Detection of mixed Plasmodium falciparum & P. vivax infections by nested-PCR in Pakistan, Iran & Afghanistan.

BACKGROUND & OBJECTIVES: Species identification and information on transmission pattern of malaria parasite in any malaria endemic area is key to success for a malaria control programme. In this investigation, malaria diagnosis using molecular method was used to assess the transmission pattern of malaria parasite in three malaria endemic regions: Afghanistan, Iran and Pakistan. METHODS: Blood samples were collected from the patients presenting with vivax malaria from Afghanistan (n=108), Iran (n=200) and Pakistan (n=199). Malaria parasite detection was made by the gold standard (microscopy) and also nested-PCR assay, using 18S small sub-unit ribosomal RNA (ssrRNA) gene. RESULTS: Based on microscopy method, the level of mixed infection was zero to 2.5 per cent; however, nested-PCR assay detected 6.5, 22 and 23.5 per cent mixed infections in samples collected from Afghanistan, Iran and Pakistan, respectively. The present results showed that the co-infection of P. vivax with P. falciparum was frequent in malaria endemic regions of Iran and Pakistan. INTERPRETATION & CONCLUSION: The present data suggest the need for improving microscopy diagnosis method and the clinician should also have careful clinical observation, along with the reports on Giemsa- stained thick blood films, particularly in summer time when P. vivax is predominant. Also sharing information on transmission pattern of mixed infection among these countries may help in designing better control strategies for malaria.

Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3alpha and pvcsp genes as molecular markers.

In this study, the diversity of Plasmodium vivax populations circulating in Pakistan and Iran has been investigated by using circumsporozoite protein (csp) and merozoite surface proteins 1 and 3alpha (msp-1 and msp-3alpha) genes as genetic markers. Infected P. vivax blood samples were collected from Pakistan (n=187) and Iran (n=150) during April to October 2008, and were analyzed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 (variable block 5) revealed the presence of type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant, in both study areas. The sequence analysis of 33 P. vivax isolates from Pakistan and 30 from Iran identified 16 distinct alleles each, with one allele (R-8) from Iran which was not reported previously. Genotyping pvcsp gene also showed that VK210 type is predominant in both countries. Moreover, based on the size of amplified fragment of pvmsp-3alpha, three major types: type A (1800bp), type B (1500bp) and type C (1200bp), were distinguished among the examined isolates that type A was predominant among Pakistani (72.7%) and Iranian (77.3%) parasites. PCR/RFLP products of pvmsp-3alpha with HhaI and AluI have detected 40 and 39 distinct variants among Pakistani and Iranian examined isolates, respectively. Based on these three studied genes, the rate of combined multiple genotypes were 30% and 24.6% for Pakistani and Iranian P. vivax isolates, respectively. These results indicate an extensive diversity in the P. vivax populations in both studies.