https://orcid.org/0000-0001-7075-9491
Figures
Abstract
Background
Zoonotic P. knowlesi and P. cynomolgi symptomatic and asymptomatic infections occur across endemic areas of Southeast Asia. Most infections are low-parasitemia, with an unknown proportion below routine microscopy detection thresholds. Molecular surveillance tools optimizing the limit of detection (LOD) would allow more accurate estimates of zoonotic malaria prevalence.
Methodology/Principal findings
An established ultra-sensitive Plasmodium genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for P. knowlesi, P. cynomolgi and P. vivax using total nucleic acid preserved (DNA/RNA Shield) isolates and archived dried blood spots (DBS). LODs for selected P. knowlesi-specific assays, and reference P. vivax- and P. cynomolgi-specific assays were determined with reverse transcription (RT). Assay specificities were assessed using clinical malaria samples and malaria-negative controls.
The use of reverse transcription improved Plasmodium species detection by up to 10,000-fold (Plasmodium genus), 2759-fold (P. knowlesi) and 1000-fold (P. vivax and P. cynomolgi).
The Kamau et al. Plasmodium genus RT-qPCR assay was highly sensitive for P. knowlesi detection with a median LOD of ≤0.0002 parasites/μL compared to 0.002 parasites/μL for P. cynomolgi and P. vivax. The LODs with RT for P. knowlesi-specific PCRs were enhanced for the Imwong et al. 18S rRNA (0.0007 parasites/μL) and Divis et al. real-time 18S rRNA (0.0002 parasites/μL) assays, but not for the Lubis et al. hemi-nested SICAvar (1.1 parasites/μL) and Lee et al. nested 18S rRNA (11 parasites/μL). The LOD for P. vivax- and P. cynomolgi-specific assays with RT were moderately improved at 0.02 and 0.002 parasites/μL, respectively (1000-fold change). For DBS P. knowlesi samples the use of RT also markedly improved the Plasmodium genus qPCR LOD from 19.89 to 0.08 parasites/μL (249-fold change); no LOD improvement was demonstrated in DBS archived beyond 6 years. The Plasmodium genus and P. knowlesi-assays were 100% specific for Plasmodium species and P. knowlesi detection, respectively, from 190 clinical infections and 48 healthy controls. Reference P. vivax-specific primers demonstrated known cross-reactivity with P. cynomolgi.
Author summary
The monkey malaria parasite Plasmodium knowlesi has been found to increasingly infect humans across Southeast Asia via the bite of its anopheline mosquito vectors. Human infections with a similar monkey parasite, Plasmodium cynomolgi, have also been reported. The diagnostic tools commonly used to detect these malaria species are often unable to detect very low-level infections. We aimed to improve surveillance detection tools and blood sample collection methods to detect these zoonotic malaria species and understand the extent of transmission and the burden of disease. This study validated and compared the use of molecular laboratory assays targeting these species. We found that with the use of reverse transcription, large improvements in the limit of detection were possible, by up to 10,000-fold for initial malaria screening, and up to 2759-fold for specific P. knowlesi detection. Findings from this study support the use of ultrasensitive detection tools to improve surveillance approaches to emerging zoonotic malaria species.
Citation: Braima KA, Piera KA, Lubis IND, Noviyanti R, Rajahram GS, Kariodimedjo P, et al. (2025) Improved limit of detection for zoonotic Plasmodium knowlesi and P. cynomolgi surveillance using reverse transcription for total nucleic acid preserved samples or dried blood spots. PLoS Negl Trop Dis 19(2): e0012129. https://doi.org/10.1371/journal.pntd.0012129
Editor: Richard Culleton, Institute of Tropical Medicine, JAPAN
Received: April 4, 2024; Accepted: January 20, 2025; Published: February 6, 2025
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by the ZOOMAL project, funded through the Australian Centre for International Agricultural Research and Indo-Pacific Centre for Health Security, DFAT, Australian Government (#LS-2019-116 to MJG), the National Institutes of Health, USA (#R01AI160457-01 to GSR), and DOD-DHA-Global Emerging Infections Surveillance program project P0097_22_N2 – to AGL). Funding support was also through the National Health and Medical Research Council, Australia (Grant Numbers #1037304 and #1045156 to NMA, fellowship to NMA [#1042072], Emerging Leadership 2 Investigator Grants to MJG [#2017436] and BEB [#2016792]), and the Ministry of Health, Malaysia (#BP00500/117/1002) awarded to GSR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors declare that they have no competing interests.
