{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"medRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T12:16:44Z","timestamp":1768565804885,"version":"3.49.0"},"posted":{"date-parts":[[2023,10,31]]},"group-title":"Infectious Diseases (except HIV\/AIDS)","reference-count":53,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,10,31]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>\n                    <jats:italic>P. ovale<\/jats:italic>\n                    spp. infections are endemic across multiple African countries and are caused by two distinct non-recombining species,\n                    <jats:italic>P. ovale curtisi<\/jats:italic>\n                    (\n                    <jats:italic>Poc<\/jats:italic>\n                    ) and\n                    <jats:italic>P. ovale wallikeri<\/jats:italic>\n                    (\n                    <jats:italic>Pow<\/jats:italic>\n                    ). 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\n                    <jats:italic>Poc<\/jats:italic>\n                    and\n                    <jats:italic>Pow<\/jats:italic>\n                    that can be used to improve our understanding of these parasites.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>\n                    Repetitive sequence motifs were identified in available\n                    <jats:italic>Poc<\/jats:italic>\n                    and\n                    <jats:italic>Pow<\/jats:italic>\n                    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\n                    <jats:italic>P. ovale<\/jats:italic>\n                    spp. samples and 40 non-ovale\n                    <jats:italic>Plasmodium<\/jats:italic>\n                    samples from the DRC.\n                    <jats:italic>Poc<\/jats:italic>\n                    and\n                    <jats:italic>Pow<\/jats:italic>\n                    prevalence among symptomatic individuals sampled across three provinces of the DRC were estimated.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                    The best-performing\n                    <jats:italic>Poc<\/jats:italic>\n                    and\n                    <jats:italic>Pow<\/jats:italic>\n                    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\/\u00b5l for\n                    <jats:italic>Poc<\/jats:italic>\n                    and\n                    <jats:italic>Pow<\/jats:italic>\n                    , respectively. Species was determined in 80% of all\n                    <jats:italic>P. ovale<\/jats:italic>\n                    spp.-positive field samples and 100% of those with &gt;10 parasites\/\u00b5l. Most\n                    <jats:italic>P. ovale<\/jats:italic>\n                    spp. field samples from the DRC were found to be\n                    <jats:italic>Poc<\/jats:italic>\n                    infections.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>\n                    We identified promising multi-copy targets for molecular detection and differentiation of\n                    <jats:italic>Poc<\/jats:italic>\n                    and\n                    <jats:italic>Pow<\/jats:italic>\n                    and used them to develop a new duplex real-time PCR assay that performed well when applied to diverse field samples. Though low-density\n                    <jats:italic>Pow<\/jats:italic>\n                    infections are not reliably detected, the assay is highly specific and can be used for high-throughput studies of\n                    <jats:italic>P. ovale<\/jats:italic>\n                    spp. epidemiology among symptomatic cases in malaria-endemic countries like the DRC.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Author Summary<\/jats:title>\n                  <jats:p>\n                    Non-falciparum malaria is gaining attention, especially in settings where\n                    <jats:italic>P. falciparum<\/jats:italic>\n                    transmission is declining.\n                    <jats:italic>Plasmodium ovale curtisi<\/jats:italic>\n                    (\n                    <jats:italic>Poc<\/jats:italic>\n                    ) and\n                    <jats:italic>wallikeri<\/jats:italic>\n                    (\n                    <jats:italic>Pow<\/jats:italic>\n                    ) 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\n                    <jats:italic>Poc<\/jats:italic>\n                    and\n                    <jats:italic>Pow<\/jats:italic>\n                    and are not well-suited for high-throughput use, hindering our understanding of\n                    <jats:italic>P. ovale<\/jats:italic>\n                    spp. epidemiology. Mining recently available\n                    <jats:italic>Poc<\/jats:italic>\n                    and\n                    <jats:italic>Pow<\/jats:italic>\n                    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\n                    <jats:italic>Pow<\/jats:italic>\n                    infections, this new assay can be used for high-throughput studies of symptomatic\n                    <jats:italic>P. ovale<\/jats:italic>\n                    spp. infections in malaria-endemic countries. We apply this tool to samples collected during a large study conducted in the DRC and investigate\n                    <jats:italic>P. ovale<\/jats:italic>\n                    spp. epidemiology across health centers in three provinces.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2023.10.31.23297819","type":"posted-content","created":{"date-parts":[[2023,11,1]],"date-time":"2023-11-01T06:27:33Z","timestamp":1698820053000},"source":"Crossref","is-referenced-by-count":1,"title":["A novel duplex qualitative real-time PCR assay for the detection and differentiation of Plasmodium ovale curtisi and Plasmodium ovale wallikeri malaria"],"prefix":"10.64898","author":[{"given":"Wenqiao","family":"He","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rachel","family":"Sendor","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Varun 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