Both the QuantiFast™ and abTES™ real-time PCR assays evaluated in this study demonstrated high diagnostic accuracy in detecting P. knowlesi and other Plasmodium species monoinfections when compared against the reference nested PCR. The experimentally determined LOD for P. knowlesi of 20 parasites/µL for QuantiFast™ and ≤0.125 parasites/µL for abTES™ were also both below typical microscopic malaria detection limits , further highlighting their utility for confirmatory referral-laboratory diagnostic and surveillance purposes when conducted on point-of-care malaria microscopy-positive samples. Although results suggested a trend towards superior performance of the abTES™ assay and the single reaction required to conduct this assay using current protocols has logistical advantages, abTES™ (~USD $15.40 per reaction) has a 3-fold higher cost than QuantiFast™ (~USD $5.19 per reaction). Therefore, this study supports the use of the current malaria diagnostic and surveillance algorithm used by the Sabah State Public Health Laboratory in an area approaching elimination of human-only Plasmodium species, whereby all microscopy positive malaria patients are tested initially using the QuantiFast™ assay, with abTES™ used for any subsequent negative or mixed Plasmodium species infection results.
Multiple sensitive molecular methods for P. knowlesi detection have been published to date including nested [4, 22], single-step [27-29], and real-time PCR [30, 31], and loop-mediated isothermal amplification (LAMP) [32-35]. Although these molecular methods are directed against a range of different P. knowlesi gene targets, their reported detection limits are all below that of routine microscopic examination of malaria blood films. Real-time PCR has a number of advantages compared to conventional nested PCR, including the ability to simultaneously detect multiple Plasmodium species in a single amplification round, with higher throughput potential, and does not require manual quantification of end-points using gel electrophoresis . However, real-time PCR requires expensive customized hydrolysis probes in addition to the pre-selected primers for common targets such as P. knowlesi-specific 18S SSU rRNA , as utilized by the QuantiFastTM assay. This validated gene target is also commonly used for both real-time and nested PCR methods for other Plasmodium species differentiation , due to a unique Plasmodium genus core sequence and a separate highly conserved Plasmodium species-specific region , resulting in improved diagnostic specificity. In this study, the target gene sequences of the abTES™ method are undisclosed, however gel electrophoresis visualization of the respective Plasmodium species-specific PCR products showed amplicon lengths consistent with standard Plasmodium 18S SSU rRNA gene sequences .
The use of confirmatory molecular detection methods have enabled accurate reporting of malaria trends in Malaysia demonstrating increasing P. knowlesi incidence , and have also provided reliable data on national and sub-national progress towards achieving elimination of other human-only Plasmodium species . In other co-endemic settings in Southeast Asia, the incorporation of P. knowlesi detection into existing nucleic acid-based detection protocols would improve their use in targeted malaria surveillance strategies and accuracy of case reporting, particularly on those reported as P. malariae or indeterminate Plasmodium species infections from point-of-care microscopy [13, 19]. Additionally, this would allow improved understanding of regional diversity in the epidemiology of P. knowlesi transmission, and assist in the design of appropriate local preventive public health interventions. The use of molecular detection methods have also enabled evaluation and improvements of local treatment guidelines for knowlesi malaria, including recommending early intravenous artesunate for those with parasitaemia ≥20,000/µL , and artemisinin-based combination therapy (ACT) for uncomplicated disease in Malaysia [40, 41]. Finally, molecular detection methods may aid surveillance for another zoonotic monkey parasite, Plasmodium cynomolgi, with increasing case-reports highlighting spill-over infections occurring in humans in Sabah , Peninsular Malaysia  and Cambodia . Plasmodium cynomolgi is morphologically similar to P. vivax on microscopic blood film evaluation , and due to being closely genetically related to P. vivax, previous PCR detection methods have also demonstrated cross-reactivity between these Plasmodium species . This may have implications for accuracy of P. vivax case reporting, and potential underestimation of P. cynomolgi incidence in Southeast Asia .
The QuantiFast™ assay evaluated in this study is currently favoured by the Sabah State Public Health Laboratory due to its lower cost compared to abTES™, despite requiring two reactions; i.e. one monoplex and one triplex for each clinical isolate. The lengthier run time for QuantiFast™ is further compounded by a more tedious sample preparation. However, the technical issue of requiring two reactions per sample for the current QuantiFast™ laboratory protocol could be overcome by changing one of the FAM reporter dyes currently used for both P. malariae or P. knowlesi probes (Table 1), thus ensuring non-overlapping of emission wavelengths across four probes. As this study replicated the current public health surveillance protocol, the development of a new probe using a fourth colour would require additional validation. Future development of the QuantiFast™ method could, therefore, result in a single quadruplex reaction, further reducing operational cost and time, and minimization of errors, which would ideally include detection of P. cynomolgi if an appropriately validated probe becomes available.
One limitation of this study related to the secondary LOD analysis, where the lowest pre-selected parasitaemia (0.125 parasites/µL) remained above the actual limit of detection for P. knowlesi and P. vivax when using the abTES™ method. Difficulties with the conduct of PCR diagnostics in reference or research laboratory settings are evident for many pathogens, and may not reflect the ideal technical accuracy of the diagnostic assay. It is also not possible to preclude the possibility of abTES™ real-time PCR having greater sensitivity for detection of P. knowlesi as compared to the reference PCR . Although unlikely, the single positive result for abTES recorded from a healthy control in an endemic area may have been a genuine asymptomatic submicroscopic P. knowlesi infection. A single P. knowlesi sample with a parasite count of 12,968/µL, a level well above the documented LOD, was found to be falsely negative on the QuantiFast™ assay, but positive with abTES™, which may have indicated a possible error during sample loading. The specificity of the QuantiFast™ assay for P. knowlesi detection was reduced by a single false-positive mixed P. knowlesi/P. malariae result for a P. malariae monoinfection of 224 parasites/µL, the corresponding abTES™ result was positive for P. malariae only. This anomaly may have been caused by unintended annealing of P. knowlesi-specific probes onto P. malariae genomic DNA ; a plausible scenario given primers used for the QuantiFast™ P. knowlesi monoplex are not Plasmodium species-specific. The QuantiFast™ assay also demonstrated a false-positive mixed P. falciparum/P. vivax result from a P. vivax monoinfection during the LOD analysis at low-level parasitaemia (2 and 20 parasites/µL). These findings imply that in routine surveillance in Sabah, mixed Plasmodium species infections using QuantiFast™ may require further validation.