Introduction
Plasmodium knowlesi is a unicellular protozoan malaria parasite present across Southeast Asia within the geographical range of its natural monkey hosts and vector mosquitoes [1,2]. P. knowlesi is the most common cause of human malaria in Malaysia; capable of causing severe disease comparable to P. falciparum [3–5]. Human infections with other genetically similar zoonotic species, such as P. cynomolgi which share the same natural macaque hosts, have also been reported [6]. Accurate detection of emerging zoonotic species such as P. knowlesi and P. cynomolgi in co-endemic areas with other human Plasmodium species infections is necessary to understand the geographical extent of zoonotic malaria transmission and to improve regional estimates of the disease burden [7]. Improving national malaria control program detection and reporting of low-level P. knowlesi infections is also vital to demonstrate World Health Organization (WHO)-certified elimination for other non-zoonotic malaria species in Southeast Asia [8].
Conventional malaria diagnostic methods such as microscopy lack sensitivity and specificity for active surveillance of P. knowlesi due to common low-level sub-microscopic infections and an inability to accurately distinguish other morphologically similar Plasmodium species [7,9]; notably P. malariae and the early ring stages of P. falciparum [10,11]. Similarly, P. cynomolgi microscopically resembles P. vivax in human infections [12,13]. Current malaria rapid diagnostic tests which detect circulating Plasmodium species antigens also remain insufficiently sensitive for P. knowlesi passive case detection at the low parasite counts able to produce symptomatic infections [14–16].
Multiple molecular methods to detect P. knowlesi have been developed, including both quantitative and conventional qualitative PCR assays of different gene targets [7,17]. However, surveillance strategies for rare or low-level infections such as P. knowlesi require maximising initial screening limits of detection, with systematic comparisons of the lower limit of detection (LOD) of these assays currently lacking [7]. The degree to which the LOD is enhanced with a prior reverse transcription (RT) step is not well characterised despite potential benefits in improving the detection of very low-level parasitemia symptomatic or asymptomatic zoonotic Plasmodium species infections [18]. The Plasmodium genus real-time assay selected for evaluation in this study [19] aims to maximise the detection threshold for zoonotic malaria screening by targeting the known high multicopy number (5 to 10 copies per genome) of the 18S rRNA gene [20], in addition to amplification of both the A- and S- type genes and their RNA transcripts. Many of the most commonly used non-zoonotic [21] and zoonotic [22] Plasmodium species-specific assays also target the 18S rRNA gene and would fit within a clear integrated subsequent laboratory workflow, although the sensitivity of these assays that include a reverse transcription step has not been evaluated. Specificity for P. knowlesi detection also ideally needs to be validated against other macaque zoonotic Plasmodium species, including P. cynomolgi [7].
To support an improved molecular surveillance workflow for detection of low-level zoonotic Plasmodium species infections [7,23], we evaluated an established Plasmodium genus and species-specific PCR assays with the inclusion of a reverse transcription step to enhance the LOD in either total nucleic acid preserved media or more logistically feasible dried blood spot (DBS) collected samples.
Methodology
Ethics approval and consent to participate
Sample collection and diagnostic evaluation were approved as part of prospective malaria studies by the Medical Research and Ethics Committee (MREC), Ministry of Health Malaysia (NMRR-10-754-6684, NMRR-19-4109-52172 and NMRR-19-3229-49967), Universitas Sumatera Utara, Indonesia (#723/KEP/USU/2021) and by the Menzies School of Health Research, Australia (HREC-2010-1431 and HREC-2022-4417) in accordance with all applicable Federal and other regulations governing the protection of human subject research. Written informed consent was obtained from all participants, including parents/guardians of those less than 18 years of age.
