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.

IFNγ, TNFα polymorphisms and IFNγ serum levels are associated with the clearance of drug-resistant P. falciparum in Malian children.

Host immunity has been suggested to clear drug-resistant parasites in malaria-endemic settings. However, the immunogenetic mechanisms involved in parasite clearance are poorly understood. Characterizing the host's immunity and genes involved in controlling the parasitic infection can inform the development of blood-stage malaria vaccines. This study investigates host regulatory cytokines and immunogenomic factors associated with the clearance of Plasmodium falciparum carrying a chloroquine resistance genotype. Biological samples from participants of previous drug efficacy trials conducted in two Malian localities were retrieved. The P. falciparum chloroquine resistance transporter (Pfcrt) gene was genotyped using parasite DNA. Children carrying parasites with the mutant allele (Pfcrt-76T) were classified based on their ability to clear their parasites. The levels of the different cytokines were measured in serum. The polymorphisms of specific human genes involved in malaria susceptibility were genotyped using human DNA. The prevalence of the Pfcrt-76T was significantly higher in Kolle than in Bandiagara (81.6 % vs 38.6 %, p < 10-6). The prevalence of children who cleared their mutant parasites was significantly higher in Bandiagara than in Kolle (82.2 % vs 67.4 %, p < 0.05). The genotyping of host genes revealed that IFN-γ -874 T and TNF-α -308A alleles were positively associated with parasite clearance. Cytokine profiling revealed that IFN-γ level was positively associated with parasite clearance (p = 0.04). This study highlights the role of host's immunity and immunogenetic factors to clear resistant parasites, suggesting further characterization of these polymorphisms may help to develop novel approaches to antiparasitic treatment strategies.

Efficacy of Cipargamin (KAE609) in a Randomized, Phase II Dose-Escalation Study in Adults in Sub-Saharan Africa With Uncomplicated Plasmodium falciparum Malaria.

BACKGROUND: Cipargamin (KAE609) is a potent antimalarial in a phase II trial. Here we report efficacy, pharmacokinetics, and resistance marker analysis across a range of cipargamin doses. These were secondary endpoints from a study primarily conducted to assess the hepatic safety of cipargamin (hepatic safety data are reported elsewhere). METHODS: This phase II, multicenter, randomized, open-label, dose-escalation trial was conducted in sub-Saharan Africa in adults with uncomplicated Plasmodium falciparum malaria. Cipargamin monotherapy was given as single doses up to 150 mg or up to 50 mg once daily for 3 days, with artemether-lumefantrine as control. Key efficacy endpoints were parasite clearance time (PCT), and polymerase chain reaction (PCR)-corrected and uncorrected adequate clinical and parasitological response (ACPR) at 14 and 28 days. Pharmacokinetics and molecular markers of drug resistance were also assessed. RESULTS: All single or multiple cipargamin doses ≥50 mg were associated with rapid parasite clearance, with median PCT of 8 hours versus 24 hours for artemether-lumefantrine. PCR-corrected ACPR at 14 and 28 days was >75% and 65%, respectively, for each cipargamin dose. A treatment-emerging mutation in the Pfatp4 gene, G358S, was detected in 65% of treatment failures. Pharmacokinetic parameters were consistent with previous data, and approximately dose proportional. CONCLUSIONS: Cipargamin, at single doses of 50 to 150 mg, was associated with very rapid parasite clearance, PCR-corrected ACPR at 28 days of >65% in adults with uncomplicated P. falciparum malaria, and recrudescent parasites frequently harbored a treatment-emerging mutation. Cipargamin will be further developed with a suitable combination partner. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov (NCT03334747).

A Novel Ex Vivo Drug Assay for Assessing the Transmission-Blocking Activity of Compounds on Field-Isolated Plasmodium falciparum Gametocytes.

