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

Abstract

Introduction: The monkey parasite Plasmodiumknowlesi is an emerging public health issue in Southeast Asia. In Sabah, Malaysia, P. knowlesiis now the dominant cause of human malaria. Molecular detection methods for P. knowlesiare 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towards malaria elimination of other Plasmodium species.However,recent commercially available PCR malaria kits have unpublished P. knowlesigene targets or have not been evaluated against clinical samples.Methods:Two real-time PCR methods currently used in Sabahfor confirmatorymalaria diagnosis and surveillance reportingwere evaluated: theQuantiFast™ Multiplex PCR kit (Qiagen, Germany) targeting the P. knowlesi18S SSU rRNA;and the abTES™ Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosedP. knowlesi gene target. Diagnostic accuracywas evaluatedusing 52P. knowlesi, 25P. vivax, 21 P. falciparum, and 10 P. malariaeclinical isolates, and 26 malaria negative controls, and compared against a validated referencenested 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. knowlesiwas 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. knowlesiwas 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 fordetecting P. vivax, P. falciparum, and P. malariae was comparable to P. knowlesi.The abTES™ assay demonstrated a lower LOD for P. knowlesiof ≤0.125 parasites/µL compared to QuantiFast™ witha LOD of20 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, supportingits use as a second-line referral-laboratory diagnostic toolin Sabah, Malaysia.

Diagnostic accuracy and limit of detection of ten malaria parasite lactate dehydrogenase-based rapid tests for Plasmodium knowlesi and P. falciparum

“…A real-time PCR assay, QuantiFast™ targeting the 18S SSU rRNA gene was conducted once for each sample in accordance with the manufacturers’ and Sabah Public Health Laboratory protocols, using the Bio-Rad CFX96 Touch™ PCR machine (Bio-Rad, USA). QuantiFast™ real-time PCR was carried out over two separate reactions, with the first reaction for Plasmodium genus screening, followed by subsequent P. knowlesi and other human Plasmodium species-specific detection if positive (32).…”

“…One RDT (careUS TM ) was tested only on clinical samples, and another (Meriscreen OneStep TM ) on only laboratory samples. (32).…”

Universal two-dimensional labelled probe-mediated melting curve analysis based on multiplex PCR for rapid typing of plasmodium in a single closed tube

“…Initially, the amplification targets for P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi based on previous studies (10,11,20,(22)(23)(24) and available gene sequences in GenBank were analyzed. Following that, nuclear small subunit (SSU) rRNA gene was confirmed as plasmodium detection target and used for the design of plasmodium species-specific primers as a result of the availability of its sufficient copies and conservative and species-specific sequences for plasmodium detection.…”