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

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 However, recent commercially available PCR malaria kits have unpublished P. knowlesi gene targets or have not been evaluated against clinical samples.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

An Optimized Real-Time qPCR Method for the Effective Detection of Human Malaria Infections

“…The major problem is often observed for detection of simian malaria Pk that also causes infection in human. Besides, the commercial kit available to detect Pk often shows false positive result in mixed infection at low parasitemia with a LOD of 0.125 and 20 parasites/µL only [ 41 ]. Targeting high copy telomere associated repetitive element and var gene acidic terminal achieved a LOD of 0.03 to 0.15 parasites/µL for detection of Pf [ 42 ] while 18s rRNA target based approach in clinical isolates resulted in detection of Pf and Pv wih LOD of 1–10 parasites/µL with 100% sensitivity [ 43 ].…”

Plasmodium vivax malaria serological exposure markers: Assessing the degree and implications of cross-reactivity with P. knowlesi

“… 37 , 38 Patients who were not confirmed by PCR to have P. knowlesi monoinfection were retrospectively excluded, using a validated PCR assay. 58 In contrast to the ACTKNOW study, only one participant was excluded due to P. vivax infection detected by PCR (unpublished data). Plasma samples were available for the current study from 41 patients at day 0 (time of enrollment), 7, 14, 28 and 365 (these exact time-points were not always available, exact days post-enrolment are stated in the results and relevant figure legends).…”

Diagnostic performance of a 5-plex malaria immunoassay in regions co-endemic for Plasmodium falciparum, P. vivax, P. knowlesi, P. malariae and P. ovale

“…as part of prospective clinical studies in Sabah, Malaysia 20 , 21 . Venous blood was also concurrently collected from healthy asymptomatic adult controls negative for malaria on microscopy and subsequent polymerase chain reaction (PCR) testing 22 . At the time of sampling Sabah had co-endemic transmission of the five aforementioned Plasmodium species.…”