A novel duplex qualitative real-time PCR assay for the detection and differentiation of Plasmodium ovale curtisi and Plasmodium ovale wallikeri malaria

Background P. ovale spp. infections are endemic across multiple African countries and are caused by two distinct non-recombining species, P. ovale curtisi ( Poc ) and P. ovale wallikeri ( Pow ). These species are thought to differ in clinical symptomatology and latency, but existing diagnostic assays have limited ability to detect and distinguish them. In this study, we developed a new duplex assay for the detection and differentiation of Poc and Pow that can be used to improve our understanding of these parasites. Methods Repetitive sequence motifs were identified in available Poc and Pow genomes and used for assay development and validation. We evaluated the analytical sensitivity and specificity of the best-performing assay using a panel of samples from Tanzania and the Democratic Republic of the Congo (DRC), then validated its performance using 55 P. ovale spp. samples and 40 non-ovale Plasmodium samples from the DRC. Poc and Pow prevalence among symptomatic individuals sampled across three provinces of the DRC were estimated. Results The best-performing Poc and Pow targets had 9 and 8 copies within the reference genomes, respectively. Our duplex assay had 100% specificity and 95% confidence lower limits of detection of 4.2 and 41.2 parasite genome equivalents/µl for Poc and Pow , respectively. Species was determined in 80% of all P. ovale spp.-positive field samples and 100% of those with >10 parasites/µl. Most P. ovale spp. field samples from the DRC were found to be Poc infections. Conclusions We identified promising multi-copy targets for molecular detection and differentiation of Poc and Pow and used them to develop a new duplex real-time PCR assay that performed well when applied to diverse field samples. Though low-density Pow infections are not reliably detected, the assay is highly specific and can be used for high-throughput studies of P. ovale spp. epidemiology among symptomatic cases in malaria-endemic countries like the DRC. Author Summary Non-falciparum malaria is gaining attention, especially in settings where P. falciparum transmission is declining. Plasmodium ovale curtisi ( Poc ) and wallikeri ( Pow ) are neglected parasites that can cause relapsing malaria and are thought to differ in clinical symptomatology and latency. However, existing diagnostic assays have limited ability to detect and distinguish Poc and Pow and are not well-suited for high-throughput use, hindering our understanding of P. ovale spp. epidemiology. Mining recently available Poc and Pow reference genomes, we identify new multi-copy targets for molecular detection and develop a novel duplex qualitative real-time PCR assay capable of species differentiation. The assay is highly specific and requires short turn-around time. While sensitivity can be improved for low-density Pow infections, this new assay can be used for high-throughput studies of symptomatic P. ovale spp. infections in malaria-endemic countries. We apply this tool to samples collected during a large study conducted in the DRC and investigate P. ovale spp. epidemiology across health centers in three provinces. ### Competing Interest Statement 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. ### Funding Statement Yes ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approvals for these studies were obtained from the Kinshasa School of Public Health, Muhimbili University of Health and Allied Sciences, and the University of North Carolina at Chapel Hill. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data and related metadata underlying reported findings have been provided as part of the submitted article.