Study details and ethical approval
Patient demographics, clinical data, and blood samples were collected as part of an ongoing prospective malaria study in Sabah, Malaysia. Patients with positive microscopy for malaria and adult healthy controls, subsequently confirmed malaria-negative via reference PCR [21, 22], were enrolled after informed consent was obtained. The study was approved by the national Medical Research Ethics Committee of Malaysia, and Menzies School of Health Research, Australia.
Blood sample procedures
Venous whole blood was collected from all participants prior to any antimalarial treatment. Microscopic quantification of Plasmodium species parasitaemia was conducted by an experienced research microscopist in Sabah (parasites per microliter; calculated from the number of parasites per 200 white blood cells on thick blood film, multiplied by the individual patient’s total white cell count [23] obtained from routine hospital laboratory flow cytometry (Full Blood Count results); in the absence of which, an assumption of an average WBC count of 8,000/µL of blood was used). EDTA whole blood samples were stored at -80°C and transported via liquid nitrogen shipper to Darwin, Australia. Genomic DNA was subsequently extracted from 200 µL of whole blood using QIAamp DNA Blood Mini Kits (Cat. No.: 51106; QIAGEN) according to the manufacturer’s manual, with a final elution volume of 200 µL.
Detection of Plasmodium species using validated reference nested PCR
For P. knowlesi detection, a previously validated nested PCR targeting the SSU rRNA gene was utilised, which has a reported specificity of 100% against other Plasmodium species infecting humans and/or relevant macaque hosts, and a sensitivity of detection for P. knowlesi of 1-10 parasite genomes per microliter [22]. A separate validated nested PCR was conducted on clinical malaria samples to identify P. falciparum, P. vivax and P. malariae [21]. Samples were then de-identified and randomly assigned onto duplicated 96-well plates for QuantiFast™ and abTES™ PCR evaluation.
Evaluation of the QuantiFast™ and abTES™ real-time PCR kits
Real-time PCR detection of Plasmodium species was performed by laboratory research members blinded to the reference nested PCR results. Both the QuantiFast™ and abTES™ real-time PCR assays were conducted once for each clinical isolate in accordance with the manufacturers’ and Sabah Public Health Laboratory protocols, using the Bio-Rad CFX96 Touch™ PCR machine (Bio-Rad, USA) and duplicated plates of genomic DNA extracted from the same isolates.
QuantiFast™ primer and probe sequences target the Plasmodium species-specific 18S SSU rRNA gene for P. knowlesi [20] and other human-only Plasmodium species [24] (described in Table 1). QuantiFast™ real-time PCR was carried out over two separate reactions due to overlapping emission wavelengths of the reporter probe (carboxyfluorescein; FAM) used to detect both P. knowlesi and P. malariae. Each final reaction volume of 25 μl consisted of 2 μl DNA template, 12.5 μl master mix, 0.5 μl ROX solution and 1.25 μl 10x Plasmodium species-specific primer-probe mixture. In the first QuantiFast™ reaction, a monoplex real-time PCR amplification was performed to detect P. knowlesi. Cycling conditions consisted of: initial Taq activation step at 95°C for 5 min, followed by 45 two-step cycles of denaturation at 95°C for 30 s, and annealing/extension at 60°C for 30 s. In the second QuantiFast™ reaction, a triplex amplification was conducted to detect P. falciparum, P. vivax, and P. malariae. Cycling conditions included: Taq activation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 45 s, and annealing/extension at 60°C for 45 s.
The abTES™ reaction was performed using the abTES™ Malaria 5 qPCR II kit, which came with primer-probe mixtures and positive controls for detection of P. knowlesi and four human-only Plasmodium species. The reaction mixture contained 5.0 μl template DNA, 6 μl reaction mix, 2 μl of primer-probe mix, with the final volume adjusted to 25 μl with nuclease-free water. Cycling conditions included: Taq activation at 95°C for 2 min, followed by 45 cycles of amplification at 95°C for 5 s, and 60°C for 20 s. The detection channels used were QUASAR 705 (P. knowlesi), FAM (P. falciparum), ROX (P. vivax), and HEX (P. malariae), with fluorescence measured at the end of each cycle of amplification.
For both QuantiFast™ and abTES™ reactions, samples were considered positive by determining the threshold cycle number (CT) at which normalized reporter dye emission raised above background noise. If the fluorescent signal did not rise above the threshold at 40 cycles (CT 40), the sample was considered negative. Due to the lack of endemic P. ovale wallikeri or P. ovale curtisi in Malaysia, detection of these Plasmodium species was not evaluated.
Limit of detection determination for each PCR method and Plasmodium species
The two PCR kits were also systematically evaluated for their respective parasite count limit of detection (LOD) for P. knowlesi, P. falciparum and P. vivax samples. In brief, a single clinical standard isolate for each Plasmodium species with high-quality research microscopic enumeration of parasite counts was utilised after reference PCR-confirmation. Individual quantified whole blood samples were then diluted using fresh malaria-negative blood in order to achieve pre-determined parasite counts, before subsequent genomic DNA extraction. The standardised concentrations included in the final analysis for each isolate were: 200, 20, 2, 0.5, 0.25, and 0.125 parasites/µL. The same clinical isolates at each parasite count concentration were used for both PCR detection kits, enabling a direct comparison of the final measured LOD. Assays were conducted in triplicate at each parasite count concentration, with the LOD defined as the lowest concentration at which a positive result was recorded for all 3 replicates.
Statistical analysis
All statistical analyses were performed using STATA v16 (TX, USA). The primary analysis compared the diagnostic accuracy of the QuantiFast™ and abTES™ kits to detect each Plasmodium species infection against the reference PCR result. Diagnostic tests for sensitivity, specificity, and positive and negative predictive values were evaluated [25], as defined below using the number of true positive (TP), false negative (FN), false positive (FP) and true negative (TN) results:
Sensitivity: proportion of those with the malaria species correctly identified = TP/(TP+FN)
Specificity: proportion of those without the malaria species correctly identified = TN/(FP+TN)
Area under the Receiver Operating Characteristic (ROC) curve: average of sensitivity and specificity
Positive Predictive Value: probability of the patient having malaria following a positive test = TP/(TP+FP)
Negative Predictive Value: probability of the patient having malaria following a negative test = TN/(TN+FN)
Exact binomial confidence intervals of 95% for each of the above diagnostic metrics were calculated and reported. Dependent comparisons between the separate PCR kits diagnostic performance on the same patient’s sample (e.g. QuantiFast™ versus abTES™ for P. knowlesi samples) were conducted using McNemar’s test [26]. Independent comparisons using the same PCR test between patients with different Plasmodium species infections were conducted using Fisher’s exact test for equality of proportions (e.g. QuantiFast™ performance for detecting P. knowlesi versus P. vivax). Overall PCR assay performance was compared by testing equality of the receiver operating characteristic (ROC) areas. Age and parasitaemia were compared across Plasmodium species results using one-way ANOVA after transformation to a normal distribution, followed by Student’s t-test for pairwise comparisons; sex was compared using chi-squared test.