The discovery and development of transmission-blocking therapies challenge malaria elimination and necessitate standard and reproducible bioassays to measure the blocking properties of antimalarial drugs and candidate compounds. Most of the current bioassays evaluating the transmission-blocking activity of compounds rely on laboratory-adapted Plasmodium strains. Transmission-blocking data from clinical gametocyte isolates could help select novel transmission-blocking candidates for further development. Using freshly collected Plasmodium falciparum gametocytes from asymptomatic individuals, we first optimized ex vivo culture conditions to improve gametocyte viability and infectiousness by testing several culture parameters. We next pre-exposed ex vivo field-isolated gametocytes to chloroquine, dihydroartemisinin, primaquine, KDU691, GNF179, and oryzalin for 48 h prior to direct membrane feeding. We measured the activity of the drug on the ability of gametocytes to resume the sexual life cycle in Anopheles after drug exposure. Using 57 blood samples collected from Malian volunteers aged 6 to 15 years, we demonstrate that the infectivity of freshly collected field gametocytes can be preserved and improved ex vivo in a culture medium supplemented with 10% horse serum at 4% hematocrit for 48 h. Moreover, our optimized drug assay displays the weak transmission-blocking activity of chloroquine and dihydroartemisinin, while primaquine and oryzalin exhibited a transmission-blocking activity of ~50% at 1 µM. KDU691 and GNF179 both interrupted Plasmodium transmission at 1 µM and 5 nM, respectively. This new approach, if implemented, has the potential to accelerate the screening of compounds with transmission-blocking activity.

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.

Hepatic safety and tolerability of cipargamin (KAE609), in adult patients with Plasmodium falciparum malaria: a randomized, phase II, controlled, dose-escalation trial in sub-Saharan Africa.

BACKGROUND: The novel anti-malarial cipargamin (KAE609) has potent, rapid activity against Plasmodium falciparum. Transient asymptomatic liver function test elevations were previously observed in cipargamin-treated subjects in two trials: one in malaria patients in Asia and one in volunteers with experimentally induced malaria. In this study, the hepatic safety of cipargamin given as single doses of 10 to 150 mg and 10 to 50 mg once daily for 3 days was assessed. Efficacy results, frequency of treatment-emerging mutations in the atp4 gene and pharmacokinetics have been published elsewhere. Further, the R561H mutation in the k13 gene, which confers artemisinin-resistance, was associated with delayed parasite clearance following treatment with artemether-lumefantrine in Rwanda in this study. This was also the first study with cipargamin to be conducted in patients in sub-Saharan Africa. METHODS: This was a Phase II, multicentre, randomized, open-label, dose-escalation trial in adults with uncomplicated falciparum malaria in five sub-Saharan countries, using artemether-lumefantrine as control. The primary endpoint was ≥ 2 Common Terminology Criteria for Adverse Events (CTCAE) Grade increase from baseline in alanine aminotransferase (ALT) or aspartate transaminase (AST) during the 4-week trial. RESULTS: Overall, 2/135 patients treated with cipargamin had ≥ 2 CTCAE Grade increases from baseline in ALT or AST compared to 2/51 artemether-lumefantrine patients, with no significant difference between any cipargamin treatment group and the control group. Cipargamin exposure was comparable to or higher than those in previous studies. Hepatic adverse events and general safety and tolerability were similar for all cipargamin doses and artemether-lumefantrine. Cipargamin was well tolerated with no safety concerns. CONCLUSIONS: This active-controlled, dose escalation study was a detailed assessment of the hepatic safety of cipargamin, across a wide range of doses, in patients with uncomplicated falciparum malaria. Comparison with previous cipargamin trials requires caution as no clear conclusion can be drawn as to whether hepatic safety and potential immunity to malaria would differ with ethnicity, patient age and or geography. Previous concerns regarding hepatic safety may have been confounded by factors including malaria itself, whether natural or experimental infection, and should not limit the further development of cipargamin. Trial registration ClinicalTrials.gov number: NCT03334747 (7 Nov 2017), other study ID CKAE609A2202.

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.

The duration of chemoprophylaxis against malaria after treatment with artesunate-amodiaquine and artemether-lumefantrine and the effects of pfmdr1 86Y and pfcrt 76T: a meta-analysis of individual patient data.

