A human antibody epitope map of Pfs230D1 derived from analysis of individuals vaccinated with a malaria transmission-blocking vaccine.

Pfs230 domain 1 (Pfs230D1) is an advanced malaria transmission-blocking vaccine antigen demonstrating high functional activity in clinical trials. However, the structural and functional correlates of transmission-blocking activity are not defined. Here, we characterized a panel of human monoclonal antibodies (hmAbs) elicited in vaccinees immunized with Pfs230D1. These hmAbs exhibited diverse transmission-reducing activity, yet all bound to Pfs230D1 with nanomolar affinity. We compiled epitope-binning data for seventeen hmAbs and structures of nine hmAbs complexes to construct a high-resolution epitope map and revealed that potent transmission-reducing hmAbs bound to one face of Pfs230D1, while non-potent hmAbs bound to the opposing side. The structure of Pfs230D1D2 revealed that non-potent transmission-reducing epitopes were occluded by the second domain. The hmAb epitope map delineated binary hmAb combinations that synergized for extremely high-potency, transmission-reducing activity. This work provides a high-resolution guide for structure-based design of enhanced immunogens and informs diagnostics that measure the transmission-reducing response.

Population-specific variations in KCNH2 predispose patients to delayed ventricular repolarization upon dihydroartemisinin-piperaquine therapy.

Dihydroartemisinin-piperaquine is efficacious for the treatment of uncomplicated malaria and its use is increasing globally. Despite the positive results in fighting malaria, inhibition of the Kv11.1 channel (hERG; encoded by the KCNH2 gene) by piperaquine has raised concerns about cardiac safety. Whether genetic factors could modulate the risk of piperaquine-mediated QT prolongations remained unclear. Here, we first profiled the genetic landscape of KCNH2 variability using data from 141,614 individuals. Overall, we found 1,007 exonic variants distributed over the entire gene body, 555 of which were missense. By optimizing the gene-specific parametrization of 16 partly orthogonal computational algorithms, we developed a KCNH2-specific ensemble classifier that identified a total of 116 putatively deleterious missense variations. To evaluate the clinical relevance of KCNH2 variability, we then sequenced 293 Malian patients with uncomplicated malaria and identified 13 variations within the voltage sensing and pore domains of Kv11.1 that directly interact with channel blockers. Cross-referencing of genetic and electrocardiographic data before and after piperaquine exposure revealed that carriers of two common variants, rs1805121 and rs41314375, experienced significantly higher QT prolongations (ΔQTc of 41.8 ms and 61 ms, respectively, vs 14.4 ms in controls) with more than 50% of carriers having increases in QTc >30 ms. Furthermore, we identified three carriers of rare population-specific variations who experienced clinically relevant delayed ventricular repolarization. Combined, our results map population-scale genetic variability of KCNH2 and identify genetic biomarkers for piperaquine-induced QT prolongation that could help to flag at-risk patients and optimize efficacy and adherence to antimalarial therapy.

Seasonal Malaria Vaccination with or without Seasonal Malaria Chemoprevention.

BACKGROUND: Malaria control remains a challenge in many parts of the Sahel and sub-Sahel regions of Africa. METHODS: We conducted an individually randomized, controlled trial to assess whether seasonal vaccination with RTS,S/AS01E was noninferior to chemoprevention in preventing uncomplicated malaria and whether the two interventions combined were superior to either one alone in preventing uncomplicated malaria and severe malaria-related outcomes. RESULTS: We randomly assigned 6861 children 5 to 17 months of age to receive sulfadoxine-pyrimethamine and amodiaquine (2287 children [chemoprevention-alone group]), RTS,S/AS01E (2288 children [vaccine-alone group]), or chemoprevention and RTS,S/AS01E (2286 children [combination group]). Of these, 1965, 1988, and 1967 children in the three groups, respectively, received the first dose of the assigned intervention and were followed for 3 years. Febrile seizure developed in 5 children the day after receipt of the vaccine, but the children recovered and had no sequelae. There were 305 events of uncomplicated clinical malaria per 1000 person-years at risk in the chemoprevention-alone group, 278 events per 1000 person-years in the vaccine-alone group, and 113 events per 1000 person-years in the combination group. The hazard ratio for the protective efficacy of RTS,S/AS01E as compared with chemoprevention was 0.92 (95% confidence interval [CI], 0.84 to 1.01), which excluded the prespecified noninferiority margin of 1.20. The protective efficacy of the combination as compared with chemoprevention alone was 62.8% (95% CI, 58.4 to 66.8) against clinical malaria, 70.5% (95% CI, 41.9 to 85.0) against hospital admission with severe malaria according to the World Health Organization definition, and 72.9% (95% CI, 2.9 to 92.4) against death from malaria. The protective efficacy of the combination as compared with the vaccine alone against these outcomes was 59.6% (95% CI, 54.7 to 64.0), 70.6% (95% CI, 42.3 to 85.0), and 75.3% (95% CI, 12.5 to 93.0), respectively. CONCLUSIONS: Administration of RTS,S/AS01E was noninferior to chemoprevention in preventing uncomplicated malaria. The combination of these interventions resulted in a substantially lower incidence of uncomplicated malaria, severe malaria, and death from malaria than either intervention alone. (Funded by the Joint Global Health Trials and PATH; ClinicalTrials.gov number, NCT03143218.).

