two patients; the day 42 failure rate was 0.6% (95% CI 0.2-2.5) for dihydroartemisinin-piperaquine and 0 (0-1.2) for artesunate-mefloquine. Whole-blood piperaquine concentrations at day 7 were similar for patients with observed and non-observed dihydroartemisinin-piperaquine treatment. Gametocytaemia developed more frequently in patients who had received dihydroartemisinin-piperaquine than in those on artesunate-mefloquine: day 7, 18 (10%) of 188 versus five (2%) of 218; relative risk 4.2 (1.6-11.0) p=0.011. INTERPRETATION: Dihydroartemisinin-piperaquine is a highly efficacious and inexpensive treatment of multidrug-resistant falciparum malaria and is well tolerated by all age groups. The effectiveness of the unsupervised treatment, as in the usual context of use, equalled its supervised efficacy, indicating good adherence without supervision. Dihydroartemisinin-piperaquine is a good alternative to artesunate-mefloquine.

Ashley E, McGready R, Proux S, Nosten F. 2006. Malaria. Travel Med Infect Dis, 4 (3-4), pp. 159-173. | Show Abstract | Read more

Malaria is increasing worldwide due to the emergence and spread of drug resistant strains. This poses major health and economic problems for the population living in endemic areas and increases the risk of infections in travelers. The diagnosis of malaria relies on a biological proof of infection by microscopy or with a rapid test. The treatment must be initiated without delay preferably with an artemisinin containing regimen. Uncomplicated malaria can be treated with oral drugs while severe infections will be hospitalized and treated with injectables. Special attention will be given to the most susceptible groups: children and pregnant women.

White NJ, Ashley EA, Nosten F. 2006. Toxic brainstem encephalopathy after artemisinin treatment for breast cancer. Ann Neurol, 59 (4), pp. 725-726. | Read more

Hutagalung R, Htoo H, Nwee P, Arunkamomkiri J, Zwang J, Carrara VI, Ashley E, Singhasivanon P, White NJ, Nosten F. 2006. A case-control auditory evaluation of patients treated with artemether-lumefantrine. Am J Trop Med Hyg, 74 (2), pp. 211-214. | Show Abstract | Read more

Artemether-lumefantrine is the first registered, fixed, artemisinin-based combination treatment. Artemisinin derivatives are highly effective antimalarials with a favorable safety profile. Concerns remain over their potential neurotoxicity, although there has been no clinical evidence of this in humans. In animals (rats, dogs, and monkeys) artemether, a derivative of artemisinin is associated with an unusual toxicity pattern in specific brain nuclei involving the auditory and vestibular pathways. A recent report from Mozambique described a small but significant and irreversible hearing loss in patients exposed to artemether-lumefantrine. To explore this issue, we conducted a case-control study using tympanometry, audiometry and auditory brain-stem responses. We assessed 68 subjects who had been treated with artemether-lumefantrine within the previous five years and 68 age- and sex-matched controls living in the malarious region along the Thailand-Myanmar border. There were no differences in the test results between cases and controls. There was no neurophysiologic evidence of auditory brainstem toxicity that could be attributed to artemether-lumefantrine in this study population.

McGready R, Ashley EA, Tan SO, Brabin B, Nosten F. 2006. Re: Malaria in pregnancy. BJOG, 113 (2), pp. 246. | Read more

Carrara VI, Sirilak S, Thonglairuam J, Rojanawatsirivet C, Proux S, Gilbos V, Brockman A, Ashley EA, McGready R, Krudsood S et al. 2006. Deployment of early diagnosis and mefloquine-artesunate treatment of falciparum malaria in Thailand: the Tak Malaria Initiative. PLoS Med, 3 (6), pp. e183. | Show Abstract | Read more

