Development of new real-time PCR assays for detection and species differentiation of Plasmodium ovale

Wenqiao He; Rachel Sendor; Varun R Potlapalli; Melchior M Kashamuka; Antoinette K Tshefu; Fernandine Phanzu; Albert Kalonji; Billy Ngasala; Kyaw Lay Thwai; Jonathan J Juliano; Jessica T Lin; Jonathan B Parr

doi:10.1371/journal.pntd.0011759

. 2024 Sep 10;18(9):e0011759. doi: 10.1371/journal.pntd.0011759

Development of new real-time PCR assays for detection and species differentiation of Plasmodium ovale

Wenqiao He ^1,², Rachel Sendor ³, Varun R Potlapalli ¹, Melchior M Kashamuka ⁴, Antoinette K Tshefu ⁴, Fernandine Phanzu ⁵, Albert Kalonji ⁵, Billy Ngasala ⁶, Kyaw Lay Thwai ¹, Jonathan J Juliano ^1,³, Jessica T Lin ¹, Jonathan B Parr ^1,^*

Editor: Georges Snounou⁷

¹Division of Infectious Diseases and Institute for Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America

²Department of Epidemiology, School of Public Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, Southern Medical University, Guangzhou, China

³Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America

⁴Kinshasa School of Public Health, Kinshasa, Democratic Republic of the Congo

⁵SANRU Asbl, Kinshasa, Democratic Republic of the Congo

⁶Muhimbili University of Health and Allied Sciences, Dar Es Salaam, Tanzania

⁷Institut Pasteur, FRANCE

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.

^✉

* E-mail: jonathan_parr@med.unc.edu

Roles

Wenqiao He: Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing

Rachel Sendor: Funding acquisition, Investigation, Writing – review & editing

Varun R Potlapalli: Investigation, Writing – review & editing

Melchior M Kashamuka: Funding acquisition, Resources, Writing – review & editing

Antoinette K Tshefu: Funding acquisition, Resources, Writing – review & editing

Fernandine Phanzu: Funding acquisition, Resources, Writing – review & editing

Albert Kalonji: Funding acquisition, Resources, Writing – review & editing

Billy Ngasala: Funding acquisition, Resources, Writing – review & editing

Kyaw Lay Thwai: Investigation, Writing – review & editing

Jonathan J Juliano: Funding acquisition, Methodology, Writing – review & editing

Jessica T Lin: Funding acquisition, Methodology, Writing – review & editing

Jonathan B Parr: Conceptualization, Funding acquisition, Methodology, Project administration, Writing – review & editing

Georges Snounou: Editor

PMCID: PMC11414980 PMID: 39255325

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.

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).

Fig 1 — 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.

Table 1. Best performing primers and probes for P. ovalecurtisi and P. ovalewallikeri detection.

Name	Sequence (5’→3’)
Poc_Fwd	GTTRCCAAATATGCTATCACTTAC
Poc_Rev	GTARCACAAAACGACGAGAC
Poc_Probe	FAM—TACATCTTCTTCAAAGTTGYCATAYGCAT—BHQ1
Pow_Fwd	GRRTCTTCTGAACTTTGRAATG
Pow_Rev	CATCAAGGRTATCCATTTCA
Pow_Probe	VIC—AACAAYCACTTCAACATCAA—BHQ1

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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).

Fig 2 — 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).

Fig 3 — 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.

(DOCX)

pntd.0011759.s001.docx^{(21.9KB, docx)}

S2 Table. Candidate primer and probe sets evaluated for the detection of P. ovalecurtisi and P. ovalewallikeri.

(DOCX)

pntd.0011759.s002.docx^{(15.8KB, docx)}

S3 Table. Sequences contained in synthetic plasmids to determine assay analytical sensitivity.

(DOCX)

pntd.0011759.s003.docx^{(14.4KB, 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.

(DOCX)

pntd.0011759.s004.docx^{(15.9KB, 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.

(DOCX)

pntd.0011759.s005.docx^{(15.9KB, 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.

(TIF)

pntd.0011759.s006.tif^{(1.1MB, 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.

(TIF)

pntd.0011759.s007.tif^{(1.7MB, 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.

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 Statement

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.

