Development and Evaluation of PCR Based Detecton of Plasmodium species and Antimalarial Resistant Genes in Various Clinical Samples

Abstract

newlineMalaria is still an endemic disease in many parts of the world. Among the five species of genus Plasmodium, P.falciparum, P.vivax, P.ovale, P.knowlesi and P.malariae are known to cause infections [1]. P.vivax and P.falciparum are commonly reported in India, the second one is associated with complications like cerebral malaria. An accurate diagnosis of malaria is necessary to prevent morbidity and mortality [2]. The subsidized cost of chloroquine has made it to be the most acceptable drug for routine empirical therapy throughout the world (3,4). Antimalarial resistance has been a major obstacle to the successful treatment of malaria globally and has contributed to its mortality. This work is aimed to develop and evaluate a rapid multiplex PCR-based kit for the detection of Plasmodium species and antimalarial resistant genes. Blood, urine and, saliva samples were included for the detection of malaria by microscopy and rapid antigen detection method and the blood samples alone were used for standardization of molecular methods. The identification of four species of Plasmodium and resistant genes like Pvcrt-o, Pvmdr1, Pfcrt-o were individually evaluated initially using uniplex PCR, followed by standardization of single tube reaction mixture and multiplex PCR respectively. The standardized protocol was evaluated for the development of multiplex PCR kit, and the sensitive markers like Cox III and Cyt b genes were used for species identification. On the other hand, the resistant genes were assessed by using conventional PCR, and gene expression study was done using RT-PCR. One blood sample from a migratory construction worker was found positive for P.vivax, which showed the presence of resistant genes pvcrt-o and pvmdr 1, as well as Cox III and Cyt b, which were further investigated by whole genomic sequencing and phylogenetic analysis.