Molecular surveillance for operationally relevant genetic polymorphisms in Plasmodium falciparum in Southern Chad, 2016-2017.

BACKGROUND: Resistance to anti-malarials is a serious threat to the efforts to control and eliminate malaria. Surveillance based on simple field protocols with centralized testing to detect molecular markers associated with anti-malarial drug resistance can be used to identify locations where further investigations are needed. METHODS: Dried blood spots were collected from 398 patients (age range 5-59 years, 99% male) with Plasmodium falciparum infections detected using rapid diagnostic tests over two rounds of sample collection conducted in 2016 and 2017 in Komé, South-West Chad. Specimens were genotyped using amplicon sequencing or qPCR for validated markers of anti-malarial resistance including partner drugs used in artemisinin-based combination therapy (ACT). RESULTS: No mutations in the pfk13 gene known to be associated with artemisinin resistance were found but a high proportion of parasites carried other mutations, specifically K189T (190/349, 54.4%, 95%CI 49.0-59.8%). Of 331 specimens successfully genotyped for pfmdr1 and pfcrt, 52% (95%CI 46.4-57.5%) carried the NFD-K haplotype, known to be associated with reduced susceptibility to lumefantrine. Only 20 of 336 (6.0%, 95%CI 3.7-9.0%) had parasites with the pfmdr1-N86Y polymorphism associated with increased treatment failures with amodiaquine. Nearly all parasites carried at least one mutation in pfdhfr and/or pfdhps genes but 'sextuple' mutations in pfdhfr-pfdhps including pfdhps -A581G were rare (8/336 overall, 2.4%, 95%CI 1.2-4.6%). Only one specimen containing parasites with pfmdr1 gene amplification was detected. CONCLUSIONS: These results provide information on the likely high efficacy of artemisinin-based combinations commonly used in Chad, but suggest decreasing levels of sensitivity to lumefantrine and high levels of resistance to sulfadoxine-pyrimethamine used for seasonal malaria chemoprevention and intermittent preventive therapy in pregnancy. A majority of parasites had mutations in the pfk13 gene, none of which are known to be associated with artemisinin resistance. A therapeutic efficacy study needs to be conducted to confirm the efficacy of artemether-lumefantrine.

Malaria epidemic in humanitarian crisis settings the case of South Kordofan state, Sudan.

INTRODUCTION: Malaria remains one of the most common public health problems worldwide, especially in Sudan. With this short communication we aimed at reporting on the latest malaria epidemic that had occurred in the humanitarian settings in South Kordofan state, south-western Sudan, during 2018 and 2019. METHODOLOGY: This is a cross-sectional study analyzing malaria surveillance reports between February 2018 to September 2019. Malaria was reported from febrile patients with confirmed malaria diagnosis using Giemsa stain. According to age, patients were distributed across three categories: less than 5 years, 5 to 15 years, and more than 15 years. RESULTS: In 2019 and 2018, 63,214 and 63,224 cases of malaria were reported, respectively, constituting around 5.5% of the state population (1,152,900). In 2018, 3,571 malaria cases were reported in February, then they decreased in August followed by increase in September-October. In 2019, 15,610 malaria cases were reported in September. Malaria cases aged less than 5 years were 21,848 and 23,561 cases in 2018 and 2019, respectively. CONCLUSIONS: The reported sudden epidemic of malaria is alarming. Therefore, identifying the risk factors associated with this epidemic is crucial to malaria prevention and control, and hence successful achievement of malaria elimination.

A snapshot of Plasmodium falciparum malaria drug resistance markers in Sudan: a pilot study.

OBJECTIVES: Malaria infection is still known to be a worldwide public health problem, especially in tropical and sub-tropical African countries like Sudan. A pilot study conducted to describe the trend of P. falciparum drug resistance markers in 2017-2018 in comparison to CQ and AS/SP eras in Sudan. The Pfcrt, Pfmdr-1, Pfdhfr, and Pfdhps genes were investigated. Data deposited by the worldwide antimalarial resistance network was consulted, and the molecular markers previously reported from Sudan were analyzed. RESULTS: Drug molecular markers analysis was successfully done on 20 P. falciparum isolates. The Pfcrt K76 showed high frequency; 16 (80%). For the Pfmdr-1, 9 (45%) isolates were carrying the N86 allele, and 11 (55%) were 86Y allele. While the Y184F of the Pfmdr-1 showed a higher frequency of 184F compared to Y184; 16 (80%) and 4 (20%), respectively. In the Pfdhfr, 51I allele showed higher frequency compared to N51; 18 (90%) and 2 (10%), respectively. For S108N, 18 (90%) were 108 N and 2 (10%) were S108. In the Pfdhps, all isolates were carrying the mutant alleles; 437G and 540E. The frequency distribution of the Pfcrt, Pfmdr-1, Pfdhfr, Pfdhps was significantly different across the whole years in Sudan.

