https://orcid.org/0000-0002-4465-9830
Figures
Abstract
Background
The parasite species Plasmodium ovalecurtisi (P. ovalecurtisi) and Plasmodium ovalewallikeri (P. ovalewallikeri), formerly known as Plasmodium ovale, are endemic across multiple African countries. These species are thought to differ in clinical symptomatology and latency, but only a small number of existing diagnostic assays can detect and distinguish them. In this study, we sought to develop new assays for the detection and differentiation of P. ovalecurtisi and P. ovalewallikeri by leveraging recently published whole-genome sequences for both species.
Methods
Repetitive sequence motifs were identified in available P. ovalecurtisi and P. ovalewallikeri genomes and used for assay development and validation. We evaluated the analytical sensitivity of the best-performing singleplex and duplex assays using synthetic plasmids. We then evaluated the specificity of the duplex assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), and validated its performance using 55 P. ovale samples and 40 non-ovale Plasmodium samples from the DRC.
Results
The best-performing P. ovalecurtisi and P. ovalewallikeri targets had 9 and 8 copies within the reference genomes, respectively. The P. ovalecurtisi assay had high sensitivity with a 95% confidence lower limit of detection (LOD) of 3.6 parasite genome equivalents/μl, while the P. ovalewallikeri assay had a 95% confidence LOD of 25.9 parasite genome equivalents/μl. A duplex assay targeting both species had 100% specificity and 95% confidence LOD of 4.2 and 41.2 parasite genome equivalents/μl for P. ovalecurtisi and P. ovalewallikeri, respectively.
Conclusions
We identified promising multi-copy targets for molecular detection and differentiation of P. ovalecurtisi and P. ovalewallikeri and used them to develop real-time PCR assays. The best performing P. ovalecurtisi assay performed well in singleplex and duplex formats, while the P. ovalewallikeri assay did not reliably detect low-density infections in either format. These assays have potential use for high-throughput identification of P. ovalecurtisi, or for identification of higher density P. ovalecurtisi or P. ovalewallikeri infections that are amenable to downstream next-generation sequencing.
Author summary
Non-falciparum malaria appears to be on the rise, especially in settings where P. falciparum transmission is declining. Plasmodium ovalecurtisi and Plasmodium ovalewallikeri are neglected parasites that can cause relapsing malaria and are thought to differ in clinical symptomatology and latency. However, few existing diagnostic assays can detect and distinguish them. Most target the 18S rRNA gene of both P. ovalecurtisi and P. ovalewallikeri with potential for cross-reactivity at higher parasite densities, and are not well-suited for high-throughput use, hindering our understanding of their epidemiology. Mining recently available P. ovalecurtisi and P. ovalewallikeri reference genomes, we identify new multi-copy targets for molecular detection and develop novel singleplex and duplex real-time PCR assays capable of species differentiation. These assays are highly specific and require short turn-around time. The P. ovalecurtisi assay performed well, while the P. ovalewallikeri assay did not reliably detect low-density infections. These assays provide new options for high-throughput studies of P. ovalecurtisi infection, as well as identification of higher density infections amenable to next-generation sequencing of both species.
Citation: He W, Sendor R, Potlapalli VR, Kashamuka MM, Tshefu AK, Phanzu F, et al. (2024) Development of new real-time PCR assays for detection and species differentiation of Plasmodium ovale. PLoS Negl Trop Dis 18(9): e0011759. https://doi.org/10.1371/journal.pntd.0011759
Editor: Georges Snounou, Institut Pasteur, FRANCE
Received: October 30, 2023; Accepted: August 15, 2024; Published: September 10, 2024
Copyright: © 2024 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data and analysis R code underlying reported findings have been provided as part of the submitted article and https://github.com/Wenqiao33/P.ovale_assays.
Funding: This study was funded by the US National Institutes of Health (NIH R21AI148579 to JBP and JTL). It was partly supported by the Global Fund to Fight AIDS, Tuberculosis, and Malaria (MK, AT, FP, AK; DRC sample collection); NIH R01AI137395 (JTL and BN; Tanzania sample collection), K24AI134990 (JJJ), and T32AI070114 (RS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: JBP reports research support from Gilead Sciences, non-financial support from Abbott Laboratories, and consulting for Zymeron Corporation, all outside the scope of the manuscript. All other authors declare no competing interests.
