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Abstract Background Chloroquine (CQ) was the main malaria therapy worldwide from the 1940s until the 1990s. Following the emergence of CQ-resistant Plasmodium falciparum, most African countries discontinued the use of CQ, and now promote artemisinin-based combination therapy as the first-line treatment. This change was generally initiated during the last decade in West and Central Africa. The aim of this study is to describe the changes in CQ susceptibility in this African region, using travellers returning from this region as a sentinel system. Methods The study was conducted by the Malaria National Reference Centre, France. The database collated the pfcrtK76T molecular marker for CQ susceptibility and the in vitro response to CQ of parasites from travellers’ isolates returning from Senegal, Mali, Ivory Coast or Cameroon. As a proxy of drug pressure, data regarding CQ intake in febrile children were collated for the study period. Logistic regression models were used to detect trends in the proportions of CQ resistant isolates. Results A total of 2874 parasite isolates were genotyped between 2000–2011. The prevalence of the pfcrt76T mutant genotype significantly decreased for Senegal (from 78% to 47%), Ivory Coast (from 63% to 37%), Cameroon (from 90% to 59%) and remained stable for Mali. The geometric mean of the 50% inhibitory concentration (IC50) of CQ in vitro susceptibility and the proportion of resistant isolates (defining resistance as an IC50 value > 100 nM) significantly decreased for Senegal (from 86 nM (59%) to 39 nM (25%)), Mali (from 84 nM (50%) to 51 nM (31%)), Ivory Coast (from 75 nM (59%) to 29 nM (16%)) and Cameroon (from 181 nM (75%) to 51 nM (37%)). Both analyses (molecular and in vitro susceptibility) were performed for the 2004–2011 period, after the four countries had officially discontinued CQ and showed an accelerated decline of the resistant isolates for the four countries. Meanwhile, CQ use among children significantly deceased in this region (fixed effects slope = −0.3, p -3). Conclusions An increase in CQ susceptibility following official withdrawal of the drug was observed in travellers returning from West and Central African countries. The same trends were observed for molecular and in vitro analysis between 2004-2011and they correlated to the decrease of the drug pressure.

Abstract Background For the results of clinical trials to have external validity, the patients included in the study must be representative of the population presenting in the general clinical settings. A scoping literature review was performed to evaluate how the eligibility criteria used in anti-malarial efficacy and safety trials translate into patient selection. Methods A search of the WorldWide Antimalarial Resistance Network (WWARN) Clinical Trials Publication Library, MEDLINE, The Cochrane Library, and clinicaltrials.gov was conducted to identify trials investigating anti-malarial efficacy and safety, published between 14th April 2001 and 31st December 2017. An updated search using the WWARN Clinical Trial Publication Library was undertaken to identify eligible publications from 1st January 2018 to 31st July 2021. The review included studies in patients of any age with uncomplicated malaria and any pharmaceutical therapeutic intervention administered. The proportion of trials with malaria-positive patients excluded was calculated and linked to the reported reason for exclusion. A subgroup analysis on eligibility criteria and trial baseline demographics was conducted to assess whether criteria are complied with when recruiting patients. Results Out of 847 studies, 176 (21%) trials were included in the final synthesis, screening a total of 157,516 malaria-positive patients, of whom 56,293 (36%) were enrolled and treated. Across the 176 studies included, 84 different inclusion and exclusion criteria were identified. The reason for exclusion of patients who tested positive for malaria was reported in 144 (82%) studies. Three criteria account for about 70% of malaria-positive patients excluded: mixed-species malaria infections or other specific Plasmodium species, parasite counts outside the set study ranges, and refusal of consent. Conclusions Nearly two-thirds of the malaria-positive subjects who present to health facilities are systematically excluded from anti-malarial treatment trials. Reasons for exclusions are largely under-reported. Anti-malarial treatment in the general population is informed by studies on a narrow selection of patients who do not fully represent the totality of those seeking antimalarial treatment in routine practice. While entry criteria ensure consistency across trials, pragmatic trials are also necessary to supplement the information currently available and improve the external validity of the findings of malaria clinical trials.

