Assessment of Microscopic Detection of Malaria with Nested Polymerase Chain Reaction in War-Torn Federally Administered Tribal Areas of Pakistan

Introduction Diagnostic accuracy of malaria is critical for early treatment, control, and elimination of malaria, especially in war-affected malaria-endemic areas. Microscopic detection of Plasmodium species has been the gold standard in remote malaria-endemic regions. However, the diagnostic accuracy is still questioned, especially in discriminating mixed and submicroscopic parasitic levels. This study was designed to evaluate the diagnostic performance of microscopic examination against nested PCR analysis in war-torn malaria-endemic Federally Administered Tribal Areas (FATA) of Pakistan. Methods Venous blood samples were collected from symptomatic patients for microscopic examination and nested PCR analysis from January 2016–December 2016 from five Agencies (Bajaur, Mohmand, Khyber, Orakzai and Kurram Agency) and four Frontier Regions (Peshawar, Kohat, Bannu, and Dera Ismail Khan Frontier Region) of FATA. Malaria-positive isolates were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes) for speciation. Results Among enrolled participants, 762 were found positive for malaria parasite on microscopic examination of the blood film. Plasmodium vivax was found in 623, Plasmodium falciparum in 132 and 7 were diagnosed with mixed infection ( P. vivax and P. falciparum coinfection). Nested PCR detected Plasmodium infection in 679 samples (523 P. vivax , 121 P. falciparum, and 35 mixed infections). Compared with microscopy, the sensitivity of nested PCR was 98.94%, and specificity was 98.27%, while the sensitivity and specificity of slide microscopy 89.34% and 87.99% respectively. Conclusion The conventional microscopy method has low sensitivity to detect the mixed infection as compared to nested PCR. High sensitivity and specificity observed in nested PCR make this molecular tool a useful technique for monitoring, controlling, and eliminating malaria-endemic regions.