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

• Published in: PloS one 2012-12, Vol.7 (12), p.e52719-e52719
• Main Authors: Sandeu, Maurice Marcel, Moussiliou, Azizath, Moiroux, Nicolas, Padonou, Gilles G, Massougbodji, Achille, Corbel, Vincent, Ndam, Nicaise Tuikue
• Format: Article
• Language: English
• Subjects: Analysis; Animal genetics; Animals; Anopheles; Anopheles - parasitology; Anopheles funestus; Anopheles gambiae; Anophelinae; Aquatic insects; Assaying; Benin; Biology; Circumsporozoite protein; Culicidae; Deoxyribonucleic acid; Diagnostic software; Diagnostic systems; Diptera; Discordance; DNA; DNA probes; Enzyme-Linked Immunosorbent Assay; Enzymes; Erythrocytes; Evolution; Female; Gene expression; Genetics; Health aspects; Immunization; Infection; Infections; Insect Vectors - parasitology; Life Sciences; Malaria; Medical research; Medicine; Microscopy; Mixed infection; Mosquitoes; Multiplexing; Parasites; Plasmodium; Plasmodium - genetics; Plasmodium falciparum; Plasmodium malariae; Plasmodium vivax; Polymerase chain reaction; Populations; Primers; Protozoan Proteins - genetics; Real time; Real-Time Polymerase Chain Reaction - methods; Reproducibility of Results; Sensitivity; Sensitivity and Specificity; Speciation; Species; Target detection; Tuberculosis; Vector-borne diseases; Vectors
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0052719

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. 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. 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