BACKGROUND: The majority of Plasmodium falciparum malaria cases in Africa are treated with the artemisinin combination therapies artemether-lumefantrine (AL) and artesunate-amodiaquine (AS-AQ), with amodiaquine being also widely used as part of seasonal malaria chemoprevention programs combined with sulfadoxine-pyrimethamine. While artemisinin derivatives have a short half-life, lumefantrine and amodiaquine may give rise to differing durations of post-treatment prophylaxis, an important additional benefit to patients in higher transmission areas. METHODS: We analyzed individual patient data from 8 clinical trials of AL versus AS-AQ in 12 sites in Africa (n = 4214 individuals). The time to PCR-confirmed reinfection after treatment was used to estimate the duration of post-treatment protection, accounting for variation in transmission intensity between settings using hidden semi-Markov models. Accelerated failure-time models were used to identify potential effects of covariates on the time to reinfection. The estimated duration of chemoprophylaxis was then used in a mathematical model of malaria transmission to determine the potential public health impact of each drug when used for first-line treatment. RESULTS: We estimated a mean duration of post-treatment protection of 13.0 days (95% CI 10.7-15.7) for AL and 15.2 days (95% CI 12.8-18.4) for AS-AQ overall. However, the duration varied significantly between trial sites, from 8.7-18.6 days for AL and 10.2-18.7 days for AS-AQ. Significant predictors of time to reinfection in multivariable models were transmission intensity, age, drug, and parasite genotype. Where wild type pfmdr1 and pfcrt parasite genotypes predominated (<=20% 86Y and 76T mutants, respectively), AS-AQ provided ~ 2-fold longer protection than AL. Conversely, at a higher prevalence of 86Y and 76T mutant parasites (> 80%), AL provided up to 1.5-fold longer protection than AS-AQ. Our simulations found that these differences in the duration of protection could alter population-level clinical incidence of malaria by up to 14% in under-5-year-old children when the drugs were used as first-line treatments in areas with high, seasonal transmission. CONCLUSION: Choosing a first-line treatment which provides optimal post-treatment prophylaxis given the local prevalence of resistance-associated markers could make a significant contribution to reducing malaria morbidity.

Relationship between weight status and anti-malarial drug efficacy and safety in children in Mali.

BACKGROUND: Anti-malarial treatments effectiveness remains a critical challenge for control programmes. However, when drug efficacy is established, the dose is calculated based on a predefined weight according to the patient age. Based on the hypothesis that the standard assumption of weight according to the age when administering the drug could lead to a therapeutic failure potentially due to under-dosing (in the case of overweight) or over-dosing (in case of underweight). In this study, the relationship between weight status and malaria drug efficacy in clearing current Plasmodium falciparum infection and preventing reinfection after treatment was investigated. METHODS: Data were drown from a clinical trial conducted previously to investigate malaria drug efficacy in 749 children from Mali (2002-2004). Participants were treated either with artesunate + amodiaquine (AS + AQ, n1 = 250), artesunate + sulfadoxine-pyrimethamine (AS + SP, n2 = 248) or artesunate (AS, n3 = 251) and followed for 28 days after treatment. The World Health Organization (WHO) z-score was used to define weight status. A Chi square test was used to compare outcomes according to drugs, weight status and the dynamic of ALAT, ASAT, creatinine and haemoglobin level. Logistic regression models were developed to determine the effect of baseline parameters (weight status, aspartate transaminase, alanine aminotransferase, creatinine and haemoglobin level) on drug efficacy as per WHO criteria. RESULTS: Without molecular correction, in AS + AQ arm, the rate of adequate clinical and parasitological response (ACPR) was higher in the group of underweight children 94.74% compared to children with normal and overweight (91.24% and 80.43% respectively, p = 0.03). After PCR correction, treatment efficacy was similar in the three groups of patients and was above 98% (p = 0.4). Overweight was observed to have no impact on recrudescence. However, it was associated with an increased risk of new infections in the (AS + AQ) arm (OR = 0.21, 95% CI [0.06; 0.86], p = 0.03). CONCLUSIONS: The findings suggest that weight deficiency has no deleterious effect on anti-malarial drug efficacy. An increase in the rate of reinfection in overweight children treated by AS + AQ should be further explored in larger studies.

Plasmodium falciparum Plasmepsin 2 Duplications, West Africa.