The efficacy of dihydroartemisinin-piperaquine and artemether-lumefantrine with and without primaquine on Plasmodium vivax recurrence: A systematic review and individual patient data meta-analysis.

BACKGROUND: Artemisinin-based combination therapy (ACT) is recommended for uncomplicated Plasmodium vivax malaria in areas of emerging chloroquine resistance. We undertook a systematic review and individual patient data meta-analysis to compare the efficacies of dihydroartemisinin-piperaquine (DP) and artemether-lumefantrine (AL) with or without primaquine (PQ) on the risk of recurrent P. vivax. METHODS AND FINDINGS: Clinical efficacy studies of uncomplicated P. vivax treated with DP or AL and published between January 1, 2000, and January 31, 2018, were identified by conducting a systematic review registered with the International Prospective Register of Systematic Reviews (PROSPERO): CRD42016053310. Investigators of eligible studies were invited to contribute individual patient data that were pooled using standardised methodology. The effect of mg/kg dose of piperaquine/lumefantrine, ACT administered, and PQ on the rate of P. vivax recurrence between days 7 and 42 after starting treatment were investigated by Cox regression analyses according to an a priori analysis plan. Secondary outcomes were the risk of recurrence assessed on days 28 and 63. Nineteen studies enrolling 2,017 patients were included in the analysis. The risk of recurrent P. vivax at day 42 was significantly higher in the 384 patients treated with AL alone (44.0%, 95% confidence interval [CI] 38.7-49.8) compared with the 812 patients treated with DP alone (9.3%, 95% CI 7.1-12.2): adjusted hazard ratio (AHR) 12.63 (95% CI 6.40-24.92), p < 0.001. The rates of recurrence assessed at days 42 and 63 were associated inversely with the dose of piperaquine: AHRs (95% CI) for every 5-mg/kg increase 0.63 (0.48-0.84), p = 0.0013 and 0.83 (0.73-0.94), p = 0.0033, respectively. The dose of lumefantrine was not significantly associated with the rate of recurrence (1.07 for every 5-mg/kg increase, 95% CI 0.99-1.16, p = 0.0869). In a post hoc analysis, in patients with symptomatic recurrence after AL, the mean haemoglobin increased 0.13 g/dL (95% CI 0.01-0.26) for every 5 days that recurrence was delayed, p = 0.0407. Coadministration of PQ reduced substantially the rate of recurrence assessed at day 42 after AL (AHR = 0.20, 95% CI 0.10-0.41, p < 0.001) and at day 63 after DP (AHR = 0.08, 95% CI 0.01-0.70, p = 0.0233). Results were limited by follow-up of patients to 63 days or less and nonrandomised treatment groups. CONCLUSIONS: In this study, we observed the risk of P. vivax recurrence at day 42 to be significantly lower following treatment with DP compared with AL, reflecting the longer period of post-treatment prophylaxis; this risk was reduced substantially by coadministration with PQ. We found that delaying P. vivax recurrence was associated with a small but significant improvement in haemoglobin. These results highlight the benefits of PQ radical cure and also the provision of blood-stage antimalarial agents with prolonged post-treatment prophylaxis.

Deployment and utilization of next-generation sequencing of Plasmodium falciparum to guide anti-malarial drug policy decisions in sub-Saharan Africa: opportunities and challenges.