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011759.r001

Decision Letter 0

Georges Snounou, Paul O Mireji

6 Feb 2024

Dear Dr. Parr,

Thank you very much for submitting your manuscript "A novel duplex qualitative real-time PCR assay for the detection and differentiation of Plasmodium ovale curtisi and Plasmodium ovale wallikeri malaria" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

I concur with the comments of the three reviewers. The concerns of reviewers 1 and 3 with respect to the lower sensitivity for P. ovalewallikeri is particularly important: 40 P/µL is very close to the limit of detection by routine thick smear examination, and this is likely to lead to false negative results for many of the infections found in the field, especially in asymptomatic individuals. These two reviewers also suggest that the central claim that the assay represents an improvement on other current molecular assays is not justified. The significance of the epidemiological analyses has also been questioned by these two reviewers.

I suggest that significant major amendments are made to any revised manuscript in response to the detailed comments made by the reviewers.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Georges Snounou, Ph.D.

Guest Editor

PLOS Neglected Tropical Diseases

Paul Mireji

Section Editor

PLOS Neglected Tropical Diseases

***********************

I suggest that significant major amendments are made to any revised manuscript in response to the detailed comments made by the reviewers.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: See attachment

Reviewer #3: The objectives and study design are appropriate. The sample size for the validation of the novel assay is appropriate, though limited to geographically restricted samples, while that for the epidemiological analyses is not adequate.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: See attachment

Reviewer #3: The results and analyses are clearly presented, as are the figures

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: See attachment

Reviewer #3: The limitations listed by the authors are sufficient to weaken some of the conclusions presented and the relevance of the new assay to public health.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: See attachment

Reviewer #3: Minor comments

Line 73 Reference 1 is not adequate in this context.

Line 316/Line 322 LSA3 might be non-essential for blood-stage parasites, but it is essential for hepatic parasites. Furthermore, there is no evidence that LSA3 is involved in antigenic variation.

Line 331 In reference 18 the cut-off of 50 plasmid copies was established for a Ct of 38 or less.

A correction to the naming of the two species has been recently made (Snounou et al. Trends in Parasitology 2004 40:21), and it would be suitable to adopt the terms P. ovalecurtisi and P. ovalewallikeri in a revised manuscript.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: In this simple report, He and colleagues describe an RTqPCR assay for the detection and differentiation of Plasmodium ovalecurtisi and P. ovalewallikeri. They argue that their assay is ‘less complex’ and ‘streamlined’ compared to other assays, and advocate its use in field studies.

Major points.

Given that their assay is of equal or less sensitivity than other previously reported assays, it is difficult to see the circumstances in which it may replace those currently in use. This assay can only be used in well-funded laboratories with access to real time PCR facilities, and such laboratories would presumably fair better using the more sensitive current methodologies. Of particular concern is the fact that their P. ovalewallikeri assay is ten times less sensitive than their P. ovalecurtisi assay, rendering it, in my opinion, useless in its ability to assay the relative prevalence of the two species, or, indeed, to identify P. ovalecurtisi in relatively low parasite density infections. The authors state in their discussion that their ‘assays sensitivity at lower parasite densities could be further improved’ (line 381). If it can be further improved, then it is perhaps better to do so before publishing and advocating its use.

My second major concern is that the assay was developed using only sequences from a limited region of central Africa. It is possible, therefore, that it may not be suitable for use outside that region, especially the P. ovalecurtisi assay, which targets a non-coding region (and so is potentially polymorphic). It would be nice to see some attempt to address this, either just through analysis of polymorphism of the targets in a more diverse pool of P. ovale parasites, or through wet lab testing of isolates from outside central Africa.

Other points

Nomenclature. Throughout the manuscript, the two ovale species are referred to either as “P. ovale curtisi” and ”P. ovale wallikeri” or as “Poc” and “Pow”. It has recently been suggested that the names “P. ovalecurtisi” and “P. ovalewallikeri” are preferable, as binomials reflect the species nature of the two parasites. I see no need to abbreviate to ‘Poc’ and ‘Pow’ (we don’t call P. falciparum “P. fal”, or P. malariae “P. mal”). I suggest correcting to “P. ovalecurtisi” and “P. ovalewallikeri”.

Line 25. ‘Species’ are, by definition, ‘non-recombining’. There is no need to add this phrase before the word ‘species’.

Line 59. The evidence for this isn’t convincing at the moment, and is somewhat contradictory.