BACKGROUND: Early diagnosis and treatment with artesunate-mefloquine combination therapy (MAS) have reduced the transmission of falciparum malaria dramatically and halted the progression of mefloquine resistance in camps for displaced persons along the Thai-Burmese border, an area of low and seasonal transmission of multidrug-resistant Plasmodium falciparum. We extended the same combination drug strategy to all other communities (estimated population 450,000) living in five border districts of Tak province in northwestern Thailand. METHODS AND FINDINGS: Existing health structures were reinforced. Village volunteers were trained to use rapid diagnostic tests and to treat positive cases with MAS. Cases of malaria, hospitalizations, and malaria-related deaths were recorded in the 6 y before, during, and after the Tak Malaria Initiative (TMI) intervention. Cross-sectional surveys were conducted before and during the TMI period. P. falciparum malaria cases fell by 34% (95% confidence interval [CI], 33.5-34.4) and hospitalisations for falciparum malaria fell by 39% (95% CI, 37.0-39.9) during the TMI period, while hospitalisations for P. vivax malaria remained constant. There were 32 deaths attributed to malaria during, and 22 after the TMI, a 51.5% (95% CI, 39.0-63.9) reduction compared to the average of the previous 3 y. Cross-sectional surveys indicated that P. vivax had become the predominant species in Thai villages, but not in populations living on the Myanmar side of the border. In the displaced persons population, where the original deployment took place 7 y before the TMI, the transmission of P. falciparum continued to be suppressed, the incidence of falciparum malaria remained low, and the in vivo efficacy of the 3-d MAS remained high. CONCLUSIONS: In the remote malarious north western border area of Thailand, the early detection of malaria by trained village volunteers, using rapid diagnostic tests and treatment with mefloquine-artesunate was feasible and reduced the morbidity and mortality of multidrug-resistant P. falciparum.

Nosten F, Hutagalung R, Carrara VI, Ashley E, White N. 2006. Letters to the editor American Journal of Tropical Medicine and Hygiene, 74 (6), pp. 940.

Ashley EA, White NJ. 2005. Artemisinin-based combinations. Curr Opin Infect Dis, 18 (6), pp. 531-536. | Show Abstract | Read more

PURPOSE OF REVIEW: Artemisinin-based combination treatments have been the mainstay of treatment for falciparum malaria in Southeast Asia for more than 10 years and are now increasingly recommended as first-line treatment throughout the rest of the world. RECENT FINDINGS: A large multicentre randomised trial conducted in East Asia has shown a 35% reduction in mortality from severe malaria following treatment with parenteral artesunate compared with quinine. There is increasing evidence that artemisinin-based combination treatments are safe and rapidly effective. Artemether-lumefantrine (six doses) has been shown to be very effective in large trials reported from Uganda and Tanzania. A once daily three-dose treatment of dihydroartemisinin piperaquine, a newer fixed combination, was a highly efficacious and well tolerated treatment for multi-drug resistant falciparum malaria in Southeast Asia. SUMMARY: Early diagnosis and treatment of uncomplicated malaria with effective drugs remains a priority as part of a comprehensive malaria control strategy. Artemisinin-based combination treatments have consistently been shown to be highly effective and safe. The challenge is to make them accessible in tropical countries.

McGready R, Ashley EA, Moo E, Cho T, Barends M, Hutagalung R, Looareesuwan S, White NJ, Nosten F. 2005. A randomized comparison of artesunate-atovaquone-proguanil versus quinine in treatment for uncomplicated falciparum malaria during pregnancy. J Infect Dis, 192 (5), pp. 846-853. | Show Abstract | Read more

BACKGROUND: There is no safe, practical, and effective treatment for pregnant women infected with multidrug-resistant Plasmodium falciparum. METHODS: We recruited pregnant Karen women in the second or third trimesters of pregnancy who had uncomplicated falciparum malaria for a randomized, open-label trial with a restricted sequential trial design of 7 days of supervised quinine (SQ7) versus 3 days of artesunate-atovaquone-proguanil (AAP). RESULTS: Eight-one pregnant women entered the study between December 2001 and July 2003; 42 were treated with SQ7 and 39 were treated with AAP. Fever, parasite clearance, and duration of anemia were significantly better with AAP; the treatment failure rate was 7 times lower (5% [2/39] vs. 37% [15/41]; relative risk, 7.1 [95% confidence interval, 1.7-29.2]; P = .001). There were no significant differences in birth weight, duration of gestation, or congenital abnormality rates in newborns or in growth and developmental parameters of infants monitored for 1 year. CONCLUSION: AAP is a well-tolerated, effective, practical, but expensive treatment for multidrug-resistant falciparum malaria during the second or third trimesters of pregnancy. Despite the small number of subjects, our results add to the growing body of evidence that AAP is safe for the mother and the fetus.