Introduction
Malaria remains a major global health concern despite decades of sustained investment in elimination efforts. Though most malaria control programs within Africa prioritize Plasmodium falciparum, the parasite species responsible for most deaths, increasing evidence confirms co-circulation of other neglected Plasmodium species that cause human malaria [1–4]. Recent surveys reveal a previously unappreciated burden of Plasmodium ovalecurtisi and Plasmodium ovalewallikeri in multiple African countries [5,6], where relapsing malaria caused by these parasites may prove to be an obstacle to malaria elimination efforts [7,8]. P. ovalecurtisi and P. ovalewallikeri (previously known as P. ovale curtisi and P. ovale wallikeri), which were formerly known as Plasmodium ovale [9–12], have potential differences in clinical symptomatology and latency [13], but few existing diagnostic assays have ability to detect and distinguish them. Some require separate PCR runs, multiple steps (nested assays, agarose gel electrophoresis, and/or sequencing), or prolonged cycling time that increases risk of false-positive results [14–19].
Differentiation of P. ovalecurtisi and P. ovalewallikeri is not currently possible using microscopy, the gold standard for malaria diagnosis in the field [20]. P. ovalecurtisi and P. ovalewallikeri infections often occur as mixed infections at low density, and are morphologically indistinguishable on blood slides [21,22]. Furthermore, widely used malaria rapid diagnostic tests (RDTs) fail to detect samples with low parasite densities and cannot distinguish parasite species other than P. falciparum and Plasmodium vivax [23,24]. Thus, alternative methods are required to identify these neglected species.
Molecular methods (S1 Table) [14–19,25–28] are more sensitive and specific for P. ovale detection than microscopic examination or RDTs, but most existing assays target the 18S rRNA gene of both P. ovalecurtisi and P. ovalewallikeri, leading to potential cross-reactivity and a lack of complete species specificity. A duplex real-time PCR assay targeting the reticulocyte-binding protein homologue (porbp2) gene for P. ovalecurtisi and P. ovalewallikeri detection was published in 2011; however, results of the melt-curve analysis can be hard to interpret [15]. A nested PCR assay developed in 2013 targets the tryptophan-rich antigen (potra) gene and can detect samples with 2–10 parasites/μl [16], but this assay requires multiple steps (nested assay, agarose gel electrophoresis, and sequencing) and long turnaround time. Nested PCR targeting the Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene can also differentiate species, but it requires agarose gel electrophoresis and sequencing [18]. Available single-target quantitative real-time PCR assays require separate runs to distinguish P. ovalecurtisi and P. ovalewallikeri [14,17,19].
Because of the limitations of the existing assays, most studies have not distinguished P. ovalecurtisi and P. ovalewallikeri [29]. However, recently released P. ovalecurtisi and P. ovalewallikeri genomes (PocGH01 and PowCR01) provide opportunities for improved molecular assay development [30]. To improve our understanding of the epidemiology of P. ovalecurtisi and P. ovalewallikeri malaria, we mined publicly available P. ovalecurtisi and P. ovalewallikeri genomes to identify novel multi-copy targets and developed new qualitative real-time PCR assays. Our new assays have high specificity and can be duplexed. Performance of the P. ovalecurtisi assay was superior to the P. ovalewallikeri assay, limiting the duplex assay’s ability to investigate their relative prevalence. These assays offer new options for high-throughput P. ovalecurtisi epidemiological analyses and identification of P. ovalecurtisi and P. ovalewallikeri samples amenable to downstream next-generation sequencing.
Materials and Methods
Ethics statement
Existing samples from previous studies were chosen based on convenience. DRC samples were collected as part of a 2017 study investigating malaria diagnostic test performance in three provinces, Kinshasa, Bas-Uele, and Sud-Kivu [31]. Tanzania samples were collected from participants enrolled in a malaria transmission study in rural Bagamoyo district from 2018–2019 [14,29,32]. Enrolled subjects provided written informed consent or assent; for children, written parental consent was obtained. Ethical approvals for these studies were obtained from the Kinshasa School of Public Health (ESP/CE/07B/2017), Muhimbili University of Health and Allied Sciences (MUHAS/DA.282/298/01/C), and the University of North Carolina at Chapel Hill (IRB#: 17–0155).