Line 71. I’m not sure ‘most malaria programes’ prioritise P. falciparum… which programmes are these? Elimination programmes? Control programmes? P. vivax is the major issue outside Africa. Perhaps it’s best to specify ‘malaria control programmes within Africa’?

Line 77 the phrase ‘distinct non-recombing species’ can be reduced to just ‘species’.

Line 80. There are no single step assays, surely?

Line 83. What do the authors mean by ‘conventional malaria diagnostic assays relying on microscopy’. Do they mean simply ‘microscopy’?

Line 98. ‘this assay requires multiple steps’. What does this mean? ALL assays require multiple steps… And a ‘long turnaround time’? Perhaps it would be helpful if a table is included comparing the various assays for detection and discrimination of the two species. The table could include the sensitivities, the costs, the number of ‘steps’ etc. It would be nice to include the methodology of Nundu et al in reference 47, who performed a simple nested PCR then sequenced the P. ovale positives.

Line 105. Perhaps the authors could explain here what a ‘duplex qualitative real-time PCR assay’ is for the general reader.

Line 129 What does the term ‘well-characterised’ mean here?

Line 152. What constitutes a ‘replicate’ in this context?

Line 154 What does the term ‘well-characterised’ mean here?

Line 187. Does this mean that of the 64 previously P. ovale positive samples, only 44 were found to be positive on repeated analysis? This is quite a significant result – could the authors speculate as to the reason of this significant discrepancy?

Line 232. It is specified that 3 uL of DNA template was used in the assay. Could the authors explain how much total blood volume this is equivalent to? How much blood was used for DNA extraction? Without this data, it is impossible to assess the actual sensitivity of the assay.

Line 239, as above. ‘equivalents/uL’… uL of what? Blood or DNA solution? The approximate volume of blood assayed in each reaction needs to be given.

Line 242. What was the negative control for these assays? Was it uninfected blood extracted in the same way as the samples?

Line 255 is rather an odd sentence. It is in effect saying that sensitivity was excellent when there was lots of parasite DNA present. That’s not a good measure of ‘sensitivity’.

Line 258: the number of parasites per ‘uL’ is given again – it is essential to know whether this is uL of DNA solution or of original blood.

Line 259 – the assays sensitivity of 80% is compared to what? Blood smear positive? Previous qPCR results?

Line 267. The 64 P. ovale spp positive samples had been determined as positive using a previous assay, I think. The authors could only confirm 44 of these to be truly P. ovale positive using the same assay… if I’ve got this right, then the epidemiological analysis should only be performed on the 44 confirmed P. ovale ceases, surely?

Line 267 onwards. The epidemiology section seems somewhat shoe-horned into the paper, and doesn’t really fit with the rest of it – as the risk factor calculations were not split into P. ovalewallikeri and P. ovalecurtisi, this analysis does not depend on the new assay under discussion, and is based on a previously carried out analysis, it seems superfluous to the paper as a whole. It would be much better to just give the breakdown of the two species compositions in the original sample set. Related to this, I fail to see the significance of the performance of an ‘inverted probability weighting analysis’ to extrapolate from 37 samples to 44 (?).

Line 301. The authors describe their assay as “highly specific’. Yet, the P. ovalecurtisi component is 10 times more sensitive than the Pow component. I can’t reconcile these two statements. They state it can be used to ‘improve our understanding of their epidemiology in malaria-endemic countries’ yet it is less sensitive than other RTqPCR assays and conventional nested PCR? How will it, therefore, improve our understanding?

Line 305 “distinct, non-recombining species” This suggests that there are indistinct, recombining species in existence….

Line 307. Why are higher volumes of DNA required in other assays? The author’s assay is less sensitive, so would require a larger volume of DNA to reach the same level of sensitivity.

Line 308. ‘multiple steps’ is vague. Again, a table comparing the methods alluded to with the authors’ assay would be useful.

Line 319. In world terms, the samples weren’t collected from particularly ‘geographically diverse sites’, all of them being in central Africa.

Line 325. Could the ‘advantageous performance characteristics’ be given here?

Line 345. How were these prevalences determined?

Line 354 “Existing evidence indicates that the most prevalent P. ovale spp. vary across different countries’. Could the authors rewrite this sentence for clarity…?