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

Abstract Background In 2005, the Democratic Republic of Congo (DRC) adopted artesunate and amodiaquine (ASAQ) as first-line anti-malarial treatment. In order to compare the efficacy of the fixed-dose formulation ASAQ versus artemether-lumefantrine (AL), a randomized, non-inferiority open-label trial was conducted in Katanga. Methods Children aged six and 59 months with uncomplicated Plasmodium falciparum malaria were enrolled and randomly allocated into one of the two regimens. The risk of recurrent parasitaemia by day 42, both unadjusted and adjusted by PCR genotyping to distinguish recrudescence from new infection, was analysed. Results Between April 2008 and March 2009, 301 children were included: 156 with ASAQ and 145 with AL. No early treatment failures were reported. Among the 256 patients followed-up at day 42, 32 patients developed late clinical or parasitological failure (9.9% (13/131) in the ASAQ group and 15.2% (19/125) in the AL group). After PCR correction, cure rates were 98.3% (95%CI, 94.1-99.8) in the ASAQ group and 99.1% (95%CI, 94.9-99.9) in the AL group (difference −0.7%, one sided 95% CI −3.1). Kaplan-Meier PCR-adjusted cure rates were similar. Both treatment regimens were generally well tolerated. Conclusion Both ASAQ and AL are highly effective and currently adequate as the first-line treatment of uncomplicated falciparum malaria in this area of Katanga, DRC. However, in a very large country, such as DRC, and because of possible emergence of resistance from other endemic regions, surveillance of efficacy of artemisinin-based combination treatments, including other evaluations of the resistance of ASAQ, need to be done in other provinces. Trial registration The protocol was registered with the clinicaltrials.gov, open clinical trial registry under the identifier number NCT01567423.

Abstract Background Data on efficacy of artemisinin-based combination therapy (ACT) to treat Plasmodium falciparum during pregnancy in sub-Saharan Africa is scarce. A recent open label, randomized controlled trial in Mbarara, Uganda demonstrated that artemether-lumefantrine (AL) is not inferior to quinine to treat uncomplicated malaria in pregnancy. Haemozoin can persist in the placenta following clearance of parasites, however there is no data whether ACT can influence the amount of haemozoin or the dynamics of haemozoin clearance. Methods Women attending antenatal clinics with weekly screening and positive blood smears by microscopy were eligible to participate in the trial and were followed to delivery. Placental haemozoin deposition and inflammation were assessed by histology. To determine whether AL was associated with increased haemozoin clearance, population haemozoin clearance curves were calculated based on the longitudinal data. Results Of 152 women enrolled in each arm, there were 97 and 98 placental biopsies obtained in the AL and quinine arms, respectively. AL was associated with decreased rates of moderate to high grade haemozoin deposition (13.3% versus 25.8%), which remained significant after correcting for gravidity, time of infection, re-infection, and parasitaemia. The amount of haemozoin proportionately decreased with the duration of time between treatment and delivery and this decline was greater in the AL arm. Haemozoin was not detected in one third of biopsies and the prevalence of inflammation was low, reflecting the efficacy of antenatal care with early detection and prompt treatment of malaria. Conclusions Placental haemozoin deposition was decreased in the AL arm demonstrating a relationship between pharmacological properties of drug to treat antenatal malaria and placental pathology at delivery. Histology may be considered an informative outcome for clinical trials to evaluate malaria control in pregnancy. Trial registration REGISTRY: http://clinicaltrials.gov/ct2/show/NCT00495508

Abstract Background Plasmodium falciparum malaria remains a major public health problem. A vital component of malaria control rests on the availability of good quality artemisinin-derivative based combination therapy (ACT) at the correct dose. However, there are increasing reports of poor quality anti-malarials in Africa. Methods Seven collections of artemisinin derivative monotherapies, ACT and halofantrine anti-malarials of suspicious quality were collected in 2002/10 in eleven African countries and in Asia en route to Africa. Packaging, chemical composition (high performance liquid chromatography, direct ionization mass spectrometry, X-ray diffractometry, stable isotope analysis) and botanical investigations were performed. Results Counterfeit artesunate containing chloroquine, counterfeit dihydroartemisinin (DHA) containing paracetamol (acetaminophen), counterfeit DHA-piperaquine containing sildenafil, counterfeit artemether-lumefantrine containing pyrimethamine, counterfeit halofantrine containing artemisinin, and substandard/counterfeit or degraded artesunate and artesunate+amodiaquine in eight countries are described. Pollen analysis was consistent with manufacture of counterfeits in eastern Asia. These data do not allow estimation of the frequency of poor quality anti-malarials in Africa. Conclusions Criminals are producing diverse harmful anti-malarial counterfeits with important public health consequences. The presence of artesunate monotherapy, substandard and/or degraded and counterfeit medicines containing sub-therapeutic amounts of unexpected anti-malarials will engender drug resistance. With the threatening spread of artemisinin resistance to Africa, much greater investment is required to ensure the quality of ACTs and removal of artemisinin monotherapies. The International Health Regulations may need to be invoked to counter these serious public health problems.