Mining and selection of multi-copy targets in P. ovalecurtisi and P. ovalewallikeri genomes
Using the publicly available P. ovalecurtisi (PocGH01) and P. ovalewallikeri (PowCR01) reference genomes obtained from the NIH National Center for Biotechnology Information (NCBI) database, we identified sequence motifs of 100 base-pairs (bp) in length with ≥ 6 copies using Jellyfish (version 2.2.10) [33] (Fig 1). Sequences with low GC content (< 25%) and highly repetitive short sequences were excluded. The remaining multi-copy targets were aligned to NCBI nt database using blastn to investigate their specificity. Sequences aligned to other Plasmodium parasites were excluded. We then re-aligned the remaining targets to the P. ovalecurtisi and P. ovalewallikeri genomes separately using blastn to investigate their copy numbers in each genome. Candidate diagnostic assay targets for P. ovalecurtisi and P. ovalewallikeri were selected based on species-specificity and copy numbers. Primer and probe sets were designed manually using Oligo Calc [34] and DNAMAN (version 9, Lynnon BioSoft, Quebec City, Canada) to estimate primer and probe melting temperatures and to avoid self-complementarity and primer dimers (S2 Table).
Figure created using Biorender.com. Maps of Africa and the DRC were created using R software. Abbreviations: Poc = P. ovalecurtisi; Pow = P. ovalewallikeri.
Assay development and optimization
A panel of 15 well-characterized P. ovalecurtisi and P. ovalewallikeri field samples and six non-ovale Plasmodium laboratory controls were selected for assay development and analytical specificity analysis. Field samples included 11 P. ovalecurtisi and four P. ovalewallikeri leukodepleted blood samples and dried blood spot (DBS) samples from Tanzania and the Democratic Republic of the Congo (DRC); species identification was conducted using the published assays [14]. Laboratory controls included two P. falciparum, one P. malariae, two P. vivax, and one P. knowlesi dried blood spot samples from an external quality assurance program [35]. DNA from dried blood spot (DBS) samples, each spot containing approximately 70μl whole blood, was extracted using Chelex 100 (Bio-Rad, Fishers, Indiana, USA) and eluted into 150μl final volume [36]. DNA from leukodepleted blood samples was extracted using the QIAamp DNA Mini Kit (Qiagen, Mettmann, North Rhine-Westphalia, Germany) according to manufacture instructions. Parasite densities were estimated using a semi-quantitative real-time PCR assay targeting the 18S rRNA gene of both P. ovalecurtisi and P. ovalewallikeri as previously described [5]. The P. ovalecurtisi versus P. ovalewallikeri species was determined using published assays as the gold standard [14].
Primer sets with the best specificity for P. ovalecurtisi and P. ovalewallikeri versus this panel of samples were selected for further development. Singleplex assays for P. ovalecurtisi and P. ovalewallikeri detection were optimized using synthetic plasmids (Azenta Life Sciences, Indianapolis, Indiana, USA) containing targets (S3 Table) for P. ovalecurtisi and P. ovalewallikeri detection. A range of annealing temperatures and primer and probe concentrations were tested to identify the optimal reaction conditions. Finally, a duplex qualitative real-time PCR assay that combined the singleplex assays was developed, in order to detect and differentiate P. ovalecurtisi and P. ovalewallikeri in a single reaction tube. Duplex assay optimization was performed using synthetic plasmids described above. Optimal reaction conditions were determined by testing a range of annealing temperatures and of primer and probe concentrations.
All reactions were performed using a CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). All optimization analyses were performed in duplicate. Non-template controls (nuclease-free water) and serially diluted P. ovalecurtisi and P. ovalewallikeri plasmid DNA solutions were included in each real-time PCR run.
Analytical sensitivity and specificity
We determined the analytical sensitivity of the best performing singleplex and duplex assays using serially diluted plasmid DNA. A total of 129 P. ovalecurtisi and 186 P. ovalewallikeri plasmid DNA replicates were tested to determine the analytical sensitivity of the singleplex assays (S4 Table). For the duplex assay, a total of 104 P. ovalecurtisi and 161 P. ovalewallikeri plasmid replicates were used (S5 Table). Probit analysis was used to estimate the 95% confidence lower limits of detection [37]. We then determined the analytical specificity of the duplex assay using the same panel of 15 well-characterized P. ovalecurtisi and P. ovalewallikeri field samples [14] and six non-ovale Plasmodium laboratory controls in duplicate as described above.
