Merozoite surface protein 1 of (PfMSP-1) is a leading malaria vaccine candidate. However, extensive genetic diversity of this gene in field isolates of represents a major obstacle for the development of an effective vaccine against malaria. The present study was aimed at analysing genetic polymorphisms of K1, MAD20 and RO33 allelic types of MSP-1 block 2 among isolates from Sistan and Baluchestan, Iran. In this study a total of 94 infected persons from Sistan and Baluchistan Province of Iran, were included. Blood samples were collected from March 2011 to September 2012. Block 2 of the MSP-1 gene was genotyped by allele-specific nested polymerase chain reaction (PCR) after DNA extraction. Eighty-nine (94.7%) of the 94 samples were successfully amplified; 7 distinct MSP-1 genotypes were identified by size differences on agarose gels. MAD20 was the predominant MSP-1 allelic family identified in 46.1% (41/89) of the samples while RO33 family had the least frequency (7.9%). A total of 9/89 (10.1%) samples exhibited multiple infections with two alleles at PfMSP-1. The present study shows that the level of genetic diversity is relatively low in southeast of Iran and most of infections are composed of one clone, which is consistent with an area of low malaria transmission. These data are useful for malaria prevention and control in Iran.
The microscopic examination of Thick Blood Smears (TBS) remains the method of choice for the diagnosis of human malaria. Recently, alternative diagnostic methods, such as Nested PCR, have been used for the detection and identification of malaria parasites. The aim of this study was to compare the sensitivity and specificity of Nested PCR accomplished using DNA extracted from whole blood against fixed stained slides. 125 blood samples including 76(60.8%) male and 49(40.2%) female accomplished examinations. The percentage of the parasitaemia on positive samples was calculated from a total count of 200 leukocytes counted in a Giemsa stained thin blood films. The nested PCR assay carried out on DNA Extracted samples by specific primers to amplify 18ssr RNA Plasmodium gene. Of all 125 blood samples 50(40%) were positive (41(32.8%) P. vivax, 9(7.2%) P. falciparum) and 75(60%) were negative for malaria parasites using microscopy examination. Nested-PCR on whole blood specimens detected 66(52.8%) plasmodium species: 47(37.6%) P. vivax, 13(10.4%) P. falciparum, 6(4.8%) mixed infections P. vivax and P. falciparum. Nested-PCR on peripheral blood slides detected 49 (39.2%) plasmodium species: 34(27.2%) P. vivax, 10(8%) P. falciparum, 5(4%) mixed infections P. vivax and P. falciparum. The study showed that the sensitivity and specificity of nested PCR were 96% and 76%, respectively, when target DNA was extracted from blood and 78% and 86% when DNA was obtained from smears. These studies demonstrated that Plasmodium DNA might be successfully isolated from TBS indicating that this method of DNA preservation could be considered adequate and convenient for epidemiological studies.
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