Rapidly Increasing Severe Acute Respiratory Syndrome Coronavirus 2 Seroprevalence and Limited Clinical Disease in 3 Malian Communities: A Prospective Cohort Study.

BACKGROUND: The extent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure and transmission in Mali and the surrounding region is not well understood. We aimed to estimate the cumulative incidence of SARS-CoV-2 in 3 communities and understand factors associated with infection. METHODS: Between July 2020 and January 2021, we collected blood samples and demographic, social, medical, and self-reported symptoms information from residents aged 6 months and older over 2 study visits. SARS-CoV-2 antibodies were measured using a highly specific 2-antigen enzyme-linked immunosorbent assay optimized for use in Mali. We calculated cumulative adjusted seroprevalence for each community and evaluated factors associated with serostatus at each visit by univariate and multivariate analysis. RESULTS: Overall, 94.8% (2533/2672) of participants completed both study visits. A total of 31.3% (837/2672) were aged <10 years, 27.6% (737/2672) were aged 10-17 years, and 41.1% (1098/2572) were aged ≥18 years. The cumulative SARS-CoV-2 exposure rate was 58.5% (95% confidence interval, 47.5-69.4). This varied between sites and was 73.4% in the urban community of Sotuba, 53.2% in the rural town of Bancoumana, and 37.1% in the rural village of Donéguébougou. Study site and increased age were associated with serostatus at both study visits. There was minimal difference in reported symptoms based on serostatus. CONCLUSIONS: The true extent of SARS-CoV-2 exposure in Mali is greater than previously reported and may now approach hypothetical "herd immunity" in urban areas. The epidemiology of the pandemic in the region may be primarily subclinical and within background illness rates.

The Anti-Circumsporozoite Antibody Response of Children to Seasonal Vaccination With the RTS,S/AS01E Malaria Vaccine.

BACKGROUND: A trial in African children showed that combining seasonal vaccination with the RTS,S/AS01E vaccine with seasonal malaria chemoprevention reduced the incidence of uncomplicated and severe malaria compared with either intervention given alone. Here, we report on the anti-circumsporozoite antibody response to seasonal RTS,S/AS01E vaccination in children in this trial. METHODS: Sera from a randomly selected subset of children collected before and 1 month after 3 priming doses of RTS,S/AS01E and before and 1 month after 2 seasonal booster doses were tested for anti-circumsporozoite antibodies using enzyme-linked immunosorbent assay. The association between post-vaccination antibody titer and incidence of malaria was explored. RESULTS: A strong anti-circumsporozoite antibody response to 3 priming doses of RTS,S/AS01E was seen (geometric mean titer, 368.9 enzyme-linked immunosorbent assay units/mL), but titers fell prior to the first booster dose. A strong antibody response to an annual, pre-malaria transmission season booster dose was observed, but this was lower than after the primary vaccination series and lower after the second than after the first booster dose (ratio of geometric mean rise, 0.66; 95% confidence interval [CI], .57-.77). Children whose antibody response was in the upper tercile post-vaccination had a lower incidence of malaria during the following year than children in the lowest tercile (hazard ratio, 0.43; 95% CI, .28-.66). CONCLUSIONS: Seasonal vaccination with RTS,S/AS01E induced a strong booster antibody response that was lower after the second than after the first booster dose. The diminished antibody response to the second booster dose was not associated with diminished efficacy. CLINICAL TRIALS REGISTRATION: NCT03143218.

Effect of Adding Azithromycin to Seasonal Malaria Chemoprevention.

BACKGROUND: Mass administration of azithromycin for trachoma control led to a sustained reduction in all-cause mortality among Ethiopian children. Whether the addition of azithromycin to the monthly sulfadoxine-pyrimethamine plus amodiaquine used for seasonal malaria chemoprevention could reduce mortality and morbidity among African children was unclear. METHODS: We randomly assigned children 3 to 59 months of age, according to household, to receive either azithromycin or placebo, together with sulfadoxine-pyrimethamine plus amodiaquine, during the annual malaria-transmission season in Burkina Faso and Mali. The drug combinations were administered in four 3-day cycles, at monthly intervals, for three successive seasons. The primary end point was death or hospital admission for at least 24 hours that was not due to trauma or elective surgery. Data were recorded by means of active and passive surveillance. RESULTS: In July 2014, a total of 19,578 children were randomly assigned to receive seasonal malaria chemoprevention plus either azithromycin (9735 children) or placebo (9843 children); each year, children who reached 5 years of age exited the trial and new children were enrolled. In the intention-to-treat analysis, the overall number of deaths and hospital admissions during three malaria-transmission seasons was 250 in the azithromycin group and 238 in the placebo group (events per 1000 child-years at risk, 24.8 vs. 23.5; incidence rate ratio, 1.1; 95% confidence interval [CI], 0.88 to 1.3). Results were similar in the per-protocol analysis. The following events occurred less frequently with azithromycin than with placebo: gastrointestinal infections (1647 vs. 1985 episodes; incidence rate ratio, 0.85; 95% CI, 0.79 to 0.91), upper respiratory tract infections (4893 vs. 5763 episodes; incidence rate ratio, 0.85; 95% CI, 0.81 to 0.90), and nonmalarial febrile illnesses (1122 vs. 1424 episodes; incidence rate ratio, 0.79; 95% CI, 0.73 to 0.87). The prevalence of malaria parasitemia and incidence of adverse events were similar in the two groups. CONCLUSIONS: Among children in Burkina Faso and Mali, the addition of azithromycin to the antimalarial agents used for seasonal malaria chemoprevention did not result in a lower incidence of death or hospital admission that was not due to trauma or surgery than antimalarial agents plus placebo, although a lower disease burden was noted with azithromycin than with placebo. (Funded by the Joint Global Health Trials scheme; ClinicalTrials.gov number, NCT02211729.).