Dihydroartemisinin/piperaquine (DHA/PPQ) is increasingly deployed as antimalaria drug in Africa. We report the detection in Mali of Plasmodium falciparum infections carrying plasmepsin 2 duplications (associated with piperaquine resistance) in 7/65 recurrent infections within 2 months after DHA/PPQ treatment. These findings raise concerns about the long-term efficacy of DHA/PPQ treatment in Africa.

Genetic diversity of Plasmodium falciparum in human malaria cases in Mali.

BACKGROUND: In Mali, Plasmodium falciparum malaria is highly endemic and remains stable despite the implementation of various malaria control measures. Understanding P. falciparum population structure variations across the country could provide new insights to guide malaria control programmes. In this study, P. falciparum genetic diversity and population structure in regions of varying patterns of malaria transmission in Mali were analysed. METHODS: A total of 648 blood isolates adsorbed onto filter papers during population surveillance surveys (December 2012-March 2013, October 2013) in four distinct sites of Mali were screened for the presence of P. falciparum via quantitative PCR (qPCR). Multiple loci variable number of tandem repeats analysis (MLVA) using eight microsatellite markers was then performed on positive qPCR samples. Complete genotypes were then analysed for genetic diversity, genetic differentiation and linkage disequilibrium. RESULTS: Of 156 qPCR-positive samples, complete genotyping of 112 samples was achieved. The parasite populations displayed high genetic diversity (mean He = 0.77), which was consistent with a high level of malaria transmission in Mali. Genetic differentiation was low (FST < 0.02), even between sites located approximately 900 km apart, thereby illustrating marked gene flux amongst parasite populations. The lack of linkage disequilibrium further revealed an absence of local clonal expansion, which was corroborated by the genotype relationship results. In contrast to the stable genetic diversity level observed throughout the country, mean multiplicity of infection increased from north to south (from 1.4 to 2.06) and paralleled malaria transmission levels observed locally. CONCLUSIONS: In Mali, the high level of genetic diversity and the pronounced gene flux amongst P. falciparum populations may represent an obstacle to control malaria. Indeed, results suggest that parasite populations are polymorphic enough to adapt to their host and to counteract interventions, such as anti-malarial vaccination. Additionally, the panmictic parasite population structure imply that resistance traits may disseminate freely from one area to another, making control measures performed at a local level ineffective.

Variation in neutrophil levels and artemisinin-based combination therapy efficacy in West-Africa.

INTRODUCTION: Polymorphonuclear neutrophils (PMN) are involved in pathogen clearance by phagocytosis. However, the role of PMNs in the efficacy of artemisinin-based combination therapy (ACT) is poorly understood. METHODOLOGY: In a prospective longitudinal in vivo study, neutrophil rates were compared with malaria carriage after treatment with different ACTs: Artemether - lumefantrine (AL), Artesunate - amodiaquine (ASAQ), Dihydroartemisinin - piperaquine (DP) or Pyronaridine artesunate (PA). The study cases were classified as having neutropenia, normal neutrophil levels or neutrophilia depending on the level of neutrophils in the blood. This study included 3148 patients and was analyzed using R. RESULTS: On day 7, only four patients in the neutropenia group and treated with AL had a malaria positive blood smear based on microscopy. On day 28, the rate of recurrent parasitemia in the AL arm was significantly higher in neutropenia patients (50.9%) than in patients with normal rates of neutrophils (43.1%) or in those with neutrophilia (6.0%) (p < 0.001). In ASAQ arm, the rate of recurrent Plasmodium falciparum parasitemia was 58.8% in the neutropenia group versus 29.4% in patients with normal rates of neutrophils and 11.8% in patients with neutrophilia (p < 0.001). No patient treated with DP with normal neutrophil counts or neutrophilia was carrying malaria parasites on day 28. Among the 15 patients with parasitemia on day 28 in the PA arm, 11 (73.33%) had neutropenia while 4 (26.67%) had a normal neutrophil count (p < 0.001). CONCLUSIONS: Patients with neutropenia had higher rates of recurrent P. falciparum parasitemia after ACT.

