Diagnosis, Clinical and Molecular Delineation of Human Plasmodium Species from Mangaluru, Southwest India

Abstract

Malaria is a global threat and a never-ending battle without appropriate identification and differentiation of the parasite species. This work compared the diagnostic methods including the thick film microscopy technique, quantitative buffy coat, and polymerase chain reaction. The inaccuracy of species determination by microscopy and the consequent treatment regime underlines the necessity to upgrade routine diagnostic methods with molecular techniques. In the study, 436 samples were collected; venous blood was processed for the quantitative buffy coat technique followed by classical Giemsa staining of thin and thick smears and nested Polymerase Chain Reaction (nPCR) for the genus-specific region of Plasmodium targeting 18S rDNA followed by species-specific identification. Of 436 samples screened for malaria, results in PCR showed 78.7% (100/127) to be P. vivax, 4.8% (6/127) as P. falciparum and 16.5% (21/127) to be mixed infection (P. vivax + P. falciparum). The prevalence of malaria was 0.29, and there was good concordance between the methods for detecting Plasmodium (Kappa:0.77). In our investigation, nested PCR and TFM exhibited a sensitivity of 97.7% and a specificity of 100% for malaria detection compared to QBC. Clinical parameters-thrombocytopenia and anemia, were compared in this study. A positive association was observed between thrombocytopenia and malaria (p<0.05), but the association between anemia and malaria infection remains unclear. Primer cross-reactions were also observed in the primer sequence of P. ovale and P. knowlesi, but sequencing confirmed it as P. vivax and the study of phylogeny paved a new way in analyzing the relatedness of the sequences.