Line 356. What does ‘Poc more prevalent in symptomatic individuals’ mean? More prevalent than in asymptomatic individuals? More prevalent than Pow in symptomatic individuals? If the latter, then this will always appear to be the case when performing the authors' assay, as the Poc sensitivity is 10 times that of Pow.

Reviewer #2: See attachment

Reviewer #3: The authors have mined the genome of the two P. ovale species and selected in each a segment of DNA that is repeated in the genome and designed sets of oligonucleotide primers and corresponding probes that were then tested in real-time PCR assays for specificity and sensitivity. One set for each species that was identified as giving the optimal results was then tested using a selected set of P. ovale-positive samples (identified in a previous study), and this new protocol is advocated to perform better/to be more practical than others and therefore to serve for high throughput surveys in endemic countries. The authors then derived some epidemiological conclusions from the data obtained from these samples.

Major Comments

1) Whereas the approach of the authors (targeting internally repeated short segments) is original for P. ovale and the methodology and data are sound, the main thrust of this manuscript is that their methodology (duplex real-time PCR) is better suited than all other published methods to date for investigations on the epidemiology/biology of these parasite species, with the main supporting argument is that it is less time-consuming (fewer steps between sample collection and assay results). However, many factors (some quoted by the authors and others not) trump this minor advantage. First, the ten-fold lower sensitivity of detection for one species (Pow) as compared to the other (Poc) is a major limitation that would bias data from all field surveys, because a) it is well-known that P. ovale infections have low parasite densities past the primary peak that often persist for long durations, and b) low parasite densities, especially in mixed and asymptomatic infections, are only detected when highly sensitive molecular assays capable of detecting very low parasite levels (1 P/µl or less).

2) It is difficult from the description given to work out the actual sensitivity that was obtained. The values (4,2 and 41,2 P/µL for Poc and Pow, respectively) quoted were from the Probit analysis using diluted plasmids. There is no indication as to the Plasmodium sequence that these plasmids contained. Moreover, it is not clear to what volume of blood the 3 µL DNA template obtained from Chelex-purified filter papers corresponds. Assuming 10 µL per filter spot, and 100 µL of DNA solution post-Chelex extraction, the 3 µL would correspond to 0,3 µL of blood. Is this the case? Furthermore, were the DNA templates used obtained from leukodepleted blood?

3) One final point concerns the potential loss of sensitivity when the reaction is multiplexed, which is the case here. The authors should conduct experiments in which different proportions of DNA template from each species are mixed to assess whether the assay is capable of detecting a population of Poc or Pow when present as a minor proportion of the parasites (for instance in samples with high P. falciparum parasitaemias and only a few P. ovale per µL of blood).

4) There is little known concerning the global diversity of the repeats in the putative lsa3 gene and for the untranslated repeats on which the assay is based. Although it is likely that potential variations would not affect sensitivity, all the samples used to validate the assay were collected from the DRC and a few from Tanzania. It would have been very useful to include samples from West Africa and Oceania.

Ultimately the authors have not provided sufficient evidence to support their claim that this novel method is superior to others for field investigations of the two P. ovale species. One might argue that the high rate of false negatives is a major disadvantage of the assay: 11 of the 55 P. ovale 5 samples used to validate the assay were not identified, i.e. a 20% failure rate, even if this was mainly in cases with low parasite burdens, A shorter processing time does not compensate for the lower sensitivity, and the techniques used still require a well-equipped laboratory.

5) The conclusions from the epidemiological analyses are at best speculative because they are based on a restricted number of samples from symptomatic cases collected from various locations in the DRC, and because of the bias in the detection of Pow. The authors acknowledge the limitations in the Discussion. I suggest that this section is omitted from the manuscript.

--------------------

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Reviewer #1: Yes: Richard Culleton

Reviewer #2: No

Reviewer #3: No

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Attachment

Submitted filename: review_PNTD-D-23-01354.docx

pntd.0011759.s008.docx^{(16.2KB, docx)}

PLoS Negl Trop Dis. 2024 Sep 10;18(9):e0011759. doi: 10.1371/journal.pntd.0011759.r002

Author response to Decision Letter 0

4 Jun 2024

Attachment

Submitted filename: comments from reviewers.docx

pntd.0011759.s009.docx^{(56.1KB, docx)}

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011759.r003

Decision Letter 1

Georges Snounou, Paul O Mireji

15 Aug 2024

Dear Dr. Parr,

We are pleased to inform you that your manuscript 'Development of new real-time PCR assays for detection and species differentiation of Plasmodium ovale' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Georges Snounou, Ph.D.

