Determination the species of plasmodium in malaria cases seen in Şanliurfa region by nested PCR and determination the sub-species of plasmodium vivax by PCR-RFLP

Malaria imposes a huge public health burden in tropical and subtropical countries with one million deaths each year. Plasmodium vivax is the most prevalent of the Plasmodium species in Southeastern Anatolia Region in Turkey. It is necessity to determine the varieties and subspecies of parasites in order to understand the global and local epidemiology of parasite strains.In this study, it was aimed to determine the validity and location of the nested PCR for malaria diagnosis. It was also aimed to detect the Plasmodium, to determine the species of Plasmodium by nested PCR and to identify the variants of P.vivax by PCR-RFLP.This methodological type of epidemiological study was carried out in malaria endemic towns of Şanlıurfa province. Fingerprick blood samples were collected from 153 malaria suspected participants working as farmworkers. Giemsa-stained thick and thin smears were examined by microscopic examination for malaria diagnosis. Plasmodium DNA samples were extracted from blood spotted filter-papers. The extracted DNA was used as template to amplify the 18S ssrRNA gene of Plasmodium with specific primers by nested PCR described by Snounou et al. The amplified PCR products were digested with restriction enzymes Alu I and Bst NI by PCR-RFLP to distinguish between two important variants of P.vivax, VK210 and VK247 (Imwong et al.). The DNA fragments obtained following nested PCR were analysed by electrophoresis in 2% agarose gels and the DNA was visualised on an ultraviolet transilluminator following ethidium bromide staining for 30 minutes.11 of 153 specimens (7.2%) were detected as Plasmodium by microscopy. 15 of 153 (9.8%) participants were detected as Plasmodium by using genus specific primers by nested PCR. 14 of 15 Plasmodium specimens were identified as P. vivax by using species-specific primers. The primers targeted circumsporozoite protein gene of P.vivax amplified by nested PCR. According to the PCR-RFLP analysis, 2 of 14 P. vivax specimens (14.3%) were VK210 and 12 of them (85.7%) were VK247.Compared to microscopy, the sensitivity, specifity and positive predictive values of nested PCR were found 100%, 97.2%, 73.3%, respectively.It is considered that, this study may be useful for rapid and accurate diagnosis of malaria, the diagnosis of P. vivax malaria cases with low parasitemia, the detection of the mixed and asemptomatic infections, the CSP-based vaccine trials of P. vivax, discriminating re-infection from relapse and recrudescence, the trials of drug resistance, in vivo drug efficacy and epidemiologic.Key words: Malaria, Plasmodium vivax, circumsporozoite, VK210, VK247