METHODS: Microscopy-based malaria notification data and polymerase chain reaction (PCR) results were obtained from the Sabah Department of Health and State Public Health Laboratory, respectively, from January 2015 to December 2017. From January 2016 this was complemented by a statewide prospective hospital surveillance study. Databases were matched, and species was determined by PCR, or microscopy if PCR was not available.
RESULTS: A total of 3867 malaria cases were recorded between 2015 and 2017, with PCR performed in 93%. Using PCR results, and microscopy if PCR was unavailable, P. knowlesi accounted for 817 (80%), 677 (88%), and 2030 (98%) malaria cases in 2015, 2016, and 2017, respectively. P. falciparum accounted for 110 (11%), 45 (6%), and 23 (1%) cases and P. vivax accounted for 61 (6%), 17 (2%), and 8 (0.4%) cases, respectively. Of those with P. knowlesi, the median age was 35 (interquartile range: 24-47) years, and 85% were male.
CONCLUSIONS: Malaysia is approaching elimination of the human-only Plasmodium species. However, the ongoing increase in P. knowlesi incidence presents a major challenge to malaria control and warrants increased focus on knowlesi-specific prevention activities. Wider molecular surveillance in surrounding countries is required.
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.