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

Abstract 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.

Abstract 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% [CI9585.4-97.3], spec 97.4% [95.9-98.3]; non-Pf: sens 78.5% [CI9571.1-84.4], 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°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.

Abstract 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.

Abstract Background Independent emergence and spread of artemisinin-resistant Plasmodium falciparum malaria have recently been confirmed in Africa, with molecular markers associated with artemisinin resistance increasingly detected. Surveillance to promptly detect and effectively respond to anti-malarial resistance is generally suboptimal in Africa, especially in low transmission settings where therapeutic efficacy studies are often not feasible due to recruitment challenges. However, these communities may be at higher risk of anti-malarial resistance. Methods From March 2018 to February 2020, a sequential mixed-methods study was conducted to evaluate the feasibility of the near-real-time linkage of individual patient anti-malarial resistance profiles with their case notifications and treatment response reports, and map these to fine scales in Nkomazi sub-district, Mpumalanga, a pre-elimination area in South Africa. Results Plasmodium falciparum molecular marker resistance profiles were linked to 55.1% (2636/4787) of notified malaria cases, 85% (2240/2636) of which were mapped to healthcare facility, ward and locality levels. Over time, linkage of individual malaria case demographic and molecular data increased to 75.1%. No artemisinin resistant validated/associated Kelch-13 mutations were detected in the 2385 PCR positive samples. Almost all 2812 samples assessed for lumefantrine susceptibility carried the wildtype mdr86ASN and crt76LYS alleles, potentially associated with decreased lumefantrine susceptibility. Conclusion Routine near-real-time mapping of molecular markers associated with anti-malarial drug resistance on a fine spatial scale provides a rapid and efficient early warning system for emerging resistance. The lessons learnt here could inform scale-up to provincial, national and regional malaria elimination programmes, and may be relevant for other antimicrobial resistance surveillance.

Abstract Background Microscopic examination of Giemsa-stained blood films remains the reference standard for malaria parasite detection and quantification, but is undermined by difficulties in ensuring high-quality manual reading and inter-reader reliability. Automated parasite detection and quantification may address this issue. Methods A multi-centre, observational study was conducted during 2018 and 2019 at 11 sites to assess the performance of the EasyScan Go, a microscopy device employing machine-learning-based image analysis. Sensitivity, specificity, accuracy of species detection and parasite density estimation were assessed with expert microscopy as the reference. Intra- and inter-device reliability of the device was also evaluated by comparing results from repeat reads on the same and two different devices. This study has been reported in accordance with the Standards for Reporting Diagnostic accuracy studies (STARD) checklist. Results In total, 2250 Giemsa-stained blood films were prepared and read independently by expert microscopists and the EasyScan Go device. The diagnostic sensitivity of EasyScan Go was 91.1% (95% CI 88.9–92.7), and specificity 75.6% (95% CI 73.1–78.0). With good quality slides sensitivity was similar (89.1%, 95%CI 86.2–91.5), but specificity increased to 85.1% (95%CI 82.6–87.4). Sensitivity increased with parasitaemia rising from 57% at 200–200,000 parasite/µL. Species were identified accurately in 93% of Plasmodium falciparum samples (kappa = 0.76, 95% CI 0.69–0.83), and in 92% of Plasmodium vivax samples (kappa = 0.73, 95% CI 0.66–0.80). Parasite density estimates by the EasyScan Go were within ± 25% of the microscopic reference counts in 23% of slides. Conclusions The performance of the EasyScan Go in parasite detection and species identification accuracy fulfil WHO-TDR Research Malaria Microscopy competence level 2 criteria. In terms of parasite quantification and false positive rate, it meets the level 4 WHO-TDR Research Malaria Microscopy criteria. All performance parameters were significantly affected by slide quality. Further software improvement is required to improve sensitivity at low parasitaemia and parasite density estimations. Trial registration ClinicalTrials.gov number NCT03512678.