The duration of protection against clinical malaria provided by the combination of seasonal RTS,S/AS01E vaccination and seasonal malaria chemoprevention versus either intervention given alone.

BACKGROUND: A recent trial of 5920 children in Burkina Faso and Mali showed that the combination of seasonal vaccination with the RTS,S/AS01E malaria vaccine (primary series and two seasonal boosters) and seasonal malaria chemoprevention (four monthly cycles per year) was markedly more effective than either intervention given alone in preventing clinical malaria, severe malaria, and deaths from malaria. METHODS: In order to help optimise the timing of these two interventions, trial data were reanalysed to estimate the duration of protection against clinical malaria provided by RTS,S/AS01E when deployed seasonally, by comparing the group who received the combination of SMC and RTS,S/AS01E with the group who received SMC alone. The duration of protection from SMC was also estimated comparing the combined intervention group with the group who received RTS,S/AS01E alone. Three methods were used: Piecewise Cox regression, Flexible parametric survival models and Smoothed Schoenfeld residuals from Cox models, stratifying on the study area and using robust standard errors to control for within-child clustering of multiple episodes. RESULTS: The overall protective efficacy from RTS,S/AS01E over 6 months was at least 60% following the primary series and the two seasonal booster doses and remained at a high level over the full malaria transmission season. Beyond 6 months, protective efficacy appeared to wane more rapidly, but the uncertainty around the estimates increases due to the lower number of cases during this period (coinciding with the onset of the dry season). Protection from SMC exceeded 90% in the first 2-3 weeks post-administration after several cycles, but was not 100%, even immediately post-administration. Efficacy begins to decline from approximately day 21 and then declines more sharply after day 28, indicating the importance of preserving the delivery interval for SMC cycles at a maximum of four weeks. CONCLUSIONS: The efficacy of both interventions was highest immediately post-administration. Understanding differences between these interventions in their peak efficacy and how rapidly efficacy declines over time will help to optimise the scheduling of SMC, malaria vaccination and the combination in areas of seasonal transmission with differing epidemiology, and using different vaccine delivery systems. TRIAL REGISTRATION: The RTS,S-SMC trial in which these data were collected was registered at clinicaltrials.gov: NCT03143218.

Effect of three years' seasonal malaria chemoprevention on molecular markers of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine and amodiaquine in Ouelessebougou, Mali.

BACKGROUND: In 2012, seasonal malaria chemoprevention (SMC) was recommended as policy for malaria control by the World Health Organization (WHO) in areas of highly seasonal malaria transmission across the Sahel sub-region in Africa along with monitoring of drug resistance. We assessed the long-term impact of SMC on Plasmodium falciparum resistance to sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) over a 3-year period of SMC implementation in the health district of Ouelessebougou, Mali. METHODS: In 8 randomly selected sub-districts of Ouelessebougou, Mali, children aged 0-5 years were randomly selected during cross-sectional surveys at baseline (August 2014) and 1, 2 and 3 years post-SMC, at the beginning and end of the malaria transmission season. Blood smears and blood spots on filter paper were obtained and frequencies of mutation in P. falciparum genes related to resistance to SP and AQ (Pfdhfr, Pfdhps, Pfmdr1, and Pfcrt) were assessed by PCR amplification on individual samples and PCR amplification followed by deep sequencing on pooled (by site and year) samples. RESULTS: At each survey, approximately 50-100 individual samples were analysed by PCR amplification and a total of 1,164 samples were analysed by deep sequencing with an average read depth of 18,018-36,918 after pooling by site and year. Most molecular markers of resistance did not increase in frequency over the period of study (2014-2016). After 3 years of SMC, the frequencies of Pfdhps 540E, Pfdhps 437G and Pfcrt K76T remained similar compared to baseline (4.0 vs 1.4%, p = 0.41; 74.5 vs 64.6%, p = 0.22; 71.3 vs 67.4%, p = 0.69). Nearly all samples tested carried Pfdhfr 59R, and this proportion remained similar 3 years after SMC implementation (98.8 vs 100%, p = 1). The frequency of Pfmdr1 N86Y increased significantly over time from 5.6% at baseline to 18.6% after 3 years of SMC (p = 0.016). Results of pooled analysis using deep sequencing were consistent with those by individual analysis with standard PCR, but also indicated for the first time the presence of mutations at the Pfdhps A581G allele at a frequency of 11.7% after 2 years of SMC, as well as the Pfdhps I431V allele at frequencies of 1.6-9.3% following 1 and 2 years of SMC, respectively. CONCLUSION: Two and 3 years of SMC implementation were associated with increased frequency of the Pfmdr1 N86Y mutation but not Pfdhps 540E, Pfdhps 437G and Pfcrt K76T. The first-time detection of the Pfdhps haplotype bearing the I431V and A581G mutations in Mali, even at low frequency, warrants further long-term surveillance.