Complexity of Plasmodium falciparum infection and genetic variations associated with differences in parasite clearance time in two Malian villages.

Background: Effective approaches to fight against malaria include disease prevention, an early diagnosis of malaria cases, and rapid management of confirmed cases by treatment with effective antimalarials. Artemisinin-based combination therapies are first-line treatments for uncomplicated malaria in endemic areas. However, cases of resistance to artemisinin have already been described in South-East Asia resulting in prolonged parasite clearance time after treatment. In Mali, though mutations in the K13 gene associated with delayed clearance in Asia are absent, a significant difference in parasite clearance time following treatment with artesunate was observed between two malaria endemic sites, Bougoula-Hameau and Faladje. Hypothetically, differences in complexity of Plasmodium falciparum infections may be accounted for this difference. Hence, the aims of this study were to assess the complexity of infection (COI) and genetic diversity of P. falciparum parasites during malaria treatment in Bougoula-Hameau and Faladje in Mali. Methods: Thirty (30) patients per village were randomly selected from 221 patients enrolled in a prospective artesunate monotherapy study conducted in Faladje and Bougoula-Hameau in 2016. All parasitemic blood samples of patients from enrollment to last positive slide were retained to assess malaria parasite COI and polymorphisms. DNA were extracted with a Qiagen kit and Pfcsp and Pfama1 encoding gene were amplified by nested PCR and sequenced using the Illumina platform. The parasite clearance time (PCT) was determined using the parasite clearance estimator of Worldwide Antimarial Resistance Network (WWARN). Data were analyzed with R®. Results: The median number of genetically distinct parasite clones was similar at enrollment, 7 (IQR of 5-9) in Faladje and 6 (IQR of 4-10) in Bougoula-Hameau (p-value = 0.1). On the first day after treatment initiation, the COI was higher in Faladje (6; CI:4-8) than in Bougoula-Hameau (4; CI:4-6) with a p-value =0. 02. Overall, COI was high with higher PCT. Finally, there was a low genetic diversity between Faladje and Bougoula-Hameau. Conclusion: This study demonstrated that the difference in PCT observed between the two villages could be due to differences in the complexity of infection of these two villages.

Ganaplacide (KAF156) plus lumefantrine solid dispersion formulation combination for uncomplicated Plasmodium falciparum malaria: an open-label, multicentre, parallel-group, randomised, controlled, phase 2 trial.

