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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.

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