Ashley EA, McGready R, Hutagalung R, Phaiphun L, Slight T, Proux S, Thwai KL, Barends M, Looareesuwan S, White NJ, Nosten F. 2005. A randomized, controlled study of a simple, once-daily regimen of dihydroartemisinin-piperaquine for the treatment of uncomplicated, multidrug-resistant falciparum malaria. Clin Infect Dis, 41 (4), pp. 425-432. | Show Abstract | Read more

BACKGROUND: Dihydroartemisinin-piperaquine (DP) is a fixed-combination antimalarial drug increasingly deployed in Southeast Asia. The current regimen involves 4 doses given over 3 days. Simplification of the dose regimen should facilitate treatment adherence and thereby increase effectiveness. METHODS: In a randomized, controlled, 3-arm trial conducted along the northwestern border of Thailand, the standard 4-dose course of DP (DP4) was compared to an equivalent dose given as a once-daily regimen (DP3) and to the standard treatment of mefloquine-artesunate (MAS3). RESULTS: A total of 499 patients were included in the study. Times to fever and parasite clearance were similar in all groups. The PCR genotyping-adjusted cure rates at day 63 after treatment initiation were 95.7% (95% confidence interval [95% CI], 92.2%-98.9%) for MAS3, 100% for DP4, and 99.4% (95% CI, 98.1%-100%) for DP3. The DP4 and DP3 cure rates were significantly higher than that for MAS3 (P=.008 and P=.03, respectively). All regimens were well tolerated. There were 3 deaths (1 in the MAS3 group and 2 in the DP3 group), all of which were considered to be unrelated to treatment. Rates of other adverse events were comparable between the groups, except for diarrhea, which was more common in the DP4 group (P=.05 vs. the MAS3 group). CONCLUSIONS: A once-daily, 3-dose regimen of DP is a highly efficacious treatment for multidrug-resistant falciparum malaria. This simple, safe, and relatively inexpensive fixed combination could become the treatment of choice for falciparum malaria.

Hutagalung R, Paiphun L, Ashley EA, McGready R, Brockman A, Thwai KL, Singhasivanon P, Jelinek T, White NJ, Nosten FH. 2005. A randomized trial of artemether-lumefantrine versus mefloquine-artesunate for the treatment of uncomplicated multi-drug resistant Plasmodium falciparum on the western border of Thailand. Malar J, 4 (1), pp. 46. | Show Abstract | Read more

BACKGROUND: The use of antimalarial drug combinations with artemisinin derivatives is recommended to overcome drug resistance in Plasmodium falciparum. The fixed combination of oral artemether-lumefantrine, an artemisinin combination therapy (ACT) is highly effective and well tolerated. It is the only registered fixed combination containing an artemisinin. The trial presented here was conducted to monitor the efficacy of the six-dose regimen of artemether-lumefantrine (ALN) in an area of multi-drug resistance, along the Thai-Myanmar border. METHODS: The trial was an open-label, two-arm, randomized study comparing artemether-lumefantrine and mefloquine-artesunate for the treatment of uncomplicated falciparum malaria with 42 days of follow up. Parasite genotyping by polymerase chain reaction (PCR) was used to distinguish recrudescent from newly acquired P. falciparum infections. The PCR adjusted cure rates were evaluated by survival analysis. RESULTS: In 2001-2002 a total of 490 patients with slide confirmed uncomplicated P. falciparum malaria were randomly assigned to receive artemether-lumefantrine (n = 245) or artesunate and mefloquine (n = 245) and were followed for 42 days. All patients had rapid initial clinical and parasitological responses. In both groups, the PCR adjusted cure rates by day 42 were high: 98.8% (95% CI 96.4, 99.6%) for artemether-lumefantrine and 96.3% (95% CI 93.1, 98.0%) for artesunate-mefloquine. Both regimens were very well tolerated with no serious adverse events observed attributable to either combination. CONCLUSION: Overall, this study confirms that these two artemisinin-based combinations remain highly effective and result in equivalent therapeutic responses in the treatment of highly drug-resistant falciparum malaria.

Nosten F, McGready R, Ashley E, White NJ. 2005. Malaria misconceptions The Lancet, 365 (9460), pp. 653-653. | Read more

Ashley EA, Krudsood S, Phaiphun L, Srivilairit S, McGready R, Leowattana W, Hutagalung R, Wilairatana P, Brockman A, Looareesuwan S et al. 2004. Randomized, controlled dose-optimization studies of dihydroartemisinin-piperaquine for the treatment of uncomplicated multidrug-resistant falciparum malaria in Thailand. J Infect Dis, 190 (10), pp. 1773-1782. | Show Abstract | Read more

