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.

Evaluation of a real-time quantitative PCR to measure the wild Plasmodium falciparum infectivity rate in salivary glands of Anopheles gambiae.

BACKGROUND: Evaluation of malaria sporozoite rates in the salivary glands of Anopheles gambiae is essential for estimating the number of infective mosquitoes, and consequently, the entomological inoculation rate (EIR). EIR is a key indicator for evaluating the risk of malaria transmission. Although the enzyme-linked immunosorbent assay specific for detecting the circumsporozoite protein (CSP-ELISA) is routinely used in the field, it presents several limitations. A multiplex PCR can also be used to detect the four species of Plasmodium in salivary glands. The aim of this study was to evaluate the efficacy of a real-time quantitative PCR in detecting and quantifying wild Plasmodium falciparum in the salivary glands of An. gambiae. METHODS: Anopheles gambiae (n=364) were experimentally infected with blood from P. falciparum gametocyte carriers, and P. falciparum in the sporozoite stage were detected in salivary glands by using a real-time quantitative PCR (qPCR) assay. The sensitivity and specificity of this qPCR were compared with the multiplex PCR applied from the Padley method. CSP-ELISA was also performed on carcasses of the same mosquitoes. RESULTS: The prevalence of P. falciparum and the intensity of infection were evaluated using qPCR. This method had a limit of detection of six sporozoites per µL based on standard curves. The number of P. falciparum genomes in the salivary gland samples reached 9,262 parasites/µL (mean: 254.5; 95% CI: 163.5-345.6). The qPCR showed a similar sensitivity (100%) and a high specificity (60%) compared to the multiplex PCR. The agreement between the two methods was "substantial" (κ = 0.63, P <0.05). The number of P. falciparum-positive mosquitoes evaluated with the qPCR (76%), multiplex PCR (59%), and CSP-ELISA (83%) was significantly different (P <0.005). CONCLUSIONS: The qPCR assay can be used to detect P. falciparum in salivary glands of An. gambiae. The qPCR is highly sensitive and is more specific than multiplex PCR, allowing an accurate measure of infective An. gambiae. The results also showed that the CSP-ELISA overestimates the sporozoite rate, detecting sporozoites in the haemolymph in addition to the salivary glands.