Insight into k13-propeller gene polymorphism and ex vivo DHA-response profiles from Cameroonian isolates.

BACKGROUND: The spread of Plasmodium falciparum resistance to artemisinin derivatives in Southeast Asia is a major source of concern and the emergence of resistance in Africa would have dramatic consequences, by increasing malaria mortality and morbidity. It is therefore urgent to implement regular monitoring in sentinel sites in sub-Saharan Africa using robust and easy-to-implement tools. The prevalence of k13-propeller mutations and the phenotypic profiles are poorly known in sub-Saharan Africa. Here, the k13-propeller polymorphism was compared to both ex vivo susceptibility to DHA and early parasitological and clinical responses to artemisinin combination therapy (ACT). METHODS: Plasmodium falciparum isolates were collected in 2015 in Yaoundé (Cameroon) from patients treated with dihydroartemisinin-piperaquine combination. Samples were analysed for their susceptibility to artemisinin using the k13-propeller sequencing, the ex vivo ring-stage survival assay, the in vivo parasite positive rate and the clinical statute at day 2. RESULTS: None of the collected isolates revealed the presence of resistance mutations in the k13-propeller sequence. The median ring-stage survival rate for all the 64 interpretable isolates after a 6-hour pulse of 700 nM dihydroartemisinin was low, 0.49% (IQR: 0-1.3). Total parasite clearance was observed for 87.5% of patients and the remaining parasitaemic isolates (12.5%) showed a high reduction of parasite load, ranging from 97.5 to 99.9%. Clinical symptoms disappeared in 92.8% of cases. CONCLUSION: This study demonstrated the absence of k13-resistant genotypes in P. falciparum isolates from Cameroon. Only synonymous mutations were found with a low prevalence (4.3%). A good association between k13 genotypes and the ex vivo ring-stage survival assay or parasitological and clinical data was obtained. These results give a baseline for the long-term monitoring of artemisinin derivative efficacy in Africa.

Antimalarial drug resistance in the Central and Adamawa regions of Cameroon: Prevalence of mutations in P. falciparum crt, Pfmdr1, Pfdhfr and Pfdhps genes.

The spread of Plasmodium falciparum resistant parasites remains one of the major challenges for malaria control and elimination in Sub Saharan Africa. Monitoring of molecular markers conferring resistance to different antimalarials is important to track the spread of resistant parasites and to optimize the therapeutic lifespan of current drugs. This study aimed to evaluate the prevalence of known mutations in the drug resistance genes Pfcrt, Pfmdr1, Pfdhfr and Pfdhps in two different epidemiological settings in Cameroon. Dried blood spots collected in 2018 and 2019 from asymptomatic individuals were used for DNA extraction and then the Plasmodium infection status was determined byPCR. Detection of SNPs was performed by nested PCR followed by allele-specific restriction analysis (ASRA). The prevalence of each genotype was compared between sites using the Chi square and Fisher's exact tests. A high prevalence of the Pfcrt K76 wild type allele was found in both sites (88.5 and 62.29% respectively; P< 0,0001). The prevalence of Pfmdr1 mutations 86Y and 1246Y was respectively 55.83 and 1.45% in Mfou and 45.87 and 5.97% in Tibati, with significant difference between the studied areas (P<0.0001). Overall, the Pfdhfr triple-mutant genotype (51I/59R/108N) was highly prevalent (> 96%), however no SNP was detected at codon 164. In Pfdhps, the prevalence of the 437G mutation reached (90%) and was at higher frequency in Mfou (P< 0.0001). Overall, the Pfdhps mutations 540E and 581G were less common (0.33 and 3.26%, respectively). The quadruple resistant genotype (Pfdhfr 51I/59R/108N+Pfdhp437G) was found almost 90% of the samples. The wild-type genotype (Pfdhfr N51/C59/S108/164I+Pfdhps A437/K540/A581) was never identified and the sextuple mutant (Pfdhfr 51I/59R/108N+Pfdhp437G/540E/581G), kwon as super resistant appeared in two samples from Tibati. These findings demonstrate declining trends in the prevalence of mutations conferring resistance to 4-aminoquinolines, especially to chloroquine. However, a high level of mutations in P. falciparum genes related to SP resistance was detected and this raises concerns about the future efficacy of IPTp-SP and SMC in Cameroon.