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.

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.

Ex Vivo Plasmodium malariae Culture Method for Antimalarial Drugs Screen in the Field.

In vitro and ex vivo cultivation of Plasmodium (P) falciparum has facilitated active research into the malaria parasite toward the quest for basic knowledge and the discovery of effective drug treatments. Such a drug discovery program is currently difficult for P. malariae simply because of the absence of in vitro and ex vivo cultivation system for its asexual blood stages supporting antimalarial evaluation. Despite availability of artemisinin combination therapies effective on P. falciparum, P. malariae is being increasingly detected in malaria endemic countries. P. malariae is responsible for chronic infections and is associated with a high burden of anemia and morbidity. Here, we optimized and adapted ex vivo conditions under which P. malariae can be cultured and used for screening antimalarial drugs. Subsequently, this enabled us to test compounds such as artemether, chloroquine, lumefantrine, and quinine for ex vivo antimalarial activity against P. malariae.

SELECTION OF PFCRT 76T AND PFMDR1 86Y MUTANT PLASMODIUM FALCIPARUM AFTER TREATMENT OF UNCOMPLICATED MALARIA WITH ARTESUNATE-AMODIAQUINE IN REPUBLIC OF GUINEA.

The use of Amodiaquine monotherapy is associated with the selection of molecular markers of Plasmodium falciparum resistance to chloroquine (pfcrt and pfmdr1). The decrease in sensitivity and the emergence of P. falciparum resistant to artemisinin-based combination therapy have been reported. Therefore, it is important to assess the impact of treatment of uncomplicated malaria with Artesunate-Amodiaquine (AS+AQ) on molecular markers of antimalarial resistance. We used standard World Health Organization (WHO) protocols to determine the in vivo efficacy of the combination (AS+AQ). In total, 170 subjects were included in the study. The molecular analysis focused on 168 dried blood spots. The aims were to determine the frequency of pfcrt 76T and pfmdr1 86Y mutations and the rates of reinfection using polymorphism markers msp1, msp2, and microsatellite markers (CA1, Ta87, TA99). Nested-PCR was used, followed in some cases by a restriction digestion. The level of P. falciparum clinical response was 92.9% (156/168) of Adequate Clinical and Parasitological Response (ACPR) before molecular correction and 97.0% (163/168) after molecular correction (P = 0.089). The frequency of mutation point pfcrt 76T was 76.2% (128/168) before treatment and 100% (7/7) after treatment (P = 0.1423). For the pfmdr1 mutation, the frequency was 28% (47/168) before treatment and 60% (6/10) after treatment (P = 0.1124). The rate of pfcrt 76T + pfmdr1 86Y was 22% (37/168) before and 50% (6/12) after treatment (P = 0.1465). Despite the presence of AS in the combination, AS+AQ selects for pfcrt 76T and pfmdr1 86Y mutant P. falciparum in Guinea.

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.

Plasmodium malariae and Plasmodium falciparum comparative susceptibility to antimalarial drugs in Mali.

OBJECTIVES: To evaluate Plasmodium malariae susceptibility to current and lead candidate antimalarial drugs. METHODS: We conducted cross-sectional screening and detection of all Plasmodium species malaria cases, which were nested within a longitudinal prospective study, and an ex vivo assessment of efficacy of a panel of antimalarials against P. malariae and Plasmodium falciparum, both PCR-confirmed mono-infections. Reference compounds tested included chloroquine, lumefantrine, artemether and piperaquine, while candidate antimalarials included the imidazolopiperazine GNF179, a close analogue of KAF156, and the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. RESULTS: We report a high frequency (3%-15%) of P. malariae infections with a significant reduction in ex vivo susceptibility to chloroquine, lumefantrine and artemether, which are the current frontline drugs against P. malariae infections. Unlike these compounds, potent inhibition of P. malariae and P. falciparum was observed with piperaquine exposure. Furthermore, we evaluated advanced lead antimalarial compounds. In this regard, we identified strong inhibition of P. malariae using GNF179, a close analogue of KAF156 imidazolopiperazines, which is a novel class of antimalarial drug currently in clinical Phase IIb testing. Finally, in addition to GNF179, we demonstrated that the Plasmodium PI4K-specific inhibitor KDU691 is highly inhibitory against P. malariae and P. falciparum. CONCLUSIONS: Our data indicated that chloroquine, lumefantrine and artemether may not be suitable for the treatment of P. malariae infections and the potential of piperaquine, as well as new antimalarials imidazolopiperazines and PI4K-specific inhibitor, for P. malariae cure.

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.

Identifying Recrudescent Plasmodium falciparum in Treated Malaria Patients by Real-time PCR and High Resolution Melt Analysis of Genetic Diversity.

