Comparative Analysis of Multiplex/Semi-nested PCR and Microscopy for Accurate Human Malaria Species Diagnosis

Abstract

Background: Iran is currently striving to eliminate malaria, making it essential to improve the accurate and rapid diagnosis of suspected cases. Objectives: This study aimed to evaluate the effectiveness of the multiplex/semi-nested polymerase chain reaction (PCR) method, using specific primers, for the precise diagnosis of human malaria species. Methods: Seventy-two blood samples from patients and suspected malaria cases were selected and stored at -80°C in the National Malaria Laboratory. DNA extraction was performed to obtain the genetic material for further analysis. Multiplex/semi-nested PCR was conducted, and the results were compared with microscopic examination. Results: Out of 72 samples, 36 were positive by microscopic analysis, which included: 16 Plasmodium falciparum, 16 P. vivax, 1 P. ovale, 1 P. malariae, 1 mixed P. falciparum - P. vivax, and 1 mixed P. falciparum - P. malariae. Thirty-three cases were diagnosed through molecular analysis: 16 P. falciparum, 13 P. vivax, 1 P. ovale, 1 P. malariae, and 2 mixed P. falciparum - P. vivax. Conclusions: Issues, such as false positives, underreporting of mixed infections, and mismatched species identified by microscopic methods need to be addressed and improved to ensure accurate diagnoses.