Phylogenetic analysis of Plasmodium vivax from clinical isolates of Rawalpindi, Islamabad and Azad Kashmir

Abstract

Malaria eradication program efforts have a significant impact on the epidemiology and parasite population dynamics. In countries aiming for malaria elimination, malaria transmission may be restricted to limited transmission hot spots, where parasite populations may be isolated from each other and experience different selection forces. No detailed studies have yet been performed to reveal the actual situation of malaria in the local population, due to which, despite of malaria being common, the epidemiological data is insufficient to elucidate the actual incidence of malaria in Pakistan and Azad Kashmir. Here we aim to examine the Plasmodium vivax population divergence in geographically isolated transmission zones in Pakistan and Kashmir. A malariometric population survey was conducted in 2013 and 2014 using whole blood samples collected from 1500 patients of all ages and genders from different localities of Pakistan and Azad Jammu Kashmir. Microscopically confirmed positive species were subjected to nested PCR for reconfirmation and detection of four species of Plasmodium causing human malaria. For characterizing P. vivax populations based on the extensive diversity. We employed the P. vivax merozoite surface protein 3β (PvMSP3β) and 3α (PvMSP3α) as a molecular marker. To examine parasite populations with different transmission levels Kashmir and Pakistan, we obtained 142 P. vivax isolates and 72 P. falciparum from Kashmir, and 184 P.vivax and 69 P. falciparum from Islamabad. From Rawalpindi, we obtained 168 P.vivax and 83 P. falciparum Positive isolates where the annual parasite incidence (API) was more than 25%. We sequenced the Pvmsp3β and Pvmsp3α gene and examined its genetic diversity and molecular evolution between the parasite populations. Using the Pvmsp3α molecular marker we characterized, 12 different genotypes of P.vivax circulating in Islamabad, 14 in Rawalpindi and 14 Predominant in Kashmir. In the case of Pvmsp3β we characterized 15 different genotypes of P.vivax circulating in Islamabad, 12 in Rawalpindi and 15 in Kashmir. The phylogenetic analyses of both the marker show a clear and distinct relation with the isolates from India, China and Brazil. We have more confirmed that Pvmsp3β and Pvmsp3α sequencing deals significantly higher power for defining genetic diversity of parasite linked with the PCR-RFLP simple method. From the current study, we concluded that malaria parasite populations in a given region may vary significantly in genetic diversity, which may be the result of control and influenced by the magnitude of malaria transmission intensity. This is an issue that should be taken into an account for the implementation of P. vivax control measures such as drug policy and vaccine development.