Optimized Pan-species and speciation duplex real-time PCR assays for Plasmodium parasites detection in malaria vectors

Abstract

Background: An accurate method for detecting malaria parasites in the mosquito's vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP) is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus.

Methods: Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin.

Results: The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6%) and specificity (98%), compared to ELISA-CSP as the referent standard. The agreement between both methods was "excellent" (κ=0.8, P<0.05). The relative quantification of Plasmodium DNA between the two Anopheles species analyzed showed no significant difference (P=0, 2). All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed.

Conclusion: This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the utility of employing an accurate molecular diagnostic tool for detecting malaria parasites in field mosquito populations.

Figure 1. Detection of minor populations by real-time PCR in unbalanced artificial plasmids mixtures.

Simultaneous detection of Plasmodium species DNA in a mixture of 18S specific plasmid constructs. A mixture of the three Plasmodium 18S rDNA targets was made with 10⁵ Pf +10² Pm+10² Po.

Figure 2. Prevalence of co-infection of Plasmodium spp in mosquitoes (An. gambiae and An. funestus) by Real-time PCR.

The figure (A) shows results of speciation analysis of 43 positive samples of An. gambiae ss by qPCR. Of the 43 positive samples, 35 were infected by P. falciparum only (81%), 7 samples showed mixed infection with P. falciparum and P. malariae (16%), and a mixed infection with P. falciparum and P.ovale was observed in 1 sample (2%). The figure (B) shows results of analysis of 22 positive samples of An. gambiae ss by qPCR. Among the 22 positive samples, mono infection with P. falciparum was found in 19 samples (86%), 1 sample showed mixed infection with P. falciparum and P. malariae (4.5%), mixed infection with P. falciparum and P. ovale was observed in 1 sample (4.5%), and in 1 sample mixed infection with 3 species (P. falciparum, P. malariae and P. ovale) was noted (4. 5%).

Figure 3. Absolute and relative quantification of Plasmodium DNA in mosquitoes.

This figure shows a not significant difference was observed in the P. falciparum densities between the two Anopheles species (P-value = 0, 2197).