Validation using field samples
The duplex assay’s clinical sensitivity and specificity were assessed using 95 dried blood spot samples selected from a large sample set from a previous study conducted in the DRC [31], including 55 P. ovalecurtisi and/or P. ovalewallikeri samples identified using published PCR assay [5], and 40 non-ovale Plasmodium samples (20 P. falciparum infections, 10 P. malariae infections, and 10 P. falciparum and P. malariae mixed infections) [31]. DNA was extracted from DBS using Chelex 100 as described above. Plasmodium species and parasite densities were identified using real-time PCR assays for both P. ovalecurtisi and P. ovalewallikeri, P. falciparum, and P. malariae as previously described [5,38,39], with samples positive in duplicate selected for use during validation of the present assay. Results of the previously published singleplex 18S rRNA real-time PCR assay for both P. ovalecurtisi and P. ovalewallikeri was used as the gold standard for clinical sensitivity and specificity calculations [5].
Statistical analysis
Statistical analysis was performed using R software (version 4.2.0; R Core Team, Vienna, Austria) in RStudio (version 2022.02.2). Maps and figures were generated using the ggplot2 (version 3.4.4), sf (version 1.0.16), rnaturalearth (version 1.0.1), and rnaturalearthdata (version 1.0.0) packages, and the study schematic was generated using BioRender. Spatial data were downloaded from the Database of Global Administrative Areas (GADM) [40].
Results
P. ovalecurtisi and P. ovalewallikeri target selection and assay development
A total of 2,585 and 3,978 sequences of 100 bp in length with ≥6 repeats were found in the P. ovalecurtisi and P. ovalewallikeri reference genomes, respectively. Targets with low GC content, highly repetitive short sequences, or aligned to other Plasmodium parasite genomes were excluded. A total of three potential assay targets with ≥8 copies in each of the P. ovalecurtisi and P. ovalewallikeri genomes were selected. Focusing on these potential targets, we designed five and three primer and probe sets for P. ovalecurtisi and P. ovalewallikeri, respectively (S2 Table). After testing all primer and probe sets using a panel of 15 well-characterized P. ovalecurtisi and P. ovalewallikeri field samples and six laboratory non-ovale Plasmodium controls, we selected two primer and probe sets with the best specificity for P. ovalecurtisi and P. ovalewallikeri, respectively, for additional laboratory testing (Table 1). The selected P. ovalecurtisi target had nine copies within putative liver stage antigen 3 (lsa3) gene on chromosome 4 (LT594585.1: 9,968–11,125), while the P. ovalewallikeri target had eight copies in a non-coding region on chromosome 14 (LT594518.1: 1,842,975–1,844,586). Short distances (< 50 bp) were noted between the repetitive P. ovalecurtisi target motifs as well as between P. ovalewallikeri target motifs.
Singleplex real-time PCR assay development
Using the primer sets and the corresponding probes with the best specificity for P. ovalecurtisi and P. ovalewallikeri, we developed singleplex assays for detection of each species. The optimized assay for P. ovalecurtisi was performed in a small volume of 10μl, including 7μl of reaction master-mix containing 2x FastStart Universal Probe Master (Rox) (Roche, Basel, Switzerland), primers and probes (240 nM of Poc_Fwd, 240 nM of Poc_Rev, 60 nM of Poc_Probe), and 3μl of DNA template (derived from approximately 1.4μl whole blood). Optimal thermocycling conditions were 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 58°C. The optimized assay for P. ovalewallikeri was also performed in a small volume of 10μl, including 7μl of reaction master-mix containing 2x FastStart Universal Probe Master (Rox), primers and probes (300 nM of Pow_Fwd, 300 nM of Pow_Rev, 200nM of Pow_Probe), and 3μl of DNA template. The optimal thermocycling conditions were 2 min at 50°C, 10 min at 95°C, followed by 45 cycles of 15 s at 95°C and 60 s at 56°C. Samples with Ct values lower than 40 and 45 were called positive for P. ovalecurtisi and P. ovalewallikeri, respectively.