Recurrent parasitaemia during follow up of clinical trials of antimalarial drug efficacy results from either recrudescence of parasites surviving treatment or from parasites newly emerging from the hepatic stage of infection. Nested PCR is used to distinguish these two possibilities and the technique is difficult to standardise. There is risk of both false positive and false negative results, leading to misclassification errors. The high-resolution melt (HRM) assay was developed with pairs of conserved primers targeting blocks of merozoite surface protein 1 and 2 (msp1 and msp2) genes, and polymorphisms were compared using sequence-confirmed Plasmodium falciparum DNA samples from laboratory isolates. In this study, the HRM dissociation profiles of msp1 and msp2 amplicons were determined and validated against parasite isolates from malaria patients. The msp1 and msp2 profiles of both laboratory and clinical isolates were reproducibly differentiated by HRM. These rapid assays are performed in a closed-tube system, and so avoid cross-contamination while increasing throughput, which are two major advantages. The HRM assays offer significant gains in simplicity, speed and interpretation of results, and reduced analysis cost, for studies that require discrimination of parasite clones. Assay performance in large-scale studies utilizing DNA samples derived from filter-paper bloodspots should now be evaluated.

Burden of malaria in mobile populations in the Greater Accra region, Ghana: a cross- sectional study.

BACKGROUND: The burden of malaria in mobile populations remains poorly documented in sub-Saharan Africa. This study determined the prevalence of malaria among hawkers and long-distance truck drivers in the Greater Accra region of Ghana. METHODS: A cross-sectional design using consecutive sampling method between June and July 2016 in Accra and Tema in Ghana was used in this study. The study population was hawkers who roam and sleep in the Market Streets, and long-distance truck drivers. Participants completed closed ended interview questionnaires on socio-demographic characteristics, primary residence and knowledge about malaria. Rapid diagnostic test and thick blood smears of each participant were stained with Giemsa and read using microscopy. Geographical position system (GPS) was used to collect the station locations of these mobile populations. RESULT: The overall prevalence of malaria was 15.1% and Plasmodium falciparum was responsible for all malaria infection. The malaria prevalence was 18.9 and 10.9% respectively among hawkers and truck drivers (p < 0.05). The hawkers, the single and the no formal educated participants were more likely to get malaria than the long-distance truck drivers (OR = 1.91, 95% CI 1.07-3.42), the married (OR = 1.94 95% CI 1.11-3.40) and the educated participants (OR = 2.56 95% CI 1.10-5.93), respectively. After controlling for other variables, marital status (OR = 2.60 95% CI 1.43- 4.73) and educational level (OR = 2.70 95% CI 1.08-6.77) were statistically significantly associated with malaria. CONCLUSION: This study demonstrated that the prevalence of malaria is high among hawkers and long distance truck drivers. Sociodemographic characteristics, such as marital status, occupation and educational level are significantly associated with malaria. The station locations as determined by GPS technology will make these mobile populations easier to reach for any malaria intervention.

Seasonal Malaria Chemoprevention with Sulphadoxine-Pyrimethamine and Amodiaquine Selects Pfdhfr-dhps Quintuple Mutant Genotype in Mali.

BACKGROUND: Seasonal malaria chemoprevention (SMC) with sulphadoxine-pyrimethamine (SP) plus amodiaquine (AQ) is being scaled up in Sahelian countries of West Africa. However, the potential development of Plasmodium falciparum resistance to the respective component drugs is a major concern. METHODS: Two cross-sectional surveys were conducted before (August 2012) and after (June 2014) a pilot implementation of SMC in Koutiala, Mali. Children aged 3-59 months received 7 rounds of curative doses of SP plus AQ over two malaria seasons. Genotypes of P. falciparum Pfdhfr codons 51, 59 and 108; Pfdhps codons 437 and 540, Pfcrt codon 76 and Pfmdr1codon 86 were analyzed by PCR on DNA from samples collected before and after SMC, and in non-SMC patient population as controls (November 2014). RESULTS: In the SMC population 191/662 (28.9%) and 85/670 (12.7%) of children were P. falciparum positive by microscopy and were included in the molecular analysis before (2012) and after SMC implementation (2014), respectively. In the non-SMC patient population 220/310 (71%) were successfully PCR analyzed. In the SMC children, the prevalence of all molecular markers of SP resistance increased significantly after SMC including the Pfdhfr-dhps quintuple mutant genotype, which was 1.6% before but 7.1% after SMC (p = 0.02). The prevalence of Pfmdr1-86Y significantly decreased from 26.7% to 15.3% (p = 0.04) while no significant change was seen for Pfcrt 76T. In 2014, prevalence of all molecular markers of SP resistance were significantly higher among SMC children compared to the non-SMC population patient (p < 0.01). No Pfdhfr-164 mutation was found neither at baseline nor post SMC. CONCLUSION: SMC increased the prevalence of molecular markers of P. falciparum resistance to SP in the treated children. However, there was no significant increase of these markers of resistance in the general parasite population after 2 years and 7 rounds of SMC.

Polymorphisms in the K13-propeller gene in artemisinin-susceptible Plasmodium falciparum parasites from Bougoula-Hameau and Bandiagara, Mali.