Impact of seasonal RTS,S/AS01E vaccination plus seasonal malaria chemoprevention on the nutritional status of children in Burkina Faso and Mali.

BACKGROUND: A recent trial in Burkina Faso and Mali showed that combining seasonal RTS,S/AS01E malaria vaccination with seasonal malaria chemoprevention (SMC) substantially reduced the incidence of uncomplicated and severe malaria in young children compared to either intervention alone. Given the possible negative effect of malaria on nutrition, the study investigated whether these children also experienced lower prevalence of acute and chronic malnutrition. METHODS: In Burkina Faso and Mali 5920 children were randomized to receive either SMC alone, RTS,S/AS01E alone, or SMC combined with RTS,S/AS01E for three malaria transmission seasons (2017-2019). After each transmission season, anthropometric measurements were collected from all study children at a cross-sectional survey and used to derive nutritional status indicators, including the binary variables wasted and stunted (weight-for-height and height-for-age z-scores below - 2, respectively). Binary and continuous outcomes between treatment groups were compared by Poisson and linear regression. RESULTS: In 2017, compared to SMC alone, the combined intervention reduced the prevalence of wasting by approximately 12% [prevalence ratio (PR) = 0.88 (95% CI 0.75, 1.03)], and approximately 21% in 2018 [PR = 0.79 (95% CI 0.62, 1.01)]. Point estimates were similar for comparisons with RTS,S/AS01E, but there was stronger evidence of a difference. There was at least a 30% reduction in the point estimates for the prevalence of severe wasting in the combined group compared to the other two groups in 2017 and 2018. There was no difference in the prevalence of moderate or severe wasting between the groups in 2019. The prevalence of stunting, low-MUAC-for-age or being underweight did not differ between groups for any of the three years. The prevalence of severe stunting was higher in the combined group compared to both other groups in 2018, and compared to RTS,S/AS01E alone in 2017; this observation does not have an obvious explanation and may be a chance finding. Overall, malnutrition was very common in this cohort, but declined over the study as the children became older. CONCLUSIONS: Despite a high burden of malnutrition and malaria in the study populations, and a major reduction in the incidence of malaria in children receiving both interventions, this had only a modest impact on nutritional status. Therefore, other interventions are needed to reduce the high burden of malnutrition in these areas. TRIAL REGISTRATION: https://www.clinicaltrials.gov/ct2/show/NCT03143218 , registered 8th May 2017.

Severe Acute Respiratory Syndrome Coronavirus 2 Seroassay Performance and Optimization in a Population With High Background Reactivity in Mali.

BACKGROUND: False positivity may hinder the utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological tests in sub-Saharan Africa. METHODS: From 312 Malian samples collected before 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and 4 other betacoronaviruses by enzyme-linked immunosorbent assay (ELISA). In a subset of samples, we assessed antibodies to a panel of Plasmodium falciparum antigens by suspension bead array and functional antiviral activity by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA using SARS-CoV-2 spike protein and receptor-binding domain developed in the United States using Malian positive and negative control samples. To optimize test performance, we compared single- and 2-antigen approaches using existing assay cutoffs and population-specific cutoffs. RESULTS: Background reactivity to SARS-CoV-2 antigens was common in prepandemic Malian samples. The SARS-CoV-2 reactivity varied between communities, increased with age, and correlated negligibly/weakly with other betacoronavirus and P falciparum antibodies. No prepandemic samples demonstrated functional activity. Regardless of the cutoffs applied, test specificity improved using a 2-antigen approach. Test performance was optimal using a 2-antigen assay with population-specific cutoffs (sensitivity, 73.9% [95% confidence interval {CI}, 51.6-89.8]; specificity, 99.4% [95% CI, 97.7-99.9]). CONCLUSIONS: We have addressed the problem of SARS-CoV-2 seroassay performance in Africa by using a 2-antigen assay with cutoffs defined by performance in the target population.

The Duration of Protection from Azithromycin Against Malaria, Acute Respiratory, Gastrointestinal, and Skin Infections When Given Alongside Seasonal Malaria Chemoprevention: Secondary Analyses of Data from a Clinical Trial in Houndé, Burkina Faso, and Bougouni, Mali.