Parasite resistance against anti-malarial drugs is a major threat to the ongoing malaria control and elimination strategies. This is especially true since resistance to the currently recommended artemisinins and partner drugs has been confirmed in South East Asia (SEA) and new anti-malarial compounds are not expected to be available in the near future. Spread from SEA or independent emergence of artemisinin resistance in sub-Saharan Africa (SSA) could reverse the achievements in malaria control that have been attained in the past two decades and derail the ongoing elimination strategies. The current surveillance of clinical efficacy and resistance to anti-malarial drugs is based on efficacy trials to assess the clinical performance of anti-malarials, in vivo/ex vivo assessment of parasite susceptibility to anti-malarials and prevalence of known molecular markers of drug resistance. Whereas clinical efficacy trials are restricted by cost and the complex logistics of patient follow-up, molecular detection of genetic mutations associated with resistance or reduced susceptibility to anti-malarials is by contrast a simple and powerful tool for early detection and monitoring of the prevalence of resistant parasites at population level. This provides needed information before clinical failure emerges, allowing policy makers to anticipate problems and respond. The various methods previously used in detection of molecular markers of drug resistance share some limitations: low-throughput, and high costs per sample and demanding infrastructure. However, recent technological advances including next-generation sequencing (NGS) methodologies promise greatly increased throughput and reduced costs, essentially providing unprecedented potential to address different research and operational questions of relevance for drug policy. This review assesses the potential role of NGS to provide comprehensive information that could guide drug policies in malaria endemic countries and looks at the foreseeable challenges facing the establishment of NGS approaches for routine surveillance of parasite resistance to anti-malarials in SSA.

The haematological consequences of Plasmodium vivax malaria after chloroquine treatment with and without primaquine: a WorldWide Antimalarial Resistance Network systematic review and individual patient data meta-analysis.

BACKGROUND: Malaria causes a reduction in haemoglobin that is compounded by primaquine, particularly in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. The aim of this study was to determine the relative contributions to red cell loss of malaria and primaquine in patients with uncomplicated Plasmodium vivax. METHODS: A systematic review identified P. vivax efficacy studies of chloroquine with or without primaquine published between January 2000 and March 2017. Individual patient data were pooled using standardised methodology, and the haematological response versus time was quantified using a multivariable linear mixed effects model with non-linear terms for time. Mean differences in haemoglobin between treatment groups at day of nadir and day 42 were estimated from this model. RESULTS: In total, 3421 patients from 29 studies were included: 1692 (49.5%) with normal G6PD status, 1701 (49.7%) with unknown status and 28 (0.8%) deficient or borderline individuals. Of 1975 patients treated with chloroquine alone, the mean haemoglobin fell from 12.22 g/dL [95% CI 11.93, 12.50] on day 0 to a nadir of 11.64 g/dL [11.36, 11.93] on day 2, before rising to 12.88 g/dL [12.60, 13.17] on day 42. In comparison to chloroquine alone, the mean haemoglobin in 1446 patients treated with chloroquine plus primaquine was - 0.13 g/dL [- 0.27, 0.01] lower at day of nadir (p = 0.072), but 0.49 g/dL [0.28, 0.69] higher by day 42 (p < 0.001). On day 42, patients with recurrent parasitaemia had a mean haemoglobin concentration - 0.72 g/dL [- 0.90, - 0.54] lower than patients without recurrence (p < 0.001). Seven days after starting primaquine, G6PD normal patients had a 0.3% (1/389) risk of clinically significant haemolysis (fall in haemoglobin > 25% to < 7 g/dL) and a 1% (4/389) risk of a fall in haemoglobin > 5 g/dL. CONCLUSIONS: Primaquine has the potential to reduce malaria-related anaemia at day 42 and beyond by preventing recurrent parasitaemia. Its widespread implementation will require accurate diagnosis of G6PD deficiency to reduce the risk of drug-induced haemolysis in vulnerable individuals. TRIAL REGISTRATION: This trial was registered with PROSPERO: CRD42016053312. The date of the first registration was 23 December 2016.

Addressing the gender-knowledge gap in glucose-6-phosphate dehydrogenase deficiency: challenges and opportunities.