Guest Editor

PLOS Neglected Tropical Diseases

Paul Mireji

Section Editor

PLOS Neglected Tropical Diseases

***********************************************************

The decisions of the reviewers are contradictory. However, I will consider that the assay you present has some value, if of limited use because of the poor sensitivity to one of the P. ovale species, and I am therefore recommending acceptance of the manuscript.

I strongly recommend that you adopt the correct nomenclature for the two species, namely P. ovalecurtisi and P. ovalewallikeri. Colin Sutherland and I are in agreement that the trinomial is misleading and contravenes the ICZN rules (as this particular nomenclature is restricted to sub-species).

With best regards and apologies for the delay in posting my recommendation.

Georges

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: See comments below

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: See comments below

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: See comments below

**********

Editorial and Data Presentation Modifications?

Reviewer #1: (No Response)

Reviewer #2: See comments below

**********

Summary and General Comments

Reviewer #1: The authors have made some modifications to their manuscript, toning down the claims of the previous version and removing the epidemiological survey component. Whilst the manuscript itself is somewhat improved, the assay still remains sub-optimal, in that the sensitivities of the two components are hugely different, and the merit of the assay versus currently used methodologies is not apparent. It is a shame that the authors have not revisited their assay in an attempt to increase the sensitivity of the P. ovalewallikeri assay, as this would make an important improvement to the work. This methodology leading to the design of this assay has potential, and it would be good to see that realised. As it stands, it feels as if the scope (and, indeed, necessity) for assay improvement renders the current manuscript more of a description of a work in progress rather than a fully optimised and practical assay.

Specific Points

Sequence Diversity

In my previous review I raised the concern that only African P. ovale spp. were used in the design and testing of this assay. In answer to this concern, the authors responded with reference to the low level of variability in the target sequences for samples from the DRC, Tanzania, Cameroon and Ethiopia. Without labouring the point, P. ovale is endemic outside Africa. If it is difficult to assess the level of diversity in non-African samples, then perhaps it should be made clear in the manuscript that this assay is optomised for African isolates, and may not be suitable for use elsewhere.

Table S1

The usefulness of this table is somewhat reduced by the omission of data for the authors’ own assay. In order to compare the assay to those previously published (the point of the previous request to include a table), it is necessary to include this data.

Table S4 and S5

These tables show the sensitivities of the assays using plasmid DNA. The ‘parasite density’ column is given as ‘parasites/ul’ (sic); I’m sorry if this is explained in the text, and I’ve missed it, but how are the authors extrapolating from ‘copy number (of target)’ to ‘parasite density’? Does each plasmid contain the same copy number of the target as a parasite genome? If not (and there are more copies per genome), then the ‘parasite density’ should be adjusted accordingly. This should be explained in a footnote to the table. Alternatively, the second column should be labelled ‘plasmid DNA copy number’. For example, there are nine copies of the P. ovalecurtisi target per parasite genome, but only one copy per plasmid. Does this mean that the “parasite density (parasites/ul)” has been multiplied nine times from the plasmid copy number sensitivity? So, for Poc, the assay is reliable only down to 10 ‘parasites/ul’; does this equate to 900 copies of the plasmid? It would be useful to clarify this.

Line 106. While differentiation of the two species is not possible, detection certainly is. Please modify sentence for clarity. Perhaps just “Differentiation of P. ovalecurtisi and P. ovalewallikeri is not possible using microscopy” is a more accurate sentence.

Nomenclature

The unanimous consensus of the malaria research community is that the trinomial names “P. ovale curtisi” and “P. ovale wallikeri”, are incorrect, confusing, and constitute ‘nomina nuda’. Snounou et al (2023a), offered a simple and positively received solution to the naming issue, to which only one objection was raised (Slapeta, 2023, who also acknowledge the incorrect status of the trinomials). This objection was subsequently shown to be erroneous and unsound (Snounou et al 2023b). In the interests of clarity, consensus and consistency in the literature pertaining to these two parasites, the use of the binomials as designated by Snounou et al (2023) is strongly advised.

