Journal of Tropical Diseases & Public Health

Abstract

Nested Polymerase Chain Reaction (PCR) on Fixed Stained Slides in Comparison to Whole Blood as a Source of DNA in Southeast of Iran

Ebrahimzadeh Adel, Mohammadi Saeed and Polshekan Mir Ali

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.