BACKGROUND: Mass drug administration (MDA) with azithromycin (AZ) is being considered as a strategy to promote child survival in sub-Saharan Africa, but the mechanism by which AZ reduces mortality is unclear. To better understand the nature and extent of protection provided by AZ, we explored the profile of protection by time since administration, using data from a household-randomized, placebo-controlled trial in Burkina Faso and Mali. METHODS: Between 2014 and 2016, 30 977 children aged 3-59 months received seasonal malaria chemoprevention (SMC) with sulfadoxine-pyrimethamine plus amodiaquine and either AZ or placebo monthly, on 4 occasions each year. Poisson regression with gamma-distributed random effects, accounting for the household randomization and within-individual clustering of illness episodes, was used to compare incidence of prespecified outcomes between SMC+AZ versus SMC+placebo groups in fixed time strata post-treatment. The likelihood ratio test was used to assess evidence for a time-treatment group interaction. RESULTS: Relative to SMC+placebo, there was no evidence of protection from SMC+AZ against hospital admissions and deaths. Additional protection from SMC+AZ against malaria was confined to the first 2 weeks post-administration (protective efficacy (PE): 24.2% [95% CI: 17.8%, 30.1%]). Gastroenteritis and pneumonia were reduced by 29.9% [21.7; 37.3%], and 34.3% [14.9; 49.3%], respectively, in the first 2 weeks postadministration. Protection against nonmalaria fevers with a skin condition persisted up to 28 days: PE: 46.3% [35.1; 55.6%]. CONCLUSIONS: The benefits of AZ-MDA are broad-ranging but short-lived. To maximize impact, timing of AZ-MDA must address the challenge of targeting asynchronous morbidity and mortality peaks from different causes.

Effectiveness of seasonal malaria chemoprevention (SMC) treatments when SMC is implemented at scale: Case-control studies in 5 countries.

BACKGROUND: Seasonal malaria chemoprevention (SMC) has shown high protective efficacy against clinical malaria and severe malaria in a series of clinical trials. We evaluated the effectiveness of SMC treatments against clinical malaria when delivered at scale through national malaria control programmes in 2015 and 2016. METHODS AND FINDINGS: Case-control studies were carried out in Mali and The Gambia in 2015, and in Burkina Faso, Chad, Mali, Nigeria, and The Gambia in 2016. Children aged 3-59 months presenting at selected health facilities with microscopically confirmed clinical malaria were recruited as cases. Two controls per case were recruited concurrently (on or shortly after the day the case was detected) from the neighbourhood in which the case lived. The primary exposure was the time since the most recent course of SMC treatment, determined from SMC recipient cards, caregiver recall, and administrative records. Conditional logistic regression was used to estimate the odds ratio (OR) associated with receipt of SMC within the previous 28 days, and SMC 29 to 42 days ago, compared with no SMC in the past 42 days. These ORs, which are equivalent to incidence rate ratios, were used to calculate the percentage reduction in clinical malaria incidence in the corresponding time periods. Results from individual countries were pooled in a random-effects meta-analysis. In total, 2,126 cases and 4,252 controls were included in the analysis. Across the 7 studies, the mean age ranged from 1.7 to 2.4 years and from 2.1 to 2.8 years among controls and cases, respectively; 42.2%-50.9% and 38.9%-46.9% of controls and cases, respectively, were male. In all 7 individual case-control studies, a high degree of personal protection from SMC against clinical malaria was observed, ranging from 73% in Mali in 2016 to 98% in Mali in 2015. The overall OR for SMC within 28 days was 0.12 (95% CI: 0.06, 0.21; p < 0.001), indicating a protective effectiveness of 88% (95% CI: 79%, 94%). Effectiveness against clinical malaria for SMC 29-42 days ago was 61% (95% CI: 47%, 72%). Similar results were obtained when the analysis was restricted to cases with parasite density in excess of 5,000 parasites per microlitre: Protective effectiveness 90% (95% CI: 79%, 96%; P<0.001), and 59% (95% CI: 34%, 74%; P<0.001) for SMC 0-28 days and 29-42 days ago, respectively. Potential limitations include the possibility of residual confounding due to an association between exposure to malaria and access to SMC, or differences in access to SMC between patients attending a clinic and community controls; however, neighbourhood matching of cases and controls, and covariate adjustment, attempted to control for these aspects, and the observed decline in protection over time, consistent with expected trends, argues against a major bias from these sources. CONCLUSIONS: SMC administered as part of routine national malaria control activities provided a very high level of personal protection against clinical malaria over 28 days post-treatment, similar to the efficacy observed in clinical trials. The case-control design used in this study can be used at intervals to ensure SMC treatments remain effective.

Persistent Submicroscopic Plasmodium falciparum Parasitemia 72 Hours after Treatment with Artemether-Lumefantrine Predicts 42-Day Treatment Failure in Mali and Burkina Faso.

A recent randomized controlled trial, the WANECAM (West African Network for Clinical Trials of Antimalarial Drugs) trial, conducted at seven centers in West Africa, found that artemether-lumefantrine, artesunate-amodiaquine, pyronaridine-artesunate, and dihydroartemisinin-piperaquine all displayed good efficacy. However, artemether-lumefantrine was associated with a shorter interval between clinical episodes than the other regimens. In a further comparison of these therapies, we identified cases of persisting submicroscopic parasitemia by quantitative PCR (qPCR) at 72 h posttreatment among WANECAM participants from 5 sites in Mali and Burkina Faso, and we compared treatment outcomes for this group to those with complete parasite clearance by 72 h. Among 552 evaluable patients, 17.7% had qPCR-detectable parasitemia at 72 h during their first treatment episode. This proportion varied among sites, reflecting differences in malaria transmission intensity, but did not differ among pooled drug treatment groups. However, patients who received artemether-lumefantrine and were qPCR positive at 72 h were significantly more likely to have microscopically detectable recurrent Plasmodium falciparum parasitemia by day 42 than those receiving other regimens and experienced, on average, a shorter interval before the next clinical episode. Haplotypes of pfcrt and pfmdr1 were also evaluated in persisting parasites. These data identify a possible threat to the parasitological efficacy of artemether-lumefantrine in West Africa, over a decade since it was first introduced on a large scale.

