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Background  The monkey parasite  Plasmodium knowlesi  is an emerging public health issue in Southeast Asia. In Sabah, Malaysia,  P. knowlesi  is now the dominant cause of human malaria. Molecular detection methods for  P. knowlesi  are essential for accurate diagnosis and in monitoring progress towards malaria elimination of other  Plasmodium  species. However, recent commercially available PCR malaria kits have unpublished  P. knowlesi  gene targets or have not been evaluated against clinical samples.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™ Multiplex PCR kit (Qiagen, Germany) targeting the  P. knowlesi  18S SSU rRNA; and the abTES™ Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosed  P. knowlesi  gene target. Diagnostic accuracy was evaluated using 52  P. knowlesi,  25  Plasmodium vivax , 21  Plasmodium falciparum , and 10  Plasmodium malariae  clinical isolates, and 26 malaria negative controls, and compared against a validated reference nested PCR assay. The limit of detection (LOD) for each PCR method and  Plasmodium  species was also evaluated.    Results  The sensitivity of the QuantiFast™ and abTES™ assays for detecting  P. knowlesi  was comparable at 98.1% (95% CI 89.7–100) and 100% (95% CI 93.2–100), respectively. Specificity of the QuantiFast™ and abTES™ for  P. knowlesi  was high at 98.8% (95% CI 93.4–100) for both assays. The QuantiFast™ assay demonstrated falsely-positive mixed  Plasmodium  species at low parasitaemias in both the primary and LOD analysis. Diagnostic accuracy of both PCR kits for detecting  P. vivax ,  P. falciparum , and  P. malariae  was comparable to  P. knowlesi . The abTES™ assay demonstrated a lower LOD for  P. knowlesi  of ≤ 0.125 parasites/µL compared to QuantiFast™ with a LOD of 20 parasites/µL. 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 detecting  P. knowlesi .    Conclusion  The QuantiFast™ and abTES™ commercial PCR kits performed well for the accurate detection of  P. knowlesi  infections. Although the QuantiFast™ kit is cheaper, the abTES™ kit demonstrated a lower LOD, supporting its use as a second-line referral-laboratory diagnostic tool in Sabah, Malaysia.

malaria journal

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