BackgroundArtemisinin-based combination therapy is the recommended first-line treatment for uncomplicated falciparum malaria worldwide. However, recent studies conducted in Mali showed an increased frequency of recurrent parasitaemia following artemether–lumefantrine (AL) treatment.MethodsStudy samples were collected during a large WANECAM study. Ex-vivo Plasmodium falciparum sensitivity to artemether and lumefantrine was assessed using the tritiated hypoxanthine-based assay. The prevalence of molecular markers of anti-malarial drug resistance (pfcrt K76T, pfmdr1 N86Y and K13-propeller) were measured by PCR and/or sequencing.ResultsOverall 61 samples were successfully analysed in ex vivo studies. Mean IC50s increased significantly between baseline and recurrent parasites for both artemether (1.6 nM vs 3.2 nM, p < 0.001) and lumefantrine (1.4 nM vs 3.4 nM, p = 0.004). Wild type Pfmdr1 N86 allele was selected after treatment (71 vs 91%, 112 of 158 vs 95 of 105, p < 0.001) but not the wild type pfcrt K76 variant (23.5 vs 24.8%, 40 of 170 vs 26 of 105, p = 0.9). Three non-synonymous K13-propeller SNPs (A522C, A578S, and G638R) were found with allele frequencies <2%.ConclusionMalian post-AL P. falciparum isolates were less susceptible to artemether and lumefantrine than baseline isolates.

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 2.8 h 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.

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.

Background Several mutations in the PF3D7_1343700 kelch propeller (K13-propeller) were recently described as associated with artemisinin resistance in vivo and in vitro in Southeast Asia. In Mali, a preliminary study on artesunate efficacy in 2011 found no delay in parasite clearance. A larger study including two sites in Mali is conducted here in the context of regular monitoring of artemisinin resistance. Methods From October 2015 to March 2016, we conducted a prospective study on artesunate monotherapy in Bougoula-Hameau and Faladje on uncomplicated malaria patients aged more than 6 months. Patients were treated for 7 days and followed up for 28 days. Blood smear was performed for parasite evaluation every 8 hours until three consecutive slides were negatives. MSP2, Ca1 and TA99 polymorphisms were used to distinguish new infections from recurrent parasites. The PfK13 mutations were genotyped using direct sequencing of PCR amplicons from dried blood spots of pre and posttreatment falciparum parasites. The results were compared with the studies conducted in a same area on 2011. Results A total of 100 and 120 patients were enrolled in Bougoula-Hameau and Faladje, respectively. The uncorrected adequate clinical and parasitological responses (ACPR) were 92.0% in Bougoula-Hameau and 78.3% in Faladje. After molecular correction, we obtained 100% cACPR in both sites. The prevalence of the non-synonymous single nucleotide polymorphisms (SNPs) K13 was 2% in Bougoula (found only at enrolment) but null in Faladje. However SNPs were 3% and 7% in Bougoula-Hameau and Faladje, respectively. Conclusions Artesunate monotherapy remains effective on P. falciparum in Mali and there are only low levels of PfK13 mutations.

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.