Glucose-6-phosphate dehyrdgoenase (G6PD) deficiency is a common X-linked genetic trait, with an associated enzyme phenotype, whereby males are either G6PD deficient or normal, but females exhibit a broader range of G6PD deficiencies, ranging from severe deficiency to normal. Heterozygous females typically have intermediate G6PD activity. G6PD deficiency has implications for the safe treatment for Plasmodium vivax malaria. Individuals with this deficiency are at greater risk of serious adverse events following treatment with the only curative class of anti-malarials, 8-aminoquinolines, such as primaquine. Quantitative diagnostic tests for G6PD deficiency are complex and require sophisticated laboratories. The commonly used qualitative tests, do not discriminate intermediate G6PD activities. This has resulted in poor understanding of the epidemiology of G6PD activity in females and its corresponding treatment ramifications. New simple-to-use quantitative tests, and a momentum to eliminate malaria, create an opportunity to address this knowledge gap. While this will require additional resources for clinical studies, adequate operational research, and appropriate pharmacovigilance, the health benefits from this investment go beyond the immediate intervention for which the G6PD status is first diagnosed.

Risk of Plasmodium vivax parasitaemia after Plasmodium falciparum infection: a systematic review and meta-analysis.

BACKGROUND: A 14-day course of primaquine is used for radical cure of Plasmodium vivax and Plasmodium ovale malaria only. We quantified the risk of P vivax parasitaemia after treatment of Plasmodium falciparum with commonly used antimalarial drugs to assess the potential benefits of radical cure for all patients with uncomplicated malaria in co-endemic regions. METHODS: In this systematic review and meta-analysis, we searched MEDLINE, Embase, Web of Science, and the Cochrane Database of Systematic Reviews for prospective clinical studies in any language, published between Jan 1, 1960, and Jan 5, 2018, assessing drug efficacy in patients with uncomplicated P falciparum malaria in countries co-endemic for P vivax. Studies were included if the presence or absence of P vivax parasitaemia was recorded after treatment. The primary outcome was the risk of P vivax parasitaemia between day 7 and day 42 after initiation of antimalarial treatment for P falciparum, with the pooled risk calculated by random-effects meta-analysis. We compared the risk of P vivax parasitaemia after treatment with different artemisinin-based combination therapies (ACTs). This study is registered with PROSPERO, number CRD42017064838. FINDINGS: 153 of 891 screened studies were included in the analysis, including 31 262 patients from 323 site-specific treatment groups: 130 (85%) studies were from the Asia-Pacific region, 16 (10%) from the Americas, and seven (5%) from Africa. The risk of P vivax parasitaemia by day 42 was 5·6% (95% CI 4·0-7·4; I2=92·0%; 117 estimates). The risk of P vivax parasitaemia was 6·5% (95% CI 4·6-8·6) in regions of short relapse periodicity compared with 1·9% (0·4-4·0) in regions of long periodicity, and was greater after treatment with a more rapidly eliminated ACT: 15·3% (5·1-29·3) for artemether-lumefantrine compared with 4·5% (1·2-9·3) for dihydroartemisinin-piperaquine and 5·2% (2·9-7·9) for artesunate-mefloquine. Recurrent parasitaemia was delayed in patients treated with ACTs containing mefloquine or piperaquine compared with artemether-lumefantrine, but by day 63 the risk of vivax parasitaemia was more than 15% for all ACTs assessed. INTERPRETATION: Our findings show a high risk of vivax parasitaemia after treatment of falciparum malaria, particularly in areas with short relapse periodicity and after rapidly eliminated treatment. In co-endemic regions, universal radical cure for all patients with uncomplicated malaria has the potential to substantially reduce recurrent malaria. FUNDING: Australian National Health and Medical Research Council, Royal Australasian College of Physicians, Wellcome Trust, and Bill & Melinda Gates Foundation.

The effect of chloroquine dose and primaquine on Plasmodium vivax recurrence: a WorldWide Antimalarial Resistance Network systematic review and individual patient pooled meta-analysis.