Duplex real-time PCR assay development
Combining the singleplex assays, we optimized a duplex, qualitative real-time PCR assay for simultaneous detection and differentiation of P. ovalecurtisi and P. ovalewallikeri in a single reaction tube. The final, optimized duplex assay was performed in a small final volume of 10μl, including 7μl of reaction master-mix containing 2x FastStart Universal Probe Master (Rox) (Roche, Basel, Switzerland), primers and probes (240 nM of Poc_Fwd, 240 nM of Poc_Rev, 60 nM of Poc_Probe, 800 nM of Pow_Fwd, 800 nM of Pow_Rev, 320nM of Pow_Probe), and 3μl of DNA template. Optimal thermocycling conditions were 2 min at 50°C, 10 min at 95°C, followed by 45 cycles of 15 s at 95°C and 60 s at 58°C, allowing for detection of parasite DNA in less than two hours. Samples with Ct values lower than 45 for either species were called positive.
Analytical sensitivity and specificity
The 95% confidence lower limits of detection of the singleplex P. ovalecurtisi and P. ovalewallikeri assays were 3.6 and 25.9 parasite genome equivalents/μl DNA template, respectively (S1 Fig and S4 Table). The 95% confidence lower limits of detection of the duplex assay were similar to the singleplex assays, at 4.2 and 41.2 parasite genome equivalents/μl DNA template, respectively (Figs 2A, S2 and S5 Table). All well-characterized P. ovalecurtisi and P. ovalewallikeri field samples were successfully detected and differentiated with no cross-reactivity between species, and no cross reactivity was found when the assay was applied to six non-ovale Plasmodium controls (Fig 2B).
A) Analytical sensitivity when applied to multiple replicates of serially diluted plasmid DNA (n = 104 and 161 total replicates for P. ovalecurtisi and P. ovalewallikeri, respectively). Points are colored to display target detection (blue) versus no detection (red). The 95% lower limit of detection (LOD) determined using probit analysis is shown for each species. B) Analytical specificity versus genomic DNA extracted from a panel of well-characterized leukodepleted blood (LDB) and dried blood spot (DBS) samples from Tanzania and the DRC with P. ovalecurtisi and P. ovalewallikeri confirmed by published real-time PCR assays, and non-ovale Plasmodium samples from an external quality assurance program. All P. ovalecurtisi and P. ovalewallikeri samples were correctly identified, and no false-positives were observed among other Plasmodium species.
Validation using field samples
The duplex assay demonstrated perfect specificity for P. ovalecurtisi and P. ovalewallikeri and high sensitivity for P. ovalecurtisi when applied to 95 field samples collected in the DRC. Parasite densities of 55 P. ovale-positive field samples included in this study ranged from 0.9 to 2,468 parasites/μl DNA template; 29 (52.7%) samples had parasite densities <10 parasites/μl. The assay’s overall sensitivity was 80%, successfully determining P. ovale species in 44 of the P. ovale-positive field samples (Fig 3A). False-negatives were limited to low-concentration samples, with 100% assay sensitivity for infections with >10 parasites/μl DNA template. The lowest parasite densities in which species could be determined were 2.0 and 20.9 parasites/μl DNA template for P. ovalecurtisi and P. ovalewallikeri, respectively. None of the 40 non-ovale Plasmodium field samples were detected by the duplex assay, consistent with 100% specificity (Fig 3B).
Gold standard species identification was performed previously using a series of semi-quantitative real-time PCR assays targeting pan-Plasmodium 18S rRNA, followed by singleplex species-specific assays. A) Detection of known P. ovale PCR-positive samples with varying parasite densities and co-infection status. Analytical 95% lower limits of detection (LOD) are represented by dashed lines. B) No detection of other Plasmodium species across a range of parasite densities. Abbreviations: P. ovale = P. ovalecurtisi and/or P. ovalewallikeri.