Cost-effectiveness of district-wide seasonal malaria chemoprevention when implemented through routine malaria control programme in Kita, Mali using fixed point distribution.

BACKGROUND: Seasonal malaria chemoprevention (SMC) is a strategy for malaria control recommended by the World Health Organization (WHO) since 2012 for Sahelian countries. The Mali National Malaria Control Programme adopted a plan for pilot implementation and nationwide scale-up by 2016. Given that SMC is a relatively new approach, there is an urgent need to assess the costs and cost effectiveness of SMC when implemented through the routine health system to inform decisions on resource allocation. METHODS: Cost data were collected from pilot implementation of SMC in Kita district, which targeted 77,497 children aged 3-59 months. Starting in August 2014, SMC was delivered by fixed point distribution in villages with the first dose observed each month. Treatment consisted of sulfadoxine-pyrimethamine and amodiaquine once a month for four consecutive months, or rounds. Economic and financial costs were collected from the provider perspective using an ingredients approach. Effectiveness estimates were based upon a published mathematical transmission model calibrated to local epidemiology, rainfall patterns and scale-up of interventions. Incremental cost effectiveness ratios were calculated for the cost per malaria episode averted, cost per disability adjusted life years (DALYs) averted, and cost per death averted. RESULTS: The total economic cost of the intervention in the district of Kita was US $357,494. Drug costs and personnel costs accounted for 34% and 31%, respectively. Incentives (payment other than salary for efforts beyond routine activities) accounted for 25% of total implementation costs. Average financial and economic unit costs per child per round were US $0.73 and US $0.86, respectively; total annual financial and economic costs per child receiving SMC were US $2.92 and US $3.43, respectively. Accounting for coverage, the economic cost per child fully adherent (receiving all four rounds) was US $6.38 and US $4.69, if weighted highly adherent, (receiving 3 or 4 rounds of SMC). When costs were combined with modelled effects, the economic cost per malaria episode averted in children was US $4.26 (uncertainty bound 2.83-7.17), US $144 (135-153) per DALY averted and US $ 14,503 (13,604-15,402) per death averted. CONCLUSIONS: When implemented at fixed point distribution through the routine health system in Mali, SMC was highly cost-effective. As in previous SMC implementation studies, financial incentives were a large cost component.

Nutritional status in young children prior to the malaria transmission season in Burkina Faso and Mali, and its impact on the incidence of clinical malaria.

BACKGROUND: Malaria and malnutrition remain major problems in Sahel countries, especially in young children. The direct effect of malnutrition on malaria remains poorly understood, and may have important implications for malaria control. In this study, nutritional status and the association between malnutrition and subsequent incidence of symptomatic malaria were examined in children in Burkina Faso and Mali who received either azithromycin or placebo, alongside seasonal malaria chemoprevention. METHODS: Mid-upper arm circumference (MUAC) was measured in all 20,185 children who attended a screening visit prior to the malaria transmission season in 2015. Prior to the 2016 malaria season, weight, height and MUAC were measured among 4149 randomly selected children. Height-for-age, weight-for-age, weight-for-height, and MUAC-for-age were calculated as indicators of nutritional status. Malaria incidence was measured during the following rainy seasons. Multivariable random effects Poisson models were created for each nutritional indicator to study the effect of malnutrition on clinical malaria incidence for each country. RESULTS: In both 2015 and 2016, nutritional status prior to the malaria season was poor. The most prevalent form of malnutrition in Burkina Faso was being underweight (30.5%; 95% CI 28.6-32.6), whereas in Mali stunting was most prevalent (27.5%; 95% CI 25.6-29.5). In 2016, clinical malaria incidence was 675 per 1000 person-years (95% CI 613-744) in Burkina Faso, and 1245 per 1000 person-years (95% CI 1152-1347) in Mali. There was some evidence that severe stunting was associated with lower incidence of malaria in Mali (RR 0.81; 95% CI 0.64-1.02; p = 0.08), but this association was not seen in Burkina Faso. Being moderately underweight tended to be associated with higher incidence of clinical malaria in Burkina Faso (RR 1.27; 95% CI 0.98-1.64; p = 0.07), while this was the case in Mali for moderate wasting (RR 1.27; 95% CI 0.98-1.64; p = 0.07). However, these associations were not observed in severely affected children, nor consistent between countries. MUAC-for-age was not associated with malaria risk. CONCLUSIONS: Both malnutrition and malaria were common in the study areas, high despite high coverage of seasonal malaria chemoprevention and long-lasting insecticidal nets. However, no strong or consistent evidence was found for an association between any of the nutritional indicators and the subsequent incidence of clinical malaria.

