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 However, recent commercially available PCR malaria kits have unpublished P. knowlesi gene targets or have not been evaluated against clinical samples.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
Plasmodium knowlesi is the major cause of zoonotic malaria in Southeast Asia. Rapid and accurate diagnosis enables effective clinical management. A novel malaria diagnostic tool, Gazelle (Hemex Health, USA) detects haemozoin, a by-product of haem metabolism found in all Plasmodium infections. A pilot phase refined the Gazelle haemozoin identification algorithm, with the algorithm then tested against reference PCR in a larger cohort of patients with P. knowlesi mono-infections and febrile malaria-negative controls. Limit-of-detection analysis was conducted on a subset of P. knowlesi samples serially diluted with non-infected whole blood. The pilot phase of 40 P. knowlesi samples demonstrated 92.5% test sensitivity. P. knowlesi-infected patients (n = 203) and febrile controls (n = 44) were subsequently enrolled. Sensitivity and specificity of the Gazelle against reference PCR were 94.6% (95% CI 90.5–97.3%) and 100% (95% CI 92.0–100%) respectively. Positive and negative predictive values were 100% and 98.8%, respectively. In those tested before antimalarial treatment (n = 143), test sensitivity was 96.5% (95% CI 92.0–98.9%). Sensitivity for samples with ≤ 200 parasites/µL (n = 26) was 84.6% (95% CI 65.1–95.6%), with the lowest parasitaemia detected at 18/µL. Limit-of-detection (n = 20) was 33 parasites/µL (95% CI 16–65%). The Gazelle device has the potential for rapid, sensitive detection of P. knowlesi infections in endemic areas.
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