Clinical specimen collection and storage
Clinical venous whole blood samples and dried blood spots were collected prior to antimalarial treatment from individuals diagnosed with malaria by routine hospital microscopy, as part of an ongoing prospective malaria study in Sabah, Malaysia between April 2013 and May 2023. Adult healthy individuals were recruited as malaria-negative controls. Additional P. vivax clinical cases were enrolled from a prospective malaria study in western Indonesia between January 2022 and August 2023. Whole blood samples were collected in ethylenediaminetetraacetic acid (EDTA) and a small subset in DNA/RNA Shield (Zymo Research, Irvine, CA, USA) before being frozen at -80°C at the time of enrolment. DBS were concurrently made using 20μL whole blood spotted on Whatman 3MM cellulose chromatography filter paper and then stored in cardboard covers in individual sealed bags with desiccant at room temperature unexposed to direct light. A single donated P. cynomolgi-infected sample [24] obtained from a macaque host was frozen in glycerolyte and stored in liquid nitrogen prior to thawing, counting and immediately placing in DNA/RNA Shield.
Microscopic parasite count quantification
Microscopic diagnosis of Plasmodium species was undertaken by experienced research microscopists using thick and thin Giemsa-stained blood films. Microscopic quantification of parasitemia (parasites per microlitre) was performed using thick blood smears calculated from the number of parasites per 200 white blood cells, multiplied by the individual patient’s total white cell count obtained from routine hospital laboratory flow cytometry [25].
Total nucleic acid extraction and PCR amplification
Total nucleic acids were directly extracted from 200 μL of whole blood using a QIAamp DNA Blood Mini Kit (QIAGEN, Cat. No. 51106), with DNA/RNA Shield samples eluted in 50μl AE buffer to account for the preservative dilution factor. DNA and RNA extraction from DBS were carried out using an in-house method, established by Zainabadi et al, 2017 [26]. Briefly, DBS equivalent to 40μl whole blood was incubated with 900μl lysis buffer at 65°C, with shaking at 250rpm for 90 minutes. Lysate was transferred to QIAamp DNA Blood Mini Kit (QIAGEN, Cat. No. 51106) columns, washed with modified buffers, dried at 65°C for 10 minutes and eluted in 40μl buffer AE. The primers, annealing temperatures and/or probe sequences for each PCR assay are described in Table 1. Real-time PCR amplifications were performed on a QuantStudio 6 Flex (Applied Biosystems). Conventional PCR was performed on a DNA thermal cycler (Bio-Rad T100™ thermal cycler). The amplified nested PCR products were separated by electrophoresis using 2% agarose gels, stained by SYBR Safe™ (Invitrogen), and visualised on a UV transilluminator (Gel Doc XR+ imaging system, Bio-Rad). Each PCR amplification included a Plasmodium species positive and negative control and molecular weight standards (Applied Biosystems). Positive (clinical malaria cases) and negative (healthy afebrile study participant) control samples were derived from venous whole blood collected in EDTA from Sabah participants with malaria status confirmed using validated reference PCR [21,22,27].
Plasmodium species confirmation using reference PCR
A validated reference PCR targeting the Plasmodium 18S rRNA genes was used to confirm the Plasmodium species using EDTA-whole blood clinical samples, consisting of an initial Plasmodium genus (hereafter abbreviated to P. genus) nest 1, followed by species-specific nest 2 for P. falciparum, P. vivax, P. malariae, and P. ovale spp. [21]. The P. knowlesi- [27] and P. cynomolgi-specific [22] reference assays used also target 18S rRNA genes. The P. knowlesi reference assay has a previously reported high sensitivity (LOD less than 10 parasite genomes) when validated without reverse transcription (RT) against four P. knowlesi strains (Malayan, H, Philippine, and Hackeri); specificity was assessed against the major human Plasmodium species in addition to other simian Plasmodium species present in Southeast Asia including P. cynomolgi, P. inui, and P. simiovale [27].