Artemisinin-based combination therapy for uncomplicated Plasmodium falciparum malaria in Mali: a systematic review and meta-analysis.

BACKGROUND: Artemisinin-based combination therapy (ACT) was deployed in 2005 as an alternative to chloroquine and is considered the most efficacious treatment currently available for uncomplicated falciparum malaria. While widespread artemisinin resistance has not been reported to date in Africa, recent studies have reported partial resistance in Rwanda. The purpose of this study is to provide a current systematic review and meta-analysis on ACT at Mali study sites, where falciparum malaria is highly endemic. METHODS: A systematic review of the literature maintained in the bibliographic databases accessible through the PubMed, ScienceDirect and Web of Science search engines was performed to identify research studies on ACT occurring at Mali study sites. Selected studies included trials occurring at Mali study sites with reported polymerase chain reaction (PCR)-corrected adequate clinical and parasite response rates (ACPRcs) at 28 days. Data were stratified by treatment arm (artemether-lumefantrine (AL), the first-line treatment for falciparum malaria in Mali and non-AL arms) and analysed using random-effects, meta-analysis approaches. RESULTS: A total of 11 studies met the inclusion criteria, and a risk of bias assessment carried out by two independent reviewers determined low risk of bias among all assessed criteria. The ACPRc for the first-line AL at Mali sites was 99.0% (95% CI (98.3%, 99.8%)), while the ACPRc among non-AL treatment arms was 98.9% (95% CI (98.3%, 99.5%)). The difference in ACPRcs between non-AL treatment arms and AL treatment arms was not statistically significant (p = .752), suggesting that there are potential treatment alternatives beyond the first-line of AL in Mali. CONCLUSIONS: ACT remains highly efficacious in treating uncomplicated falciparum malaria in Mali. Country-specific meta-analyses on ACT are needed on an ongoing basis for monitoring and evaluating drug efficacy patterns to guide local malaria treatment policies, particularly in the wake of observed artemisinin resistance in Southeast Asia and partial resistance in Rwanda.

Hepatic safety of repeated treatment with pyronaridine-artesunate versus artemether-lumefantrine in patients with uncomplicated malaria: a secondary analysis of the WANECAM 1 data from Bobo-Dioulasso, Burkina Faso.

BACKGROUND: The use of pyronaridine-artesunate (PA) has been associated with scarce transaminitis in patients. This analysis aimed to evaluate the hepatic safety profile of repeated treatment with PA versus artemether-lumefantrine (AL) in patients with consecutive uncomplicated malaria episodes in Bobo-Dioulasso, Burkina Faso. METHODS: This study analysed data from a clinical trial conducted from 2012 to 2015, in which participants with uncomplicated malaria were assigned to either PA or AL arms and followed up to 42 days. Subsequent malaria episodes within a 2-years follow up period were also treated with the same ACT initially allocated. Transaminases (AST/ALT), alkaline phosphatase (ALP), total and direct bilirubin were measured at days 0 (baseline), 3, 7, 28 and on some unscheduled days if required. The proportions of non-clinical hepatic adverse events (AEs) following first and repeated treatments with PA and AL were compared within study arms. The association of these AEs with retreatment in each arm was also determined using a logistic regression model. RESULTS: A total of 1379 malaria episodes were included in the intention to treat analysis with 60% of all cases occurring in the AL arm. Overall, 179 non-clinical hepatic AEs were recorded in the AL arm versus 145 in the PA arm. Elevated ALT was noted in 3.05% of treated malaria episodes, elevated AST 3.34%, elevated ALP 1.81%, and elevated total and direct bilirubin in 7.90% and 7.40% respectively. Retreated participants were less likely to experience elevated ALT and AST than first episode treated participants in both arms. One case of Hy's law condition was recorded in a first treated participant of the PA arm. Participants from the retreatment group were 76% and 84% less likely to have elevated ALT and AST, respectively, in the AL arm and 68% less likely to present elevated ALT in the PA arm. In contrast, they were almost 2 times more likely to experience elevated total bilirubin in both arms. CONCLUSIONS: Pyronaridine-artesunate and artemether-lumefantrine showed similar hepatic safety when used repeatedly in participants with uncomplicated malaria. Pyronaridine-artesunate represents therefore a suitable alternative to the current first line anti-malarial drugs in use in endemic areas. Trial registration Pan African Clinical Trials Registry. PACTR201105000286876.

Selection of pfcrt K76 and pfmdr1 N86 Coding Alleles after Uncomplicated Malaria Treatment by Artemether-Lumefantrine in Mali.