BACKGROUND: Emergence of drug resistance demands novel antimalarial drugs with new mechanisms of action. We aimed to identify effective and well tolerated doses of ganaplacide plus lumefantrine solid dispersion formulation (SDF) in patients with uncomplicated Plasmodium falciparum malaria. METHODS: This open-label, multicentre, parallel-group, randomised, controlled, phase 2 trial was conducted at 13 research clinics and general hospitals in ten African and Asian countries. Patients had microscopically-confirmed uncomplicated P falciparum malaria (>1000 and <150 000 parasites per µL). Part A identified the optimal dose regimens in adults and adolescents (aged ≥12 years) and in part B, the selected doses were assessed in children (≥2 years and <12 years). In part A, patients were randomly assigned to one of seven groups (once a day ganaplacide 400 mg plus lumefantrine-SDF 960 mg for 1, 2, or 3 days; ganaplacide 800 mg plus lumefantrine-SDF 960 mg as a single dose; once a day ganaplacide 200 mg plus lumefantrine-SDF 480 mg for 3 days; once a day ganaplacide 400 mg plus lumefantrine-SDF 480 mg for 3 days; or twice a day artemether plus lumefantrine for 3 days [control]), with stratification by country (2:2:2:2:2:2:1) using randomisation blocks of 13. In part B, patients were randomly assigned to one of four groups (once a day ganaplacide 400 mg plus lumefantrine-SDF 960 mg for 1, 2, or 3 days, or twice a day artemether plus lumefantrine for 3 days) with stratification by country and age (2 to <6 years and 6 to <12 years; 2:2:2:1) using randomisation blocks of seven. The primary efficacy endpoint was PCR-corrected adequate clinical and parasitological response at day 29, analysed in the per protocol set. The null hypothesis was that the response was 80% or lower, rejected when the lower limit of two-sided 95% CI was higher than 80%. This study is registered with EudraCT (2020-003284-25) and ClinicalTrials.gov (NCT03167242). FINDINGS: Between Aug 2, 2017, and May 17, 2021, 1220 patients were screened and of those, 12 were included in the run-in cohort, 337 in part A, and 175 in part B. In part A, 337 adult or adolescent patients were randomly assigned, 326 completed the study, and 305 were included in the per protocol set. The lower limit of the 95% CI for PCR-corrected adequate clinical and parasitological response on day 29 was more than 80% for all treatment regimens in part A (46 of 50 patients [92%, 95% CI 81-98] with 1 day, 47 of 48 [98%, 89-100] with 2 days, and 42 of 43 [98%, 88-100] with 3 days of ganaplacide 400 mg plus lumefantrine-SDF 960 mg; 45 of 48 [94%, 83-99] with ganaplacide 800 mg plus lumefantrine-SDF 960 mg for 1 day; 47 of 47 [100%, 93-100] with ganaplacide 200 mg plus lumefantrine-SDF 480 mg for 3 days; 44 of 44 [100%, 92-100] with ganaplacide 400 mg plus lumefantrine-SDF 480 mg for 3 days; and 25 of 25 [100%, 86-100] with artemether plus lumefantrine). In part B, 351 children were screened, 175 randomly assigned (ganaplacide 400 mg plus lumefantrine-SDF 960 mg once a day for 1, 2, or 3 days), and 171 completed the study. Only the 3-day regimen met the prespecified primary endpoint in paediatric patients (38 of 40 patients [95%, 95% CI 83-99] vs 21 of 22 [96%, 77-100] with artemether plus lumefantrine). The most common adverse events were headache (in seven [14%] of 51 to 15 [28%] of 54 in the ganaplacide plus lumefantrine-SDF groups and five [19%] of 27 in the artemether plus lumefantrine group) in part A, and malaria (in 12 [27%] of 45 to 23 [44%] of 52 in the ganaplacide plus lumefantrine-SDF groups and 12 [50%] of 24 in the artemether plus lumefantrine group) in part B. No patients died during the study. INTERPRETATION: Ganaplacide plus lumefantrine-SDF was effective and well tolerated in patients, especially adults and adolescents, with uncomplicated P falciparum malaria. Ganaplacide 400 mg plus lumefantrine-SDF 960 mg once daily for 3 days was identified as the optimal treatment regimen for adults, adolescents, and children. This combination is being evaluated further in a phase 2 trial (NCT04546633). FUNDING: Novartis and Medicines for Malaria Venture.

Differential Incidence of Malaria in Neighboring Villages in a High-Transmission Setting of Southern Mali.

Throughout a phase IIIb/IV efficacy study of repeated treatment with four artemisinin-based combination therapies, significant heterogeneity was found in the number of clinical episodes experienced by individuals during the 2-year follow-up. Several factors, including host, parasite, and environmental factors, may contribute to the differential malaria incidence. We aimed to identify risk factors of malaria incidence in the context of a longitudinal study of the efficacy of different artemisinin-based combination therapy regimens in Bougoula-Hameau, a high-transmission setting in Mali. Risk factors including age, residence, and treatment regimen were compared among individuals experiencing eight or more clinical episodes of malaria ("high-incidence group") and individuals experiencing up to three clinical episodes ("low-incidence group"). Consistent with the known association between age and malaria risk in high-transmission settings, individuals in the high incidence group were significantly younger than individuals in the low-risk group (mean age, 7.0 years versus 10.6 years, respectively; t-test, P < 0.0001). Compared with individuals receiving artemether-lumefantrine, those receiving artesunate-amodiaquine had greater odds of being in the high-incidence group (odds ratio [OR], 2.24; 95% CI, 1.03 - 4.83, P = 0.041), while individuals receiving dihydroartemisinin-piperaquine had a lower odds of being in high incidence group (OR: 0.30, 95% CI, 0.11-0.85; P = 0.024). Individuals residing in the forested areas of Sokourani and Karamogobougou had significantly greater odds of being in the high-incidence group compared with individuals residing in the semi-urban area of Bougoula-Hameau 1 (Karamogobougou: OR, 3.68; 95% CI, 1.46-9.31; P = 0.0059; Sokourani: OR, 11.46; 95% CI, 4.49-29.2; P < 0.0001). This study highlights the importance of fine-mapping malaria risks even at sub-district levels for targeted and customized interventions.