Selection of PCR assays for LOD evaluation
A reverse transcriptase real-time hydrolysis probe (RT-qPCR) assay designed to detect the P. lasmodium genus was evaluated for its utility in enhancing LOD (Fig 1A) [19]. Three published P. knowlesi species-specific assays, which have not previously had their LODs evaluated using reverse transcription, were compared to the reference assay (Fig 1B). Test A is a qPCR assay [28] in current use in the State Public Health Laboratory (Makmal Kesihatan Awam; MKA) Sabah, Malaysia, for routine P. knowlesi malaria detection [30]. Test B is a hemi-nested PCR targeting the multicopy SICAvar genes, previously validated for P. knowlesi detection against P. falciparum, P. vivax, P. malariae, and P. ovale spp., in addition to clinical P. knowlesi isolates from Sarawak, Malaysia [29]. Test C is a nested conventional PCR assay targeting 18S rRNA genes specific for P. knowlesi that has been robustly validated against P. vivax and other zoonotic Plasmodium species [22].
Abbreviations: DBS = dried blood spot; LOD = limit of detection; qPCR = real-time quantitative PCR; Pk = P. knowlesi; Pf = P. falciparum, Pv = P. vivax; Pcyn = P. cynomolgi; RT = reverse transcription.
Limit of detection evaluation for PCR assays with and without RT
The initial microscopy-quantified Plasmodium species infected samples collected in DNA/RNA Shield were diluted with malaria-negative whole blood (also at the same manufacturer recommended DNA/RNA Shield ratio) to prepare individual parasite count dilutions ranging between 20 to 0.0002 parasites/μL. Total nucleic acids from samples at each serial parasite count dilution were then extracted and duplicate aliquots prepared. High-capacity cDNA reverse transcription (Applied Biosystems, Thermo Fisher Scientific, MA, USA) was then performed on one aliquot from each pair according to manufacturer’s recommendations. PCR was performed on the paired aliquots to detect P. genus by the qPCR assay [19], followed by species-specific assays for: P. knowlesi by the reference hemi-nested assay [27] and test assays A, B, C [22,28,29]; P. vivax [21] and P. cynomolgi [22] by reference assays. The LOD was expressed as the lowest parasite count per microlitre of whole blood detected by an individual PCR assay in both amplification replicates (Fig 1C). All P. genus qPCR assays were run in duplicate for individual samples. The nested species-specific assays were run in singlicate for each sample.
Limit of detection of P. genus qPCR assay for dried blood spots (DBS) with and without RT
The LOD for the P. genus qPCR assay was also evaluated using archived DBS from P. knowlesi clinical infections with and without RT (Fig 1C). Nucleic acids were extracted from the DBS samples, with 10μl immediately reverse transcribed into cDNA. P. genus qPCR was conducted on serial 1:10 DNA and cDNA dilutions run in duplicate with the LOD calculated using the initial enumerated parasitemia divided by the corresponding dilution level.
Evaluation of PCR assay specificity on clinical malaria samples
For the analysis of P. knowlesi, P. falciparum, and P. vivax clinical isolates, in addition to a P. cynomolgi macaque-derived isolate, all samples were individually tested using the P. genus qPCR assay and the three P. knowlesi-specific PCR assays. Results were compared against the reference PCR. Specificity for the P. genus assay was evaluated for malaria detection (any Plasmodium species) versus malaria-negative controls, and for species-specific assays using the corresponding Plasmodium species infection versus other Plasmodium species and malaria-negative controls combined. Clinical whole blood samples collected in EDTA likely had RNA degradation upon thawing; therefore, reverse transcription was not performed for this part of the analysis.