Abstract 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.

Abstract Microscopy performed on stained films of peripheral blood for detection, identification and quantification of malaria parasites is an essential reference standard for clinical trials of drugs, vaccines and diagnostic tests for malaria. The value of data from such research is greatly enhanced if this reference standard is consistent across time and geography. Adherence to common standards and practices is a prerequisite to achieve this. The rationale for proposed research standards and procedures for the preparation, staining and microscopic examination of blood films for malaria parasites is presented here with the aim of improving the consistency and reliability of malaria microscopy performed in such studies. These standards constitute the core of a quality management system for clinical research studies employing microscopy as a reference standard. They can be used as the basis for the design of training and proficiency testing programmes as well as for procedures and quality assurance of malaria microscopy in clinical research.

Abstract To limit the spread and impact of anti-malarial drug resistance and react accordingly, surveillance systems able to detect and track in real-time its emergence and spread need to be strengthened or in some places established. Currently, surveillance of anti-malarial drug resistance is done by any of three approaches: (1) in vivo studies to assess the efficacy of drugs in patients; (2) in vitro/ex vivo studies to evaluate parasite susceptibility to the drugs; and/or (3) molecular assays to detect validated gene mutations and/or gene copy number changes that are associated with drug resistance. These methods are complementary, as they evaluate different aspects of resistance; however, standardization of methods, especially for in vitro/ex vivo and molecular techniques, is lacking. The World Health Organization has developed a standard protocol for evaluating the efficacy of anti-malarial drugs, which is used by National Malaria Control Programmes to conduct their therapeutic efficacy studies. Regional networks, such as the East African Network for Monitoring Antimalarial Treatment and the Amazon Network for the Surveillance of Antimalarial Drug Resistance, have been set up to strengthen regional capacities for monitoring anti-malarial drug resistance. The Worldwide Antimalarial Resistance Network has been established to collate and provide global spatial and temporal trends information on the efficacy of anti-malarial drugs and resistance. While exchange of information across endemic countries is essential for monitoring anti-malarial resistance, sustainable funding for the surveillance and networking activities remains challenging. The technology landscape for molecular assays is progressing quite rapidly, and easy-to-use and affordable new techniques are becoming available. They also offer the advantage of high throughput analysis from a simple blood spots obtained from a finger prick. New technologies combined with the strengthening of national reference laboratories in malaria-endemic countries through standardized protocols and training plus the availability of a proficiency testing programme, would contribute to the improvement and sustainability of anti-malarial resistance surveillance networks worldwide.

Abstract 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.

Abstract Background Considering the uncertainty of safety of anti-malarial drugs in pregnancy, efficacy studies are one of the few sources of clinical safety data. Complete safety evaluation is not usually incorporated in efficacy studies due to financial and human resource constraints. This review reports the methods used for the assessment of safety of artemisinin-based and quinine-based treatments in efficacy studies in pregnancy. Methods Methodology of assessment and reporting of safety in efficacy studies of artemisinin-based and quinine-based treatment in pregnancy was reviewed using seven databases and two clinical trial registries. The protocol was registered to PROSPERO (CRD42017054808). Results Of 48 eligible efficacy studies the method of estimation of gestational age was reported in only 32 studies (67%, 32/48) and ultrasound was used in 18 studies (38%, 18/48). Seventeen studies (35%, 17/48) reported parity, 9 (19%, 9/48) reported gravidity and 13 (27%, 13/48) reported both. Thirty-eight studies (79%, 38/48) followed participants through to pregnancy outcome. Fetal loss was assessed in 34 studies (89%, 34/38), but the definition of miscarriage and stillbirth were defined only in 11 (32%, 11/34) and 7 (21%, 7/34) studies, respectively. Preterm birth was assessed in 26 studies (68%, 26/38) but was defined in 16 studies (62%, 16/26). Newborn weight was assessed in 30 studies (79%, 30/38) and length in 10 studies (26%, 10/38). Assessment of birth weight took gestational age into account in four studies (13%, 4/30). Congenital abnormalities were reported in 32 studies (84%, 32/38). Other common risk factors for adverse pregnancy outcomes were not well-reported. Conclusion Incomplete reporting and varied methodological assessment of pregnancy outcomes in anti-malarial drug efficacy studies limits comparison across studies. A standard list of minimal necessary parameters to assess and report the safety component of efficacy studies of anti-malarials in pregnancy is proposed.