Artemisinin-resistant Plasmodium falciparum malaria has been documented in southeast Asia and may already be spreading in that region. Molecular markers are important tools for monitoring the spread of antimalarial drug resistance. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain were shown to be associated with artemisinin resistance in vivo and in vitro. The prevalence and role of K13-propeller mutations are poorly known in sub-Saharan Africa. K13-propeller mutations were genotyped by direct sequencing of nested polymerase chain reaction (PCR) amplicons from dried blood spots of pre-treatment falciparum malaria infections collected before and after the use of artemisinin-based combination therapy (ACT) as first-line therapy in Mali. Although K13-propeller mutations previously associated with delayed parasite clearance in Cambodia were not identified, 26 K13-propeller mutations were identified in both recent samples and pre-ACT infections. Parasite clearance time was comparable between infections with non-synonymous K13-propeller mutations and infections with the reference allele. These findings suggest that K13-propeller mutations are present in artemisinin-sensitive parasites and that they preceded the wide use of ACTs in Mali.

Efficacy of sulphadoxine-pyrimethamine + artesunate, sulphadoxine-pyrimethamine + amodiaquine, and sulphadoxine-pyrimethamine alone in uncomplicated falciparum malaria in Mali.

BACKGROUND: Plasmodium falciparum resistance to artemisinin has been reported in South-East Asia. Long half-life drugs are increasingly being used for malaria prevention. The potential spread of parasite resistance to these regimens is real and makes regular efficacy surveillance a priority. METHODS: From August to December 2004 and July to December 2005, a randomized open label trial of sulphadoxine-pyrimethamine (SP) + artesunate (AS) versus SP + amodiaquine (AQ), and SP alone, was conducted in two villages of Mali. PCR was used to distinguish new infections from recrudescent P. falciparum infections. Patients were followed for 28 days to assess treatment efficacy. RESULTS: Overall 912 children aged between six to 59 months, with uncomplicated P. falciparum malaria were recruited. Baseline characteristics were similar in the three treatment arms. Crude ACPRs were 94.9%; 98.6% and 93.5% for SP + AS; SP + AQ and SP alone arms respectively (SP + AS versus SP + AQ, p = 0.01; SP + AS versus SP, p = 0.5; SP + AQ versus SP, p = 0.001). After PCR adjustment, cACPRs were 99%; 100% and 97.2% for SP + AS; SP + AQ and SP alone arms, respectively (SP + AS versus SP + AQ, p = 0.25; SP + AS versus SP, p = 0.12; SP + AQ versus SP, p = 0.007). CONCLUSION: Sulphadoxine-pyrimethamine + amodiaquine therapy was as efficacious as sulphadoxine-pyrimethamine + artesunate, but more efficacious than sulphadoxine-pyrimethamine alone in the treatment of uncomplicated P. falciparum malaria in Mali.

Quinine treatment selects the pfnhe-1 ms4760-1 polymorphism in Malian patients with Falciparum malaria.

BACKGROUND: The mechanism of Plasmodium falciparum resistance to quinine is not known. In vitro quantitative trait loci mapping suggests involvement of a predicted P. falciparum sodium-hydrogen exchanger (pfnhe-1) on chromosome 13. METHODS: We conducted prospective quinine efficacy studies in 2 villages, Kollé and Faladié, Mali. Cases of clinical malaria requiring intravenous therapy were treated with standard doses of quinine and followed for 28 days. Treatment outcomes were classified using modified World Health Organization protocols. Molecular markers of parasite polymorphisms were used to distinguish recrudescent parasites from new infections. The prevalence of pfnhe-1 ms4760-1 among parasites before versus after quinine treatment was determined by direct sequencing. RESULTS: Overall, 163 patients were enrolled and successfully followed. Without molecular correction, the mean adequate clinical and parasitological response (ACPR) was 50.3% (n = 163). After polymerase chain reaction correction to account for new infections, the corrected ACPR was 100%. The prevalence of ms4760-1 increased significantly, from 26.2% (n = 107) before quinine treatment to 46.3% (n = 54) after therapy (P = .01). In a control sulfadoxine-pyrimethamine study, the prevalence of ms4760-1 was similar before and after treatment. CONCLUSIONS: This study supports a role for pfnhe-1 in decreased susceptibility of P. falciparum to quinine in the field.

Toxoplasma gondii seroprevalence in Mali.

The protozoan parasite Toxoplasma gondii is globally distributed, with considerable local variation in prevalence based on behavioral and environmental factors. To assess prevalence and estimate risk in Mali, we conducted a survey of 760 serum samples previously collected for malaria studies. A modified agglutination test detected antibodies in ∼27% of the adult population, with no significant differences between men and women, or between urban and rural study sites. In the village of Kolle, seroprevalence rose from 0% in infants (<1 yr, but after weaning of maternal immunoglobulin G) to 0.8% (1-5 yr), 2.7% (6-10), 11.3% (11-15), and 26.8% (>15); differences between the <10-, 11-15-, and >15-yr age groups were highly significant (P ≤ 0.01). We also observed an increase in anti- T. gondii antibody titers with age. Modeling the observed age distribution suggests a seroconversion rate of ∼1%/yr, indicating that congenital toxoplasmosis may be an under-appreciated public health concern in Mali.