Statistical analyses
Parasite counts for each clinical Plasmodium species infection were summarised using median and interquartile range (IQR). The median whole blood LOD was calculated with and without reverse transcription for the P. knowlesi and P. vivax isolates. To calculate the LOD fold-change, the LOD without RT was divided by the LOD with RT. One-way ANOVA was used to test for differences in parasite count distribution across Plasmodium species, followed by Student’s t-test for pairwise comparisons of log-transformed data. Results of PCR assays evaluated against reference PCR were defined as true positive (TP), false negative (FN), true negative (TN), and false positive (FP), enabling calculation of diagnostic sensitivity (TP/TP+FN) and specificity (TN/TN+FP) with exact binomial 95% confidence intervals. All statistical analyses were performed using Stata version 17.0 (StataCorp, Texas, USA).
Results
Limit of detection of P. genus PCR assays
The LOD was performed on P. knowlesi (n = 4), P. vivax (n = 4) and P. cynomolgi (n = 1) whole blood isolates. For the P. genus Kamau et al. qPCR assay, without reverse transcription, the median LOD to detect each individual Plasmodium species was 2 parasites/μL (Table 2 and Fig 2A). With reverse transcription, the assay sensitivity for P. knowlesi improved with a LOD of ≤0.0002 parasites/μL for all four isolates (10,000-fold change). The LOD for both P. vivax and P. cynomolgi improved to 0.002 parasites/μL with RT (1,000-fold change); Fig 2B. In comparison, the reference Snounu P. genus assay had a slightly higher LOD for P. vivax and P. cynomolgi without RT (0.2 parasites/μL for both); however, with RT a less pronounced improvement in LOD was demonstrated at 0.02 and 0.01 parasites/μL, respectively.
(A) The median LOD (parasites/μL) for P. genus assays using P. knowlesi, P. vivax, and P. cynomolgi samples without reverse transcription; (B) The median LOD (parasites/μL) for assays with reverse transcription. Error bars represent the IQR. Abbreviations: P. genus, Plasmodium genus; Pk, P. knowlesi; Pv, P. vivax; Pc, P. cynomolgi.
Limit of detection of Plasmodium species-specific PCR assays
The Plasmodium species-specific LOD evaluation was performed on P. knowlesi (n = 4), P. vivax (n = 1) and P. cynomolgi (n = 1) whole blood isolates previously evaluated for Plasmodium genus detection. For the P. knowlesi-specific assays, the median LOD without and with reverse transcription was 2 and 0.0007 parasites/μL, respectively, for the Imwong et al. reference assay (2759-fold change); 0.2 and 0.0002 parasites/μL, respectively, for Test A (1000-fold); 0.11 and 1.1 parasites/μL, respectively, for Test B (no improvement); and 11 parasites/μL for both (no change) for Test C (Table 2 and Fig 2A).
Without reverse transcription, the LODs using species-specific reference assays were 2 parasites/μL for P. cynomolgi and 20 parasites/μL for P. vivax. With reverse transcription, the LODs were 0.002 and 0.02 (1000-fold change) for P. cynomolgi and P. vivax, respectively. However, additional testing of the P. cynomolgi isolate using the reference P. vivax-targeted rVIV1/rVIV2 primers [21] amplified this target from the macaque-origin P. cynomolgi infection both without (0.2 parasites/μL) and with reverse transcription (0.02 parasites/μL), producing a false-positive P. vivax result (Table 2 and Fig 2B).
The LOD with reverse transcription of the P. genus assay was comparable to the LOD of the best performing species-specific assay for P. knowlesi (≤0.0002 parasites/μL) and P. cynomolgi (0.002 parasites/μL) detection. In contrast, the P. genus assay had a superior LOD compared to the reference species-specific assay for P. vivax (0.002 vs 0.02 parasites/μL respectively).
Limit of detection of P. genus qPCR for dried blood spots (DBS)
The median LOD between DNA and cDNA generated from dried blood spots (DBS) for 4 P. knowlesi extracted samples collected 8 months prior to evaluation of the P. genus Kamau 2011 assay without and with RT was 19.86 and 0.08 parasites/uL, respectively (249-fold change) (Table 3). Archived DBS samples (n = 12) collected more than 6 years (up to 11 years) prior to extraction demonstrated a similar LOD with and without RT (median 20 parasites/μL).