Discussion
We mined recently published genomes of P. ovalecurtisi and P. ovalewallikeri to develop new real-time PCR assays that can be used to improve our understanding of their epidemiology in malaria-endemic countries. Recent studies have revealed a previously unappreciated burden of P. ovalecurtisi and P. ovalewallikeri in Africa [3, 5, 15, 29]. Though P. ovalecurtisi and P. ovalewallikeri are distinct species, only a small number of existing assays can distinguish them. Many are not well-suited to large studies, requiring separate assays for each species, multiple steps (nested assays, agarose gel electrophoresis, and/or sequencing), higher input volumes of DNA solution, and long turnaround time, with potential for cross-reactivity at higher parasite densities [14–19]. Because of the limits of the existing assays, most field studies do not distinguish P. ovalecurtisi and P. ovalewallikeri, and their prevalence and clinical features remain understudied [41–43].
Our assays are highly specific for P. ovalecurtisi and P. ovalewallikeri, but we observed differences in sensitivity for detection of P. ovalecurtisi and P. ovalewallikeri. This difference in sensitivity limits the duplex assay’s use for studies of their relative prevalence. However, the P. ovalecurtisi singleplex or duplex assay is well-suited for high-throughput studies of symptomatic P. ovalecurtisi infection. In contrast, the P. ovalewallikeri assay is well-suited to identify higher-density infections that are amenable to next-generation sequencing, but other more sensitive assays should be used for epidemiological analyses because our assay does not reliably detect lower-density infections. Thus, choice of assay and format should be informed by the user’s specific objectives.
Our assay targets are distinct from those used in prior assays and take advantage of 100 bp repetitive motifs in the putative lsa3 gene on P. ovalecurtisi chromosome 4 and a non-coding region on P. ovalewallikeri chromosome 14, respectively. Studies of P. falciparum lsa3 indicate that it is an essential gene that encodes an antigen with tetrapeptide repeats of unclear function during the liver stage of infection [44–46]. Previous work confirmed conservation of P. falciparum lsa3 in isolates collected from geographically diverse sites [45]. The non-coding P. ovalewallikeri repetitive motif we targeted has unclear function, with no obvious orthologues identified in publicly available databases. These targets appear to be conserved in the limited P. ovalecurtisi and P. ovalewallikeri genomes released to-date. We leveraged the repetitive nature of these poorly understood P. ovalecurtisi and P. ovalewallikeri targets to develop highly specific assays for P. ovalecurtisi and P. ovalewallikeri, and high sensitivity for P. ovalecurtisi.
Compared to published real-time PCR assays that mostly target P. ovalecurtisi and P. ovalewallikeri 18S rRNA genes [5, 17, 19], inclusion of distinct P. ovalecurtisi and P. ovalewallikeri targets enabled development of highly specific assays. The targets’ copy numbers in our study are in the same range as those reported for 18S rRNA genes in Plasmodium genomes [47–49]. Similar limits of detection of P. ovalecurtisi were found between the published 18S rRNA PCR assay (1.5 parasites/μl) and our P. ovalecurtisi singleplex and duplex assays, while we observed inferior limits of detection for P. ovalewallikeri compared to some published assays (S1 Table). It is possible that the short distances between our P. ovalecurtisi targets and between P. ovalewallikeri targets decrease the PCR efficiency, offsetting sensitivity that might otherwise be achieved from their copy number.
We further evaluated the duplex assay using field samples from the DRC. Validation using field samples from the DRC confirmed robust species differentiation when the duplex assay was applied to P. ovale samples with >10 parasites/μl and 100% specificity across all parasite densities. Though its ability to identify P. ovalewallikeri in particular was limited at lower parasite densities, the simultaneous amplification of P. ovalecurtisi and P. ovalewallikeri DNA in a single reaction tube allows our assay to have shorter turnaround time and require less materials compared to published singleplex assays [17, 19]. The duplex assay had high specificity, high sensitivity for P. ovalecurtisi detection, short turnaround time, and capacity for high-throughput use.