BACKGROUND: Chloroquine remains the mainstay of treatment for Plasmodium vivax malaria despite increasing reports of treatment failure. We did a systematic review and meta-analysis to investigate the effect of chloroquine dose and the addition of primaquine on the risk of recurrent vivax malaria across different settings. METHODS: A systematic review done in MEDLINE, Web of Science, Embase, and Cochrane Database of Systematic Reviews identified P vivax clinical trials published between Jan 1, 2000, and March 22, 2017. Principal investigators were invited to share individual patient data, which were pooled using standardised methods. Cox regression analyses with random effects for study site were used to investigate the roles of chloroquine dose and primaquine use on rate of recurrence between day 7 and day 42 (primary outcome). The review protocol is registered in PROSPERO, number CRD42016053310. FINDINGS: Of 134 identified chloroquine studies, 37 studies (from 17 countries) and 5240 patients were included. 2990 patients were treated with chloroquine alone, of whom 1041 (34·8%) received a dose below the target 25 mg/kg. The risk of recurrence was 32·4% (95% CI 29·8-35·1) by day 42. After controlling for confounders, a 5 mg/kg higher chloroquine dose reduced the rate of recurrence overall (adjusted hazard ratio [AHR] 0·82, 95% CI 0·69-0·97; p=0·021) and in children younger than 5 years (0·59, 0·41-0·86; p=0·0058). Adding primaquine reduced the risk of recurrence to 4·9% (95% CI 3·1-7·7) by day 42, which is lower than with chloroquine alone (AHR 0·10, 0·05-0·17; p<0·0001). INTERPRETATION: Chloroquine is commonly under-dosed in the treatment of vivax malaria. Increasing the recommended dose to 30 mg/kg in children younger than 5 years could reduce substantially the risk of early recurrence when primaquine is not given. Radical cure with primaquine was highly effective in preventing early recurrence and may also improve blood schizontocidal efficacy against chloroquine-resistant P vivax. FUNDING: Wellcome Trust, Australian National Health and Medical Research Council, and Bill & Melinda Gates Foundation.

Molecular markers of resistance to amodiaquine plus sulfadoxine-pyrimethamine in an area with seasonal malaria chemoprevention in south central Niger.

BACKGROUND: In Niger, malaria transmission is markedly seasonal with most of the disease burden occurring in children during the rainy season. Seasonal malaria chemoprevention (SMC) with amodiaquine plus sulfadoxine-pyrimethamine (AQ + SP) is recommended in the country to be administered monthly just before and during the rainy season. Moreover, clinical decisions on use of SP for intermittent preventive treatment in pregnancy (IPTp) now depend upon the validated molecular markers for SP resistance in Plasmodium falciparum observed in the local parasite population. However, little is known about molecular markers of resistance for either SP or AQ in the south of Niger. To address this question, clinical samples which met clinical and biological criteria, were collected in Gabi, Madarounfa district, Maradi region, Niger in 2011-2012 (before SMC implementation). Molecular markers of resistance to pyrimethamine (pfdhfr), sulfadoxine (pfdhps) and amodiaquine (pfmdr1) were assessed by DNA sequencing. RESULTS: Prior to SMC implementation, the samples showed a high proportion of clinical samples that carried the pfdhfr 51I/59R/108N haplotype associated with resistance to pyrimethamine and pfdhps 436A/F/H and 437G mutations associated with reduced susceptibility to sulfadoxine. In contrast mutations in codons 581G, and 613S in the pfdhps gene, and in pfmdr1, 86Y, 184Y, 1042D and 1246Y associated with resistance to amodiaquine, were less frequently observed. Importantly, pfdhfr I164L and pfdhps K540E mutations shown to be the most clinically relevant markers for high level clinical resistance to SP were not detected in Gabi. CONCLUSIONS: Although parasites with genotypes associated with the highest levels of resistance to AQ + SP are not yet common in this setting, their importance for deployment of SMC and IPTp dictates that monitoring of these markers of resistance should accompany these interventions. This study also highlights the parasite heterogeneity within a small spatial area and the need to use caution when extrapolating results from surveys of molecular markers of resistance in a single site to inform regional policy decisions.

Artemether-Lumefantrine and Dihydroartemisinin-Piperaquine Exert Inverse Selective Pressure on Plasmodium Falciparum Drug Sensitivity-Associated Haplotypes in Uganda.