BACKGROUND: Artemether-lumefantrine is a highly effective artemisinin-based combination therapy that was adopted in Mali as first-line treatment for uncomplicated Plasmodium falciparum malaria. This study was designed to measure the efficacy of artemether-lumefantrine and to assess the selection of the P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multi-drug resistance 1 (pfmdr1) genotypes that have been associated with drug resistance. METHODS: A 28-day follow-up efficacy trial of artemether-lumefantrine was conducted in patients aged 6 months and older suffering from uncomplicated falciparum malaria in four different Malian areas during the 2009 malaria transmission season. The polymorphic genetic markers MSP2, MSP1, and Ca1 were used to distinguish between recrudescence and reinfection. Reinfection and recrudescence were then grouped as recurrent infections and analyzed together by PCR-restriction fragment length polymorphism (RFLP) to identify candidate markers for artemether-lumefantrine tolerance in the P. falciparum chloroquine resistance transporter (pfcrt) gene and the P. falciparum multi-drug resistance 1 (pfmdr1) gene. RESULTS: Clinical outcomes in 326 patients (96.7%) were analyzed and the 28-day uncorrected adequate clinical and parasitological response (ACPR) rate was 73.9%. The total PCR-corrected 28-day ACPR was 97.2%. The pfcrt 76T and pfmdr1 86Y population prevalence decreased from 49.3% and 11.0% at baseline (n = 337) to 38.8% and 0% in patients with recurrent infection (n = 85); p = 0.001), respectively. CONCLUSION: Parasite populations exposed to artemether-lumefantrine in this study were selected toward chloroquine-sensitivity and showed a promising trend that may warrant future targeted reintroduction of chloroquine or/and amodiaquine.

Zika Virus Circulation in Mali.

The circulation of Zika virus (ZIKV) in Mali has not been clearly characterized. Therefore, we conducted a serologic survey of 793 asymptomatic volunteers >15 years of age (2016), and 637 blood donors (2013) to assess the seroprevalence of ZIKV infection in 2 ecoclimatic regions of Mali, tropical savannah and warm semiarid region, using ELISA and seroneutralization assays. The overall seroprevalence was ≈12% and increased with age, with no statistical difference between male and female participants. In the warm semiarid study sites we detected immunological markers of an outbreak that occurred in the late 1990s in 18% (95% CI 13%-23%) of participants. In tropical savannah sites, we estimated a low rate of endemic transmission, with 2.5% (95% CI 2.0%-3.1%) of population infected by ZIKV annually. These data demonstrate the circulation of ZIKV in Mali and provide evidence of a previously unidentified outbreak that occurred in the late 1990s.

Evaluation of seasonal malaria chemoprevention in two areas of intense seasonal malaria transmission: Secondary analysis of a household-randomised, placebo-controlled trial in Houndé District, Burkina Faso and Bougouni District, Mali.

BACKGROUND: Seasonal malaria chemoprevention (SMC) is now widely deployed in the Sahel, including several countries that are major contributors to the global burden of malaria. Consequently, it is important to understand whether SMC continues to provide a high level of protection and how SMC might be improved. SMC was evaluated using data from a large, household-randomised trial in Houndé, Burkina Faso and Bougouni, Mali. METHODS AND FINDINGS: The parent trial evaluated monthly SMC plus either azithromycin (AZ) or placebo, administered as directly observed therapy 4 times per year between August and November (2014-2016). In July 2014, 19,578 children aged 3-59 months were randomised by household to study group. Children who remained within the age range 3-59 months in August each year, plus children born into study households or who moved into the study area, received study drugs in 2015 and 2016. These analyses focus on the approximately 10,000 children (5,000 per country) under observation each year in the SMC plus placebo group. Despite high coverage and high adherence to SMC, the incidence of hospitalisations or deaths due to malaria and uncomplicated clinical malaria remained high in the study areas (overall incidence rates 12.5 [95% confidence interval (CI): 11.2, 14.1] and 871.1 [95% CI: 852.3, 890.6] cases per 1,000 person-years, respectively) and peaked in July each year, before SMC delivery began in August. The incidence rate ratio comparing SMC within the past 28 days with SMC more than 35 days ago-adjusted for age, country, and household clustering-was 0.13 (95% CI: 0.08, 0.20), P < 0.001 for malaria hospitalisations and deaths from malaria and 0.21 (95% CI 0.20, 0.23), P < 0.001 for uncomplicated malaria, indicating protective efficacy of 87.4% (95% CI: 79.6%, 92.2%) and 78.3% (95% CI: 76.8%, 79.6%), respectively. The prevalence of malaria parasitaemia at weekly surveys during the rainy season and at the end of the transmission season was several times higher in children who missed the SMC course preceding the survey contact, and the smallest prevalence ratio observed was 2.98 (95% CI: 1.95, 4.54), P < 0.001. The frequency of molecular markers of sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) resistance did not increase markedly over the study period either amongst study children or amongst school-age children resident in the study areas. After 3 years of SMC deployment, the day 28 PCR-unadjusted adequate clinical and parasitological response rate of the SP + AQ regimen in children with asymptomatic malaria was 98.3% (95% CI: 88.6%, 99.8%) in Burkina Faso and 96.1% (95% CI: 91.5%, 98.2%) in Mali. Key limitations of this study are the potential overdiagnosis of uncomplicated malaria by rapid diagnostic tests and the potential for residual confounding from factors related to adherence to the monthly SMC schedule. CONCLUSION: Despite strong evidence that SMC is providing a high level of protection, the burden of malaria remains substantial in the 2 study areas. These results emphasise the need for continuing support of SMC programmes. A fifth monthly SMC course is needed to adequately cover the whole transmission season in the study areas and in settings with similar epidemiology. TRIAL REGISTRATION: The AZ-SMC trial in which these data were collected was registered at clinicaltrials.gov: NCT02211729.