Different Plasmodium falciparum clearance times in two Malian villages following artesunate monotherapy.

BACKGROUND: Artemisinin resistance described as increased parasite clearance time (PCT) is rare in Africa. More sensitive methods such as qPCR might better characterize the clearance phenotype in sub-Saharan Africa. METHODS: PCT is explored in Mali using light microscopy and qPCR after artesunate for uncomplicated malaria. In two villages, patients were followed for 28 days. Blood smears and spots were collected respectively for microscopy and qPCR. Parasitemia slope half-life was calculated after microscopy. Patient residual parasitemia were measured by qPCR. RESULTS: Uncorrected adequate clinical and parasitological responses (ACPR) observed in Faladje and Bougoula-Hameau were 78% and 92%, respectively (p=0.01). This reached 100% for both after molecular correction. Proportions of 24H microscopy positive patients in Faladje and Bougoula-Hameau were 97.2% and 72%, respectively (p<0.0001). Slope half-life was 2.8h in Faladje vs 2H in Bougoula-Hameau (p<0.001) and Proportions of 72H patients with residual parasitemia were 68.5% and 40% in Faladje and Bougoula-Hameau, respectively (p=0.003). The mean residual parasitemia was 2.9 in Faladje vs. 0.008 in Bougoula-Hameau (p=0.002). Although artesunate is efficacious in Mali, the longer parasite clearance time with submicroscopic parasitemia observed may represent early signs of developing P. falciparum resistance to artemisinins.

Efficacy of chloroquine, amodiaquine and sulphadoxine-pyrimethamine for the treatment of uncomplicated falciparum malaria: revisiting molecular markers in an area of emerging AQ and SP resistance in Mali.

BACKGROUND: To update the National Malaria Control Programme of Mali on the efficacy of chloroquine, amodiaquine and sulphadoxine-pyrimethamine in the treatment of uncomplicated falciparum malaria. METHODS: During the malaria transmission seasons of 2002 and 2003, 455 children--between six and 59 months of age, with uncomplicated malaria in Kolle, Mali, were randomly assigned to one of three treatment arms. In vivo outcomes were assessed using WHO standard protocols. Genotyping of msp1, msp2 and CA1 polymorphisms were used to distinguish reinfection from recrudescent parasites (molecular correction). RESULTS: Day 28 adequate clinical and parasitological responses (ACPR) were 14.1%, 62.3% and 88.9% in 2002 and 18.2%, 60% and 85.2% in 2003 for chloroquine, amodiaquine and sulphadoxine-pyrimethamine, respectively. After molecular correction, ACPRs (cACPR) were 63.2%, 88.5% and 98.0% in 2002 and 75.5%, 85.2% and 96.6% in 2003 for CQ, AQ and SP, respectively. Amodiaquine was the most effective on fever. Amodiaquine therapy selected molecular markers for chloroquine resistance, while in the sulphadoxine-pyrimethamine arm the level of dhfr triple mutant and dhfr/dhps quadruple mutant increased from 31.5% and 3.8% in 2002 to 42.9% and 8.9% in 2003, respectively. No infection with dhps 540E was found. CONCLUSION: In this study, treatment with sulphadoxine-pyrimethamine emerged as the most efficacious on uncomplicated falciparum malaria followed by amodiaquine. The study demonstrated that sulphadoxine-pyrimethamine and amodiaquine were appropriate partner drugs that could be associated with artemisinin derivatives in an artemisinin-based combination therapy.

Baseline in vitro efficacy of ACT component drugs on Plasmodium falciparum clinical isolates from Mali.