BACKGROUND: Altered sensitivity to multiple antimalarial drugs is mediated by polymorphisms in pfmdr1, which encodes the Plasmodium falciparum multidrug resistance transporter. In Africa the N86Y and D1246Y polymorphisms have been shown to be selected by treatment, with artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DP) selecting for wild-type and mutant alleles, respectively. However, there has been little study of pfmdr1 haplotypes, in part because haplotype analyses are complicated by multiclonal infections. METHODS: We fit a haplotype frequency estimation model, which accounts for multiclonal infections, to the polymorphic pfmdr1 N86Y, Y184F, and D1246Y alleles in samples from a longitudinal trial comparing AL and DP to treat uncomplicated P falciparum malaria in Tororo, Uganda from 2007 to 2012. We regressed estimates onto covariates of trial arm and selective drug pressure. RESULTS: Yearly trends showed increasing frequency estimates for haplotypes with wild type pfmdr1 N86 and D1246 alleles and decreasing frequency estimates for haplotypes with the mutant pfmdr1 86Y allele. Considering days since prior therapy, we saw evidence suggestive of selection by AL for haplotypes with N86 combined with 184F, D1246, or both, and against all haplotypes with 86Y, and evidence suggestive of selection by DP for 86Y only when combined with Y184 and 1246Y (haplotype YYY) and against haplotypes NFD and NYY. CONCLUSIONS: Based on our model, AL selected several haplotypes containing N86, whereas DP selection was haplotype specific, demonstrating the importance of haplotype analyses. Inverse selective pressure of AL and DP on the complementary haplotypes NFD and YYY suggests that rotating artemisinin-based antimalarial combination regimens may be the best treatment option to prevent resistance selection.

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.

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.

Benefits of a pharmacology antimalarial reference standard and proficiency testing program provided by the Worldwide Antimalarial Resistance Network (WWARN).

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

Estimation of malaria haplotype and genotype frequencies: a statistical approach to overcome the challenge associated with multiclonal infections.

BACKGROUND: Reliable measures of anti-malarial resistance are crucial for malaria control. Resistance is typically a complex trait: multiple mutations in a single parasite (a haplotype or genotype) are necessary for elaboration of the resistant phenotype. The frequency of a genetic motif (proportion of parasite clones in the parasite population that carry a given allele, haplotype or genotype) is a useful measure of resistance. In areas of high endemicity, malaria patients generally harbour multiple parasite clones; they have multiplicities of infection (MOIs) greater than one. However, most standard experimental procedures only allow measurement of marker prevalence (proportion of patient blood samples that test positive for a given mutation or combination of mutations), not frequency. It is misleading to compare marker prevalence between sites that have different mean MOIs; frequencies are required instead. METHODS: A Bayesian statistical model was developed to estimate Plasmodium falciparum genetic motif frequencies from prevalence data collected in the field. To assess model performance and computational speed, a detailed simulation study was implemented. Application of the model was tested using datasets from five sites in Uganda. The datasets included prevalence data on markers of resistance to sulphadoxine-pyrimethamine and an average MOI estimate for each study site. RESULTS: The simulation study revealed that the genetic motif frequencies that were estimated using the model were more accurate and precise than conventional estimates based on direct counting. Importantly, the model did not require measurements of the MOI in each patient; it used the average MOI in the patient population. Furthermore, if a dataset included partially genotyped patient blood samples, the model imputed the data that were missing. Using the model and the Ugandan data, genotype frequencies were estimated and four biologically relevant genotypes were identified. CONCLUSIONS: The model allows fast, accurate, reliable estimation of the frequency of genetic motifs associated with resistance to anti-malarials using prevalence data collected from malaria patients. The model does not require per-patient MOI measurements and can easily analyse data from five markers. The model will be a valuable tool for monitoring markers of anti-malarial drug resistance, including markers of resistance to artemisinin derivatives and partner drugs.

High-throughput analysis of antimalarial susceptibility data by the WorldWide Antimalarial Resistance Network (WWARN) in vitro analysis and reporting tool.

Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here we describe a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets. Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs were collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC50s) were determined by a modified sigmoid Emax model-fitting algorithm, allowing standardized analysis of 7,350 concentration-inhibition assays involving 1,592 isolates. Examination of concentration-inhibition data revealed evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more-reliable IC50s based on estimated confidence intervals and growth ratios improved correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis showed that maximal drug inhibition was higher for artemisinins than for other drugs, particularly in ELISA (enzyme-linked immunosorbent assay)-based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle. This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing in vitro susceptibility to these drugs.

Implementation of a reference standard and proficiency testing programme by the World Wide Antimalarial Resistance Network (WWARN).

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

Global resistance surveillance: ensuring antimalarial efficacy in the future.