Specificity of P. genus and individual Plasmodium species PCR assays
A total of 239 whole blood samples were included in the clinical evaluation of test specificity without RT, consisting of 96 P. knowlesi, 50 P. vivax, 44 P. falciparum, and 1 P. cynomolgi infected samples, and 48 malaria-negative controls. The median parasitemia was 1,957/μL (IQR 261–5,762; range 27–210,100 parasites/μL) for P. knowlesi, 3,246/μL (IQR 1,588–7,306; range 77–20,064 parasites/μL) for P. vivax, and 14,015/μL (IQR 2,193–33,413; range 34–297,000 parasites/μL) for P. falciparum.
The P. genus qPCR screening assay was both 100% specific and sensitive for the detection of Plasmodium species overall compared to the reference PCR, with all 48 samples from uninfected controls confirmed as malaria-negative.
P. knowlesi- assays for Tests A [28] and B [29] correctly identified all 96 P. knowlesi and 95 non-P. knowlesi samples, resulting in 100% specificity and sensitivity. Test C [22] was negative for a single P. knowlesi isolate with a parasite count of 1,535 parasites/μL, resulting in 99% (95% CI 94.3–100.0) sensitivity and 100% specificity. The reference assays for P. falciparum and P. vivax [21], and for P. cynomolgi [22] were negative for all P. knowlesi clinical isolates tested (Table 2).
Discussion
Malaria-susceptible countries in most of Southeast Asia, including those approaching or achieving WHO elimination of major human-only Plasmodium species, remain at-risk for zoonotic malaria transmission [31,32]. Understanding regional heterogeneity in P. knowlesi transmission intensity and disease morbidity will require the selective deployment of highly sensitive and specific molecular detection tools for both diagnostic and surveillance purposes [23]. Our major finding demonstrated that the use of a reverse-transcription step after extraction of preserved total nucleic acids in clinical samples considerably improves the limit of detection of both the selected P. genus RT-qPCR screening assay, and P. knowlesi-specific assays (reference and Test A; both >1000-fold) by additionally amplifying ribosomal RNA sequences. The enhanced limit of detection was consistent across both field-stable DNA/RNA Shield samples and to a lesser degree in recent (although not older) dried blood spots. The second key finding of this study was the excellent performance of the P. genus screening assay, originally developed and validated for use in an African context for P. falciparum [19], to detect previously unvalidated species including P. knowlesi, P. cynomolgi and P. vivax. The specificity of each of the P. knowlesi-targeted assays to exclude P. cynomolgi and non-zoonotic Plasmodium species was confirmed to be excellent. Together these findings highlight the potential utility of incorporating these assays in a molecular surveillance approach to detect both human and zoonotic Plasmodium species that are well below the reported parasite count detection limits for current conventional PCR, loop-mediated isothermal amplification, microscopy or parasite lactate-dehydrogenase-based rapid diagnostic tests [23].
The community-based detection of submicroscopic P. knowlesi and P. cynomolgi infections, both symptomatic or asymptomatic, requires ultrasensitive molecular tools to understand the true extent of population-level transmission [23]. Recent studies in areas of both Peninsular Malaysia and the East Malaysian state of Sarawak have reported human infections in local communities living in or near forested areas with other macaque malaria species, including P. inui, P. coatneyi, P. fieldi and possibly P. simiovale, in addition to P. knowlesi and P. cynomolgi [33]. It is unclear whether or to what extent these low-level zoonotic infections may facilitate onward transmission to humans [34,35], as occurs with low parasitemia P. falciparum and P. vivax infections [36,37], although sustained human-to-human transmission of P. knowlesi has not been evident to date [22,38].
