Evaluation of Negative Malaria Giemsa-Stained Smears Referred from Malaria Centers in Sistan and Balouchestan Using Nested Polymerase Chain Reaction

Malaria is one of the most important infectious diseases worldwide and also in Iran. Reports announced by Controlling Diseases Center (CDC) prove the presence of two species including Plasmodium vivax and Plasmodium falciparum in this province. P. falciparum cases diagnosis in purpose of true treatment and following up patients is necessary. This study is intending to extract DNA from negative microscopic smears using the newly invented method by research group and afterwards to evaluate negative malaria Giemsa-stained smears using nested polymerase chain reaction. Aiming to diagnose malaria, nested PCR amplification was accomplished using DNA extracted from fixed negatively diagnosed microscopic smears and Giemsa-stained. The extracted DNA was used as a pattern to amplify the specific sequence of P. falciparum and P. vivax using the small subunit of ribosomal RNA [18ssrRNA]. This retrospective study was implemented on 500 malaria negative microscopic smears storing from 6 months to 1 year. By accomplishing PCR amplification on 500 microscopic smears, 54 (10.8%) malaria positive samples were diagnosed which were incorrectly diagnosed to be negative previously. Of all specimens revealed to be positive, 34 (6.8%) cases were P. vivax while 20 (4%) cases were P. falciparum. This study reveals the priority of nested-PCR method to microscopic examination method and the possibility of using old microscopic smears in epidemiologic and retrospective studies clearly.