BACKGROUND: Dihydroartemisinin-piperaquine (DP) is a new and relatively inexpensive artemisinin-containing fixed-combination antimalarial treatment. An adult treatment course contained 6.4 mg/kg dihydroartemisinin (DHA), which is >40% lower than the level in most artemisinin-containing combinations. This raised the possibility that the efficacy of the current coformulation may not be optimal in the treatment of multidrug-resistant falciparum malaria. METHODS: In 2 large randomized, controlled studies in Thailand, the recommended dose of DP was compared with a regimen with additional artemisinin derivative (12 mg/kg; DP+) and with mefloquine plus artesunate (MAS3). RESULTS: A total of 731 patients were included: 201 in a hospital-based study and 530 in a community study. Day-28 cure rates in the hospital-based study were 100% (95% confidence interval [CI], 93.9%-100%) in the MAS3 and DP+ groups and 98.3% (95% CI, 91%-99.7%) in the DP group, with a single recrudescence on day 21. In the community study, polymerase chain reaction genotyping-adjusted cure rates on day 63 were 96.1% (95% CI, 92.6%-99.7%) in the DP group, 98.3% (95% CI, 96.1%-100%) in the DP+ group, and 94.9% (95% CI, 91.2%-98.6%) in the MAS3 group (P=.2). Adverse events were few, with an excess of mild abdominal pain in the DP group. CONCLUSIONS: The current dosage of DP (6.4 mg/kg DHA and 51.2 mg/kg piperaquine phosphate) given over the course of 48 h is highly effective, safe, and well tolerated for the treatment of multidrug-resistant falciparum malaria, and its efficacy is not improved by the addition of more DHA.

McGready R, Ashley EA, Nosten F. 2004. Malaria and the pregnant traveller. Travel Med Infect Dis, 2 (3-4), pp. 127-142. | Show Abstract | Read more

Malaria in pregnancy contributes to significant maternal and foetal mortality and morbidity in women in the tropics. Adverse effects for non-immune travellers are potentially devastating for mother and foetus. Women travellers should always be strongly advised against visiting malarious areas if they are pregnant or intend to get pregnant. Chemoprophylactic and treatment options for pregnant women (or those planning to conceive) are extremely limited and lag behind what can currently be offered to non-pregnant travellers. This is because of spread of multi-resistant strains of P. falciparum. Personal protection from malaria vectors remains essential. Mosquito-net and skin repellents (DEET (20%)) are effective. Diagnosis of malaria in travellers is difficult and is more likely to be missed in pregnant travellers due to lower parasitaemia. Pregnant women can succumb rapidly to severe malaria. Should the returned traveller survive an episode of malaria in pregnancy and go on to deliver, the adverse effects on the infant are potentially irreversible. These risks need to be clearly communicated.

Price RN, Uhlemann A-C, Brockman A, McGready R, Ashley E, Phaipun L, Patel R, Laing K, Looareesuwan S, White NJ et al. 2004. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet, 364 (9432), pp. 438-447. | Show Abstract | Read more

BACKGROUND: The borders of Thailand harbour the world's most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known. METHODS: The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism. FINDINGS: Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6.3 (95% CI 2.9-13.8, p<0.001) after mefloquine monotherapy and 5.4 (2.0-14.6, p=0.001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo. INTERPRETATION: Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria. RELEVANCE TO PRACTICE: Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days' artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.

Nacher M, Carrara VI, Ashley E, McGready R, Hutagalung R, Nguen JV, Thwai KL, Looareesuwan S, Nosten F. 2004. Seasonal variation in hyperparasitaemia and gametocyte carriage in patients with Plasmodium falciparum malaria on the Thai-Burmese border. Trans R Soc Trop Med Hyg, 98 (5), pp. 322-328. | Show Abstract | Read more

Between January 2000 and December 2002 monthly rainfall was correlated with the proportion of patients with hyperparasitaemic Plasmodium falciparum malaria and with the proportion of patients with P. falciparum gametocytes. During the observation period 6953 cases of P. falciparum malaria were treated at the Shoklo Malaria Research Unit in Maela refugee camp on the Thai-Burmese border. Three hundred and seventy-five of these patients had >/=4% of parasitized red blood cells. Although there were more monthly malaria cases in the rainy season, rainfall was negatively correlated with the proportion of patients with hyperparasitaemia (Spearman's rho = -0.59, P < 0.001 ), and the proportion of gametocyte carriers among P. falciparum cases, (Spearman's rho = -0.39, P = 0.018). After controlling for age and the origin of the patient, the odds ratio for developing hyperparasitaemia during the dry season was 1.6 (95% CI 1.14-2.2; P = 0.006). The adjusted odds ratio for gametocyte carriage during the dry season was 1.3 (95% CI 1.03-1.6; P = 0.02). Migrations, changes in transmission patterns, the haematological burden of cumulative infections, and ultraviolet immunosuppression are discussed as potential explanations for these observations.