In vitro susceptibility to antimalarial drugs of Malian Plasmodium falciparum isolates collected between 2004 and 2006 was studied. Susceptibility to chloroquine and to three artemisinin-based combination therapy (ACT) component drugs was assessed as a first, to our knowledge, in vitro susceptibility study in Mali. Overall 96 Malian isolates (51 from around Bamako and 45 collected from French travellers returning from Mali) were cultivated in a CO(2) incubator. Fifty percent inhibitory concentrations (IC(50)s) were measured by either hypoxanthine incorporation or Plasmodium lactate dehydrogenase (pLDH) ELISA. Although the two sets of data were generated with different methods, the global IC(50) distributions showed parallel trends. A good concordance of resistance phenotype with pfcrt 76T mutant genotype was found within the sets of clinical isolates tested. We confirm a high prevalence of P. falciparum in vitro resistance to chloroquine in Mali (60-69%). While some isolates showed IC(50)s close to the cut-off for resistance to monodesethylamodiaquine, no decreased susceptibility to dihydroartemisinin or lumefantrine was detected. This study provides baseline data for P. falciparum in vitro susceptibility to ACT component drugs in Mali.

Efficacy, safety, and selection of molecular markers of drug resistance by two ACTs in Mali.

We conducted a randomized single-blinded trial comparing the efficacy and safety of artesunate (AS) + amodiaquine (AQ, 3 days) versus AS (3 days) + sulfadoxine-pyrimethamine (SP, single dose) versus AS monotherapy (5 days) in Southern Mali. Uncomplicated malaria cases were followed for 28 days. Molecular markers of drug resistance were determined. After identification of recrudescences by genotyping, both artemisinin-based combination therapies (ACTs) reached nearly 100% efficacy at Day 14 and Day 28 versus 98.3% and 96.5% for AS, respectively (P > 0.05). AS + SP significantly selected DHFR and DHPS mutations associated with sulfadoxine and pyrimethamine resistance (P < 0.001), and AS + AQ equally selected PfCRT and PfMDR1 point mutations associated with chloroquine and AQ resistance (P < 0.001). No significant adverse event attributable to any of the study drugs was found. The ACTs were efficacious and safe, but the selection of markers for resistance to the partner drugs raises concerns over their lifespan in areas of intense malaria transmission.

Differential infectivity of gametocytes after artemisinin-based combination therapy of uncomplicated falciparum malaria.

BACKGROUND: Most malaria-endemic countries use artemisinin-based combination therapy (ACT) as their first-line treatment. ACTs are known to be highly effective on asexual stages of the malaria parasite. Malaria transmission and the spread of resistant parasites depend on the infectivity of gametocytes. The effect of the current ACT regimens on gametocyte infectivity is unclear. OBJECTIVES: This study aimed to determine the infectivity of gametocytes to Anopheles gambiae following ACT treatment in the field. METHODS: During a randomised controlled trial in Bougoula-Hameau, Mali, conducted from July 2005 to July 2007, volunteers with uncomplicated malaria were randomised to receive artemether-lumefantrine, artesunate-amodiaquine, or artesunate-sulfadoxine/pyrimethamine. Volunteers were followed for 28 days, and gametocyte carriage was assessed. Direct skin feeding assays were performed on gametocyte carriers before and after ACT administration. RESULTS: Following artemether-lumefantrine treatment, gametocyte carriage decreased steadily from Day 0 to Day 21 post-treatment initiation. In contrast, for the artesunate-amodiaquine and artesunate-sulfadoxine/pyrimethamine arms, gametocyte carriage increased on Day 3 and remained constant until Day 7 before decreasing afterward. Mosquito feeding assays showed that artemether-lumefantrine and artesunate-amodiaquine significantly increased gametocyte infectivity to Anopheles gambiae sensu lato (s.l.) (p < 10-4), whereas artesunate-sulfadoxine/pyrimethamine decreased gametocyte infectivity in this setting (p = 0.03). CONCLUSION: Different ACT regimens could lead to gametocyte populations with different capacity to infect the Anopheles vector. Frequent assessment of the effect of antimalarials on gametocytogenesis and gametocyte infectivity may be required for the full assessment of treatment efficacy, the potential for spread of drug resistance and malaria transmission in the field.