Comparison of Nested PCR and Conventional Analysis of <i>Plasmodium</i> Parasites in Kano, Nigeria

Abstract

Plasmodium identification represents the crucial factor in malaria diagnosis and treatment across developing countries. Conventional microscopy and the use of rapid diagnostic kits have been extensively applied towards human malaria diagnosis. Recombinant DNA techniques have been applied towards malaria diagnosis as well as in the species specific identification using Plasmodium 18s-rRNA gene. This study was undertaken amongst patients attending the Murtala Mohammed Specialist Hospital, Kano. Blood samples were collected from 350 malaria suspected patients. Microscopic analysis via Giemsa-staining revealed that 220 patients were positive for malaria. RDT analysis showed that 248 test samples were positive for Plasmodium infection. DNA products obtained from the blood samples were analyzed by nested PCR to amplify the 18S ssrRNA Plasmodium gene with genus and specific primers rPLU1/5, rPLU3/4, rVIV1/2, rFAL1/2, rMAL1/2 and rOVA1/2. Data obtained showed that 58.64% of specimens tested by microscopy were false positives while 60.62% of false positives were obtained using RDTs in comparison to nPCR which proved that on 91 out of 350 patients were infected with Plasmodium falciparum, representing 26% of tested specimen. Comparative analysis of nPCR to microscopy showed that the sensitivity and positive predictive values of the nPCR were determined as 100 and 41.36%, respectively, while against RDTs it was 100 and 39.38% respectively. nPCR was determined to be more sensitive and specific than either microscopy or RDTs. This study revealed that the accurate diagnosis of malaria by nPCR was compulsory in malaria-prone regions of Nigeria such that nPCR should be applied routinely in laboratory studies.

Challenges in Laboratory Diagnosis of Malaria in a Low-Resource Country at Tertiary Care in Eastern Nepal: A Comparative Study of Conventional vs. Molecular Methodologies

“…A PCR study in Plasmodium species conducted in the US showed real-time PCR was a reliable confirmatory tool for the diagnosis of malaria to resolve conflicting doubts between light microscopy and RDT [ 34 ]. A research work in Nigeria found the sensitivity and specificity of PCR to be 100%, whereas 58%–60% of the cases were falsely diagnosed by microscopy and RDT [ 37 ]. False diagnosis by light microscopy happens due to several factors based on experience as well as improper reagent quality [ 9 , 38 ].…”

Comparison of Different 18S rRNA Primers with Conventional PCR Methods in Determination of Plasmodium spp. and Evaluation of Nested PCR Method

“…According to their results, sensitivity and specificity of 18S rRNA based primers were 91.9% and 100%, respectively and the sensitivity and specificity of dhfr-ts based primers were 51.4% and 100%, respectively (24). Regarding research on nested PCR, there are several studies reporting that genus-specific rPLU1, rPLU5 based primers and species-specific based rFAL1, rFAL2, rVIV1, rVIV2, rOVA1, rOVA2 and rMAL1, rMAL2 primers had high sensitivity and specificity (5,7,15,(23)(24)(25). In addition, there is also a wide range of studies reporting that especially genus-specific rPLU5, rPLU6 based primers and species-specific based rFAL1, rFAL2, rVIV1, rVIV2, rOVA1, rOVA2 and rMAL1, rMAL2 primers had high sensitivity and specificity in the nested PCR (1,6).…”

Diagnostic accuracy of an automated microscope solution (miLab™) in detecting malaria parasites in symptomatic patients at point-of-care in Sudan: a case–control study

“…The sample size was calculated assuming miLab™ would have a sensitivity of 93.75% and a specificity of 95.65%, each with a 95% CI of ± 5%, based on preliminary data from the developer using PCR as the reference standard. Additionally, due to the higher sensitivity of molecular methods over microscopy, up to 30% of samples identified as negative by microscopy were expected to be false negatives when verified by PCR [ 15 ].…”