Nacher M, Carrara VI, McGready R, Ashley E, Nguen JV, Thwai KL, Looareesuwan S, Nosten F. 2004. Seasonal fluctuations in the carriage of Plasmodium vivax gametocytes in Thailand. Ann Trop Med Parasitol, 98 (2), pp. 115-120. | Show Abstract | Read more

To study the influence of season on Plasmodium vivax gametocyte carriage, the relationship between monthly rainfall and the proportion of P. vivax patients with detectable gametocytaemia was analysed. Most of the data used came from 6807 aggregated observations collected, in a refugee camp on the Thai-Burmese border, between January 2000 and December 2002. There was a positive correlation between rainfall and the incidence of P. vivax infection (Spearman's rho=+0.42; P =0.01) but the prevalence of gametocyte carriage among those with P. vivax infection was negatively correlated with rainfall (Spearman's rho=-0.58; P <0.001). The latter, negative correlation remained significant after controlling for the proportion of visitors relative to camp residents (P =0.003). Migrations, changes in transmission patterns, seasonal haematological changes, and ultraviolet immunosuppression are discussed as potential explanations for these observations.

Nosten F, Ashley E. 2004. The detection and treatment of Plasmodium falciparum malaria: time for change. J Postgrad Med, 50 (1), pp. 35-39. | Show Abstract

In most countries where malaria is endemic, P. falciparum malaria is on the rise. This is primarily due to the spread of drug-resistant strains. Drug resistance is mediated by spontaneous changes in the parasite genome that allow resistant parasites to escape the action of the drugs. The spread of drug resistance increases the transmission of malaria parasites. The consequences for the populations at risk are profound both in terms of consequences for health and economy. In order to halt the progression of drug resistance, we need to change the way antimalarials are used. As in tuberculosis and HIV/AIDS, we must use a combination of drugs for the treatment of malaria. Taking into account the pharmacokinetic and pharmacodynamic properties of the various anti-malarial agents, artemisinin-based combination therapy (ACT) seems to be the best option. This strategy should be used in conjunction with early diagnosis and appropriate vector control measures to achieve reduction in the emergence and spread of drug resistance.

McGready R, Stepniewska K, Seaton E, Cho T, Cho D, Ginsberg A, Edstein MD, Ashley E, Looareesuwan S, White NJ, Nosten F. 2003. Pregnancy and use of oral contraceptives reduces the biotransformation of proguanil to cycloguanil. Eur J Clin Pharmacol, 59 (7), pp. 553-557. | Show Abstract | Read more

OBJECTIVE: To determine the effects of late pregnancy and also oestrogen supplementation on the CYP2C19-mediated biotransformation of proguanil (PG) to its active antifol triazine metabolite cycloguanil (CG). METHODS: Case control study conducted on the NW border of Thailand; a single dose of PG (4 mg/kg) was administered to Karen women in late pregnancy and a single blood and urine sample taken 6 h later. Women were studied in late pregnancy (>36 weeks) and restudied 2 months after delivery. A separate cohort of Karen women newly attending a birth-control clinic were studied before and 3 weeks into their first course of oral contraceptives (OCP: levonorgestrel 0.15 mg and ethinyloestradiol 0.03 mg). Forty-five pregnant women and forty-two healthy OCP users were studied. RESULTS: The results were similar in both groups; pregnancy and OCP use were both associated with reduced formation of cycloguanil (CG). Impaired PG biotransformation was seen in women with the "extensive metaboliser" phenotype (urine PG/CG ratio <10). CG levels, adjusted for dose, were a median (range) 73% (-59 to 420%) higher following the pregnancy than during the pregnancy in women characterised as extensive metabolisers ( P<0.001). CG levels in women characterised as extensive metabolisers were 34% (-54 to 323%) higher before than while taking the OCP ( P<0.01). CONCLUSION: Late pregnancy and OCP use impair biotransformation of the active antimalarial metabolite CG from the parent PG. This may be mediated by oestrogen inhibition of CYP2C19 activity. The dose of PG should be increased by 50% in these groups.

Determining the optimal dose of mono-specific Daboia siamensis antivenom for systemic envenoming

Myanmar has a high incidence snake-bite, resulting in an estimated 2000 deaths annually. The Eastern Russell’s viper (Daboia siamensis) is responsible for approximately 85% of snake bites in Myanmar and commonly causes coagulopathy and acute kidney injury. Case fatality despite current management strategies remains at approximately 10%.The mainstay of management consists of early identification of systemic envenoming using clinical features and the 20-minute whole blood clotting test (20WBCT) ...

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