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

Malaysia has made significant progress towards their World Health Organization (WHO) goal of eliminating human-only malaria by 2020, with no indigenous cases of Plasmodium falciparum or Plasmodium vivax malaria reported in 2018 [1]. However, the emergence of zoonotic transmission of the monkey parasite Plasmodium knowlesi has been less tractable to conventional malaria control efforts [2]. Within Malaysia, P. knowlesi is now the most common cause of malaria in humans, accounting for almost all reported malaria cases [1, 3], including over 2000 notifications in the state of Sabah in 2017 [2]. Confirmed P. knowlesi human infections have now been reported in all areas of Southeast Asia where the primary reservoir macaque hosts and Anopheles leucosphyrus group mosquitoes are present [4‐6].

Microscopic assessment of Giemsa-stained blood smears remains the appropriate primary point-of-care method for malaria diagnosis in most P. knowlesi endemic countries, including Malaysia [7]. Ideally, differentiation of Plasmodium species via microscopy allows initiation of prompt and appropriate treatment and accurate public health reporting [8]. However, well-established limitations in the use of routine diagnostic malaria microscopy include the inability to differentiate between P. knowlesi and P. malariae due to similar morphology across all life-stages [9, 10]. Plasmodium knowlesi is also commonly misidentified as Plasmodium falciparum due to similarities in the early ring stage [11], and also with Plasmodium vivax in co-endemic areas such as Malaysia [10, 12] and Indonesia [13]. Commercially available pLDH-based rapid diagnostic tests (RDTs) developed for human-only Plasmodium species are known to be cross-reactive for P. knowlesi epitopes [14]. However, RDTs evaluated for P. knowlesi detection to date have demonstrated insufficient sensitivity and specificity to support their use for routine diagnosis [15‐18].

Molecular methods are necessary for accurate diagnostic confirmation of P. knowlesi and other Plasmodium species, and for improved public health malaria surveillance reporting in co-endemic areas in Southeast Asia [11, 19]. In Malaysia, molecular detection methods have been implemented for routine confirmation of all malaria cases since 2014 [7]. In Sabah, the primary molecular detection method at the State Public Health Laboratory (Makmal Kesihatan Awam; MKA) is a multiplex real-time PCR using the QuantiFast™ Multiplex PCR kit (QIAGEN, Germany), which requires addition of previously published primers and probes targeting the SSU rRNA gene of P. knowlesi and other human-only Plasmodium species [20]. The commercially available abTES™ Malaria 5 qPCR II Kit (AITbiotech, Singapore) is used for subsequent validation of any inconclusive results. However, the gene target is undisclosed, and there are no published data evaluating this assay on clinical P. knowlesi samples.

This study aimed to evaluate the diagnostic accuracy and limit of detection of the two commercially available multiplex real-time PCR methods used in routine molecular diagnosis and surveillance reporting of P. knowlesi and other human-only Plasmodium species in Sabah, Malaysia.

A total of 134 samples collected from Dec 2012 to Feb 2016 were included in the primary analysis evaluating the performance of the real-time PCR detection methods, including: 52 P. knowlesi, 21 P. falciparum, 25 P. vivax, and 10 P. malariae monoinfections, and 26 malaria-negative controls. The median age for those with P. knowlesi was 35 years (range 25–47) which was higher than patients infected with other Plasmodium species (p < 0.001) (Table 2). Plasmodium knowlesi-infected patients had a geometric mean parasite count of 9435/µL, similar to those with P. vivax (p = 0.99), higher than the 1023 parasites/µL seen for P. malariae (p < 0.001), and lower than the 24,631 parasites/µL for P. falciparum (p = 0.002). Performance of the screening hospital microscopy result for detecting P. knowlesi compared to the reference PCR demonstrated a sensitivity of 78.8% (95% CI 65.3–88.9) and specificity of 80.4% (95% CI 67.6–89.8), with 21.2% of samples diagnosed as P. malariae.

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 [8], 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 threefold 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 [20]. 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 [36], as utilized by the QuantiFast™ assay. This validated gene target is also commonly used for both real-time and nested PCR methods for other Plasmodium species differentiation [37], due to a unique Plasmodium genus core sequence and a separate highly conserved Plasmodium species-specific region [38], 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 [21].

The use of confirmatory molecular detection methods have enabled accurate reporting of malaria trends in Malaysia demonstrating increasing P. knowlesi incidence [3], and have also provided reliable data on national and sub-national progress towards achieving elimination of other human-only Plasmodium species [2]. 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 [39], 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 [42], Peninsular Malaysia [43] and Cambodia [44]. Plasmodium cynomolgi is morphologically similar to P. vivax on microscopic blood film evaluation [45], and due to being closely genetically related to P. vivax, previous PCR detection methods have also demonstrated cross-reactivity between these Plasmodium species [44]. This may have implications for accuracy of P. vivax case reporting, and potential underestimation of P. cynomolgi incidence in Southeast Asia [19].

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 [22]. 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 [46]; 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.

The QuantiFast™ and abTES™ methods for detection of P. knowlesi infections both performed to an appropriately high standard in the primary evaluation of this study, supporting their continued usage for confirmatory malaria diagnosis and accurate malaria surveillance reporting in Sabah, Malaysia. The QuantiFast™ kit is a cost-effective initial method, with the abTES™ kit appropriate for second-line confirmation of negative or mixed Plasmodium species infections due to potentially improved sensitivity and specificity.