Comparative evaluation of two commercial real-time PCR kits (QuantiFast™ and abTES™) for the detection of Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia

Background The monkey parasitePlasmodium knowlesiis an emerging public health issue in Southeast Asia. In Sabah, Malaysia,P. knowlesiis now the dominant cause of human malaria. Molecular detection methods forP. knowlesiare essential for accurate diagnosis and in monitoring progress towards malaria elimination of otherPlasmodiumspecies. However, recent commercially available PCR malaria kits have unpublishedP. knowlesigene targets or have not been evaluated against clinical samples. Methods Two real-time PCR methods currently used in Sabah for confirmatory malaria diagnosis and surveillance reporting were evaluated: the QuantiFast (TM) Multiplex PCR kit (Qiagen, Germany) targeting theP. knowlesi18S SSU rRNA; and the abTES (TM) Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosedP. knowlesigene target. Diagnostic accuracy was evaluated using 52P. knowlesi,25Plasmodium vivax, 21Plasmodium falciparum, and 10Plasmodium malariaeclinical isolates, and 26 malaria negative controls, and compared against a validated reference nested PCR assay. The limit of detection (LOD) for each PCR method andPlasmodiumspecies was also evaluated. Results The sensitivity of the QuantiFast (TM) and abTES (TM) assays for detectingP. knowlesiwas comparable at 98.1% (95% CI 89.7-100) and 100% (95% CI 93.2-100), respectively. Specificity of the QuantiFast (TM) and abTES (TM) forP. knowlesiwas high at 98.8% (95% CI 93.4-100) for both assays. The QuantiFast (TM) assay demonstrated falsely-positive mixedPlasmodiumspecies at low parasitaemias in both the primary and LOD analysis. Diagnostic accuracy of both PCR kits for detectingP. vivax,P. falciparum, andP. malariaewas comparable toP. knowlesi. The abTES (TM) assay demonstrated a lower LOD forP. knowlesiof <= 0.125 parasites/mu L compared to QuantiFast (TM) with a LOD of 20 parasites/mu L. Hospital microscopy demonstrated a sensitivity of 78.8% (95% CI 65.3-88.9) and specificity of 80.4% (95% CI 67.6-89.8) compared to reference PCR for detectingP. knowlesi. Conclusion The QuantiFast (TM) and abTES (TM) commercial PCR kits performed well for the accurate detection ofP. knowlesiinfections. Although the QuantiFast (TM) kit is cheaper, the abTES (TM) kit demonstrated a lower LOD, supporting its use as a second-line referral-laboratory diagnostic tool in Sabah, Malaysia.