Abstract Background There is no agreed standard method to assess the efficacy of anti-malarials for uncomplicated falciparum in pregnancy despite an increased risk of adverse outcomes for the mother and the fetus. The aim of this review is to present the currently available evidence from both observational and interventional cohort studies on anti-malarial efficacy in pregnancy and summarize the variability of assessment and reporting found in the review process. Methods Efficacy methodology and assessment of artemisinin-based treatments (ABT) and quinine-based treatments (QBT) were reviewed systematically using seven databases and two clinical trial registries (protocol registration—PROSPERO: CRD42017054808). Pregnant women in all trimesters with parasitologically confirmed uncomplicated falciparum malaria were included irrespective of symptoms. This review attempted to re-calculate proportions of treatment success applying the same definition as the standard WHO methodology for non-pregnant populations. Aggregated data meta-analyses using data from randomized control trials (RCTs) comparing different treatments were performed by random effects model. Results A total of 48 eligible efficacy studies were identified including 7279 treated Plasmodium falciparum episodes. While polymerase chain reaction (PCR) was used in 24 studies for differentiating recurrence, the assessment and reporting of treatment efficacy was heterogeneous. When the same definition could be applied, PCR-corrected treatment failure of ≥ 10% at any time points was observed in 3/30 ABT and 3/7 QBT arms. Ten RCTs compared different combinations of ABT but there was a maximum of two published RCTs with PCR-corrected outcomes for each comparison. Five RCTs compared ABT and QBT. Overall, the risk of treatment failure was significantly lower in ABT than in QBT (risk ratio 0.22, 95% confidence interval 0.07–0.63), although the actual drug combinations and outcome endpoints were different. First trimester women were included in 12 studies none of which were RCTs of ABT. Conclusions Efficacy studies in pregnancy are not only limited in number but use varied methodological assessments. In five RCTs with comparable methodology, ABT resulted in higher efficacy than QBT in the second and third trimester of pregnancy. Individual patient data meta-analysis can include data from observational cohort studies and could overcome some of the limitations of the current assessment given the paucity of data in this vulnerable group.

Abstract The Kaplan–Meier (K–M) method is currently the preferred approach to derive an efficacy estimate from anti-malarial trial data. In this approach event times are assumed to be continuous and estimates are generated on the assumption that there is only one cause of failure. In reality, failures are captured at pre-scheduled time points and patients can fail treatment due to a variety of causes other than the primary endpoint, commonly termed competing risk events. Ignoring these underlying assumptions can potentially distort the derived efficacy estimates and result in misleading conclusions. This review details the evolution of statistical methods used to derive anti-malarial efficacy for uncomplicated Plasmodium falciparum malaria and assesses the limitations of the current practices. Alternative approaches are explored and their implementation is discussed using example data from a large multi-site study.

This meeting report presents the key findings and discussion points of a 1-day meeting entitled 'Fake anti-malarials: start with the facts' held on 28th May 2015, in Geneva, Switzerland, to disseminate the findings of the artemisinin combination therapy consortium's drug quality programme. The teams purchased over 10,000 samples, using representative sampling approaches, from six malaria endemic countries: Equatorial Guinea (Bioko Island), Cambodia, Ghana, Nigeria, Rwanda and Tanzania. Laboratory analyses of these samples showed that falsified anti-malarials (<8 %) were found in just two of the countries, whilst substandard artemisinin-based combinations were present in all six countries and, artemisinin-based monotherapy tablets are still available in some places despite the fact that the WHO has urged regulatory authorities in malaria-endemic countries to take measures to halt the production and marketing of these oral monotherapies since 2007. This report summarizes the presentations that reviewed the public health impact of falsified and substandard drugs, sampling strategies, techniques for drug quality analysis, approaches to strengthen health systems capacity for the surveillance of drug quality, and the ensuing discussion points from the dissemination meeting.

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.