The selection of the major RT-qPCR P. genus screening assay aimed to maximise the detection limits for low-level zoonotic Plasmodium species infections using the known high copy number 18S rRNA target [20], and amplification of both the A- and S- type genes and their RNA transcripts [19]. The excellent LOD demonstrated with the P. genus RT-qPCR assay in the current study of <0.0002 parasites/μL for P. knowlesi detection is consistent with a previously reported extremely low LOD of ~0.0004 parasites/μL for clinical P. falciparum samples [19]. DNA-concentrated packed red blood cell samples have been demonstrated to further improve sensitivity for P. falciparum detection in population-based malaria prevalence surveys in elimination areas of Thailand [39]. Comparable performance to this P. genus RT-qPCR assay was reported with a separately established ultrasensitive quantitative PCR method (uPCR) with a limit of detection of 0.022 parasites/μL [39], however, a major advantage of the reverse transcriptase qPCR assay is the requirement for comparatively lower blood volumes [18]. Interestingly, in the current study the conventional PCR P. genus reference assay also demonstrated a large increase in analytical sensitivity after reverse transcription (~1800-fold) and may provide a more cost-effective option compared to qPCR for surveillance purposes. DNA/RNA Shield was selected as the preferred blood collection preservation method over other media due to its reported ability to stabilise DNA/RNA at ambient temperatures in field settings and compatibility with most DNA and RNA purification kits for subsequent high-throughput workflows including reverse transcription [40].
To date, only a few studies have incorporated reverse transcription in the molecular detection of Plasmodium species [41,42]. The reverse transcription step in the present study improved the analytical sensitivity of our selected assays to detect zoonotic P. knowlesi, P. cynomolgi and other human malaria infections by up to 10,000-fold (Plasmodium genus), 2759-fold (P. knowlesi), and 1000-fold (P. vivax and P. cynomolgi), respectively. The P. knowlesi-specific hemi-nested reference assay with reverse transcription demonstrated a comparable limit of detection to the qPCR Test A (which requires an expensive real-time hydrolysis probe), and was superior to both Test B targeting SICavar and Test C targeting the 18S rRNA gene. Without reverse transcription, the lowest limit of detection for P. knowlesi was seen with Test B, suggesting that the multiple chromosomal copies of the variant antigen SICAvar provide equivalent or better signal amplification than the detection of transcripts from whichever of these gene copies is activated in any particular parasite cell in the peripheral blood. Constraints on the widespread use of reverse transcription include the additional cost, laboratory time, and the usual rapid degradation of RNA molecules in field or laboratory settings. However, the degree of RNA amplification with reverse transcription was aided in our study by collecting blood samples in room-temperature stable RNA preservation media suitable for field-based surveillance, which also allows other potential downstream pathophysiological or transcriptomic analyses dependent on pathogen or host RNA transcripts.
The low reported limit of detection for the RT-qPCR P. genus assay conducted on DBS in this study (~0.08 parasites/μL with reverse transcription) suggests this type of sample collection would also enable the identification of a large proportion of submicroscopic and/or asymptomatic infections. DBS collection is logistically a more feasible, inexpensive and acceptable option particularly for asymptomatic or younger participants (given the need for fingerprick blood collection rather than venepuncture) for large-scale malaria surveillance surveys. The use of Whatman 3MM filter paper for storage and extraction of DBS with reverse transcription was justified compared to other potential media including Whatman 903 protein saver or FTA classic cards due to the much lower cost and superior detection limits seen when validated using P. falciparum mRNA gametocyte transcripts [43,44]. However, the reverse transcription step only improved the LOD in DBS samples that were collected within 8 months; older DBS stored in recommended conditions for more than 6 years did not provide any improvement in the LOD with and without RT due to likely degradation of RNA. Regardless, the P. genus LOD of around 2 to 20 parasites/μL without RT for DBS samples remains encouraging as a first-line option for surveillance screening purposes. The use of DBS would require further evaluation with Plasmodium species-specific PCR assay differentiation, similar to that conducted for the SICAvar assay on DBS for P. knowlesi which had a very low reported LOD of 0.1 parasites/μL [29].
