Evaluation of Two Multiplexed qPCR Assays for Malaria Detection and Speciation: A Comparative Study With Nested PCR and Microscopy.

Background: Malaria is a deadly vector-borne parasitic disease spread by the bite of an infective female Anopheles mosquito. In routine malaria diagnosis, microscopic examination is generally regarded as the gold standard. Our study sought to evaluate the diagnostic precision of two commercially accessible quantitative PCR (qPCR) kits, in contrast to light microscopy and nested multiplex PCR (NM-PCR). Methods: This cross-sectional study in southwest Saudi Arabia included 92 febrile patients meeting the inclusion criteria. Detection of Plasmodium species used light microscopy, NM-PCR, and qPCR kits (RealStar and Viasure). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and receiver operating characteristic (ROC) curves were calculated. Statistical analysis was performed using SPSS v25, with significance set at p ≤ 0.05. Results: Light microscopy detected 92.4% of cases, NM-PCR detected 73.9%, and RealStar and Viasure detected 92.4% and 95.7%, respectively. Viasure showed the highest sensitivity (97.6%) and NPV (50%), while NM-PCR had superior specificity (71.4%). For species identification, Plasmodium falciparum detection was highest with RealStar (85%). Mixed infections were better identified by Viasure (34.6%). RealStar excelled in Plasmodium vivax detection (area under the curve [AUC] = 90%). qPCR detected low parasitemia levels missed by microscopy. Conclusions: The qPCR kits, particularly Viasure, demonstrated superior sensitivity for detecting Plasmodium species and identifying mixed infections compared to light microscopy and NM-PCR. While light microscopy showed higher specificity and PPV, qPCR effectively detected low parasitemia levels missed by microscopy, highlighting its value in improving malaria diagnostics.