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

Abstract : Background : Evaluation of malaria sporozoite rates in the salivary glands ofAnopheles gambiaeis essential forestimating the number of infective mosquitoes, and consequently, the entomological inoculation rate (EIR). EIR is akey indicator for evaluating the risk of malaria transmission. Although the enzyme-linked immunosorbent assayspecific for detecting the circumsporozoite protein (CSP-ELISA) is routinely used in the field, it presents severallimitations. A multiplex PCR can also be used to detect the four species ofPlasmodiumin salivary glands. The aimof this study was to evaluate the efficacy of a real-time quantitative PCR in detecting and quantifying wild Plasmodium falciparumin the salivary glands ofAn. gambiae. Methods : Anopheles gambiae(n=364) were experimentally infected with blood from P. falciparum gametocyte carriers, andP. falciparumin the sporozoite stage were detected in salivary glands by using a real-time quantitativePCR (qPCR) assay. The sensitivity and specificity of this qPCR were compared with the multiplex PCR applied fromthe Padley method. CSP-ELISA was also performed on carcasses of the same mosquitoes. Results : The prevalence ofP. falciparumand the intensity of infection were evaluated using qPCR. This method hada limit of detection of six sporozoites perμL based on standard curves. The number ofP. falciparumgenomes inthe salivary gland samples reached 9,262 parasites/μL (mean: 254.5; 95% CI: 163.5-345.6). The qPCR showed a similarsensitivity (100%) and a high specificity (60%) compared to the multiplex PCR. The agreement between the twomethods was“substantial”(κ= 0.63, P <0.05). The number ofP. falciparum-positive mosquitoes evaluated with theqPCR (76%), multiplex PCR (59%), and CSP-ELISA (83%) was significantly different (P <0.005). Conclusions : The qPCR assay can be used to detectP. falciparumin salivary glands ofAn. gambiae. The qPCR ishighly sensitive and is more specific than multiplex PCR, allowing an accurate measure of infectiveAn. gambiae. The results also showed that the CSP-ELISA overestimates the sporozoite rate, detecting sporozoites in thehaemolymph in addition to the salivary glands.