The current study confirmed previous findings detailing cross-reactivity between the nested PCR primers for P. vivax (rVIV1/rVIV2) with P. cynomolgi [13]. A single mismatch in the 30 nucleotides of the rVIV2 primer sequences was reported to cross-amplify P. cynomolgi isolates [13]. The nested P. vivax-specific assay using the same primers rVIV1/rVIV2 designed to target the 18S rRNA gene also amplified P. cynomolgi in our LOD analysis [21]. The separate P. cynomolgi primers remained highly specific and did not erroneously amplify P. vivax or other closely related Plasmodium species DNA. In practice, the concurrent use of both assays would enable accurate identification of a P. vivax mono-infection, however, this approach would not be able to differentiate a P. vivax/P. cynomolgi co-infection from a P. cynomolgi mono-infection. Mis-identification of symptomatic P. cynomolgi infections as P. vivax would not result in inappropriate treatment, given both have a latent hypnozoite liver life-stage requiring additional radical cure with primaquine. Most other commonly used single-round multiplex [45] and qPCR assays [46] containing P. vivax-specific targets have also not been validated against isolates of P. cynomolgi or other closely related macaque Plasmodium species. However, a variety of sequencing approaches of targeted gene amplicons including mitochondrial COX1 and cytochrome b, SICAvar and SSU 18S rRNA followed by sequencing and reference alignment have been used to confirm unknown or mixed zoonotic Plasmodium species infections following initial ambiguous PCR results [47,48].
A limitation of this study was the inability to validate submicroscopic clinical P. knowlesi infections and other zoonotic species such as P. fieldi, P. inui and P. coatneyi for which samples were not available. Due to the increasing number of published P. knowlesi assays, we were not able to evaluate other P. knowlesi assays of possibly comparable performance within our selected workflow. There is a small inherent additional risk of contamination with the inclusion of a RT step in this type of ultra-sensitive PCR workflow. We were also unable to evaluate mixed infections of P. knowlesi, P. vivax and P. falciparum despite these cases being reported in certain areas such as Indonesia [29] and Vietnam [49]. Due to sample availability, we were also restricted to only a single P. cynomolgi sample to determine the LOD of the P. cynomolgi-specific assay [22]. The discrepancy between the LOD for the P. genus qPCR screening assay and the species-specific assay for P. cynomolgi detection may mean a proportion of very low-level P. cynomolgi infections are unable to be identified beyond a P. genus threshold using the current protocol.
Conclusions/Significance
The Plasmodium genus reverse transcriptase qPCR assay can provide highly sensitive screening for zoonotic and human malaria, including for submicroscopic infections in at-risk populations in endemic areas. The use of this molecular surveillance protocol for either whole blood or DBS collected samples in understudied areas of Southeast Asia would enable improved understanding of the regional disease burden and transmission dynamics of zoonotic malaria. Enhanced molecular tools and future iterative improvements to conventional surveillance protocols are especially critical as Southeast Asia continues to exert considerable public health efforts towards human malaria elimination despite the challenge of additional zoonotic Plasmodium species infections at an expanding human-animal-interface.
Disclaimers
The views expressed are those of the author(s) and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. Copyright Statement: NC (LCDR, MSC, USN) & AL (CAPT, MC, USN) are military service members and employees of the U.S. Government. This work was prepared as part of their official duties at U.S. Naval Medical Research Unit INDO PACIFIC. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties.
Supporting information
S1 Data. Limit of detection dataset for PCR assays.
https://doi.org/10.1371/journal.pntd.0012129.s001
(XLSX)
Acknowledgments
We thank the study participants, the IDSKKS malaria research team (Mohd Rizan Osman, Danshy Alaza, Azielia Elastiqah, Sitti Saimah binti Sakam), and Bruce Russell of the Department of Microbiology & Immunology, University of Otago, Dunedin, New Zealand, for providing the P. cynomolgi isolate. We thank the Director General of Health Malaysia for the permission to publish this article.
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