Reviewer #2: Dear editor,

Please find below, my review of the revision of the manuscript PNTD-D-23-01354-R1.

The manuscript has globally improved in its presentation following the corrections and modifications made by the authors according to the reviewer's comments. i am glad to see the 'so called' epidemiological section out.

Regarding the assay itself, as share before, its the development and setting up are correct and sounded following expected steps.

Although, as new P. ovale curtisi and P. ovale wallikeri genomes have been released recently (Higgins et al 2024), it is surprising that the authors did not attempt to verify if their assay is still functional /compatible on these new genomes and also check for possible other repetitive elements to try improve the main weakness of their assay which is a 10 fold variations between the limits of detection of the two P. ovale species.

at the end of the day, the question of the usefulness of this assay remains, as it is of strictly no interest for the medical doctors and unlikely to be adopted in resources limited settings laboratories where the potra assay a been adopted. However, this assay might still find its place in laboratories covering national programs and screening large number of samples where it could be incorporated and combined within establish workflows.

**********

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Reviewer #1: No

Reviewer #2: No

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011759.r004

Acceptance letter

Georges Snounou, Paul O Mireji

3 Sep 2024

Dear Dr. Parr,

We are delighted to inform you that your manuscript, "Development of new real-time PCR assays for detection and species differentiation of Plasmodium ovale," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Molecular assays to distinguish P. ovalecurtisi and P. ovalewallikeri.

(DOCX)

pntd.0011759.s001.docx^{(21.9KB, docx)}

S2 Table. Candidate primer and probe sets evaluated for the detection of P. ovalecurtisi and P. ovalewallikeri.

(DOCX)

pntd.0011759.s002.docx^{(15.8KB, docx)}

S3 Table. Sequences contained in synthetic plasmids to determine assay analytical sensitivity.

(DOCX)

pntd.0011759.s003.docx^{(14.4KB, 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.

(DOCX)

pntd.0011759.s004.docx^{(15.9KB, 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.

(DOCX)

pntd.0011759.s005.docx^{(15.9KB, docx)}

S1 Fig. 95% lower limits of detection for singleplex assays, determined using probit analysis.

(TIF)

pntd.0011759.s006.tif^{(1.1MB, tif)}

S2 Fig. 95% lower limits of detection for duplex assay, determined using probit analysis.

(TIF)

pntd.0011759.s007.tif^{(1.7MB, tif)}

Attachment

Submitted filename: review_PNTD-D-23-01354.docx

pntd.0011759.s008.docx^{(16.2KB, docx)}

Attachment

Submitted filename: comments from reviewers.docx

pntd.0011759.s009.docx^{(56.1KB, docx)}

Data Availability Statement

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.

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PERMALINK

Development of new real-time PCR assays for detection and species differentiation of Plasmodium ovale

Wenqiao He

Rachel Sendor

Varun R Potlapalli

Melchior M Kashamuka

Antoinette K Tshefu

Fernandine Phanzu

Albert Kalonji

Billy Ngasala

Kyaw Lay Thwai

Jonathan J Juliano

Jessica T Lin

Jonathan B Parr

Roles

Abstract

Background

Methods

Results

Conclusions

Author summary

Introduction

Materials and Methods

Ethics statement

Mining and selection of multi-copy targets in P. ovalecurtisi and P. ovalewallikeri genomes

Fig 1. Approach to develope real-time PCR assays for the detection and differentiation of P. ovalecurtisi and P. ovalewallikeri.

Assay development and optimization

Analytical sensitivity and specificity

Validation using field samples

Statistical analysis

Results

P. ovalecurtisi and P. ovalewallikeri target selection and assay development

Table 1. Best performing primers and probes for P. ovalecurtisi and P. ovalewallikeri detection.

Singleplex real-time PCR assay development

Duplex real-time PCR assay development

Analytical sensitivity and specificity

Fig 2. Duplex P. ovalecurtisi and P. ovalewallikeri assay performance.

Validation using field samples

Fig 3. Assay validation using field samples collected in the DRC.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Georges Snounou

Paul O Mireji

Roles

Author response to Decision Letter 0

Decision Letter 1

Georges Snounou

Paul O Mireji

Roles

Acceptance letter

Georges Snounou

Paul O Mireji

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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