PURPOSE OF REVIEW: Effective surveillance for and rapid identification of evolved antimalarial resistance ensures that all patients are treated with efficacious drugs. This review summarizes the current status and the challenges to effective surveillance, and suggests approaches for improvement. RECENT FINDINGS: The replacement of older drugs by artemisinin combination therapies (ACTs) as the recommended treatment for malaria has dramatically improved treatment outcomes wherever ACTs have been deployed effectively. Moreover, there has been considerable technical and organizational progress, and support for the health professionals needed to carry out this work is also increasing. As a result, the prospects for more effective surveillance of antimalarial resistance, and other vital health information are improving. However, resistance to the artemisinin component of ACTs is already suspected in Cambodia, and the current methods for tracking this resistance are not yet in place. Identification of efficient markers of ACT efficacy is a crucial challenge. SUMMARY: Technical advances alone are not sufficient. Detection of decreased drug efficacy is only the first step to producing accessible and useful information for decision makers. The translation of increased access to data on health outcomes into usable evidence for rational policy and planning requires a global coordination and communication effort.

World Antimalarial Resistance Network I: clinical efficacy of antimalarial drugs.

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.

World Antimalarial Resistance Network (WARN) III: molecular markers for drug resistant malaria.

Molecular markers for drug resistant malaria represent public health tools of great but mostly unrealized potential value. A key reason for the failure of molecular resistance markers to live up to their potential is that data on the their prevalence is scattered in disparate databases with no linkage to the clinical, in vitro and pharmacokinetic data that are needed to relate the genetic data to relevant phenotypes. The ongoing replacement of older monotherapies for malaria by new, more effective combination therapies presents an opportunity to create an open access database that brings together standardized data on molecular markers of drug resistant malaria from around the world. This paper presents a rationale for creating a global database of molecular markers for drug resistant malaria and for linking it to similar databases containing results from clinical trials of drug efficacy, in vitro studies of drug susceptibility, and pharmacokinetic studies of antimalarial drugs, in a World Antimalarial Resistance Network (WARN). This database will be a global resource, guiding the selection of first line drugs for treating uncomplicated malaria, for preventing malaria in travelers and for intermittent preventive treatment of malaria in pregnant women, infants and other vulnerable groups. Perhaps most important, a global database for molecular markers of drug resistant malaria will accelerate the identification and validation of markers for resistance to artemisinin-based combination therapies and, thereby, potentially prolong the useful therapeutic lives of these important new drugs.

World Antimalarial Resistance Network (WARN) IV: clinical pharmacology.

A World Antimalarial Resistance Network (WARN) database has the potential to improve the treatment of malaria, through informing current drug selection and use and providing a prompt warning of when treatment policies need changing. This manuscript outlines the contribution and structure of the clinical pharmacology component of this database. The determinants of treatment response are multi-factorial, but clearly providing adequate blood concentrations is pivotal to curing malaria. The ability of available antimalarial pharmacokinetic data to inform optimal dosing is constrained by the small number of patients studied, with even fewer (if any) studies conducted in the most vulnerable populations. There are even less data relating blood concentration data to the therapeutic response (pharmacodynamics). By pooling all available pharmacokinetic data, while paying careful attention to the analytical methodologies used, the limitations of small (and thus underpowered) individual studies may be overcome and factors that contribute to inter-individual variability in pharmacokinetic parameters defined. Key variables for pharmacokinetic studies are defined in terms of patient (or study subject) characteristics, the formulation and route of administration of the antimalarial studied, the sampling and assay methodology, and the approach taken to data analysis. Better defining these information needs and criteria of acceptability of pharmacokinetic-pharmacodynamic (PK-PD) studies should contribute to improving the quantity, relevance and quality of these studies. A better understanding of the pharmacokinetic properties of antimalarials and a more clear definition of what constitutes "therapeutic drug levels" would allow more precise use of the term "antimalarial resistance", as it would indicate when treatment failure is not caused by intrinsic parasite resistance but is instead the result of inadequate drug levels. The clinical pharmacology component of the WARN database can play a pivotal role in monitoring accurately for true antimalarial drug resistance and promptly correcting sub-optimal dosage regimens to prevent these contributing to the emergence and spread of antimalarial resistance.