Several limitations of our assays should be highlighted. First, the duplex assay’s relatively low sensitivity at lower parasite densities, particularly for P. ovalewallikeri detection as noted above, limits its utility in epidemiological analyses and particularly among low-density or asymptomatic infections. This limitation could be overcome in the future by combining an 18S rRNA assay capable of detecting both P. ovalecurtisi and P. ovalewallikeri (e.g. such as that used by Mitchell et al. [5]) with our P. ovalecurtisi lsa3 assay, allowing definitive identification of P. ovalecurtisi (18S rRNA assay-positive, P. ovalecurtisi lsa3-positive) and deductive identification of P. ovalewallikeri mono-infection (18S rRNA assay-positive, P. ovalecurtisi lsa3-negative). Second, the assays were optimized with high-throughput applications in mind, but lower-throughput approaches may be more appropriate in some cases. For example, users with smaller numbers of samples or willing to expend larger DNA volumes could consider increasing sample volumes to improve sensitivity. Careful validation of this approach within one’s own lab is critical to ensure assay specificity is maintained. Third, our assays target two non-essential genomic regions at risk of deletion or disruption if future treatment choices are tied to diagnosis, as has been proposed for P. falciparum and observed for Chlamydia trachomatis non-essential diagnostic targets [50, 51]. However, this hypothetical threat is unlikely to be realized any time soon. Malaria programs in Africa focus largely on P. falciparum and do not routinely offer radical cure to clear P. ovalecurtisi and P. ovalewallikeri hypnozoites. Finally, these assays were developed based on P. ovalecurtisi and P. ovalewallikeri genomes from Africa. More sequences from other regions are needed to assess for variation in the primer and probe targets.
In conclusion, we developed and validated novel, highly specific real-time PCR assays capable of detection and differentiation of P. ovalecurtisi and P. ovalewallikeri. Though its ability to identify P. ovalewallikeri was limited at lower parasite densities, the duplex assay’s streamlined work-flow reduces complexity and may be suitable for specific use cases. We recommend these assays for high-throughput analyses of symptomatic P. ovalecurtisi malaria and for identification of higher-density P. ovalecurtisi or P. ovalewallikeri infections that may be amenable to sequencing. As some countries progress toward malaria elimination, improved assays for P. ovalecurtisi and P. ovalewallikeri like those presented here will become more important and open the way to improved understanding of P. ovalecurtisi and P. ovalewallikeri epidemiology and clinical impact, and ultimately inform elimination strategies.
Supporting information
S1 Table. Molecular assays to distinguish P. ovalecurtisi and P. ovalewallikeri.
https://doi.org/10.1371/journal.pntd.0011759.s001
(DOCX)
S2 Table. Candidate primer and probe sets evaluated for the detection of P. ovalecurtisi and P. ovalewallikeri.
https://doi.org/10.1371/journal.pntd.0011759.s002
(DOCX)
S3 Table. Sequences contained in synthetic plasmids to determine assay analytical sensitivity.
https://doi.org/10.1371/journal.pntd.0011759.s003
(DOCX)
S4 Table. Limits of detection of the optimized, singleplex P. ovalecurtisi and P. ovalewallikeri assays versus serially diluted plasmid DNA.
Parasite density = plasmid DNA copy number/copy number of the target in the parasite genome.
https://doi.org/10.1371/journal.pntd.0011759.s004
(DOCX)
S5 Table. Limits of detection of the optimized, duplex P. ovalecurtisi and P. ovalewallikeri assay versus serially diluted plasmid DNA.
Parasite density = plasmid DNA copy number/copy number of the target in the parasite genome.
https://doi.org/10.1371/journal.pntd.0011759.s005
(DOCX)
S1 Fig. 95% lower limits of detection for singleplex assays, determined using probit analysis.
A) P. ovalecurtisi singleplex assay 95% lower limit of detection (3.6 parasites/μl [95% CI 2.7–6]). B) P. ovalewallikeri singleplex assay 95% lower limit of detection (25.9 parasites/μl [95% CI 22–33.6]). Confidence intervals are shown in lighter shade.
https://doi.org/10.1371/journal.pntd.0011759.s006
(TIF)
S2 Fig. 95% lower limits of detection for duplex assay, determined using probit analysis.
A) P. ovalecurtisi 95% lower limit of detection (4.2 parasites/μl [95% CI 3.1–9.5]). B) P. ovalewallikeri 95% lower limit of detection (41.2 parasites/μl [95% CI 33.3–58.3]). Confidence intervals are shown in lighter shade.
https://doi.org/10.1371/journal.pntd.0011759.s007
(TIF)
Acknowledgments
We thank the study teams and participants in the DRC and Tanzania research studies from which samples were derived. The following reagents were obtained through BEI Resources, NIAID, NIH: diagnostic plasmid containing the small subunit ribosomal RNA gene (18S) from Plasmodium ovale, MRA-180, contributed by Peter A. Zimmerman.
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