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

Background: spp. infections are endemic across multiple African countries and are caused by two distinct non-recombining species, () and (). 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 and that can be used to improve our understanding of these parasites.

Methods: Repetitive sequence motifs were identified in available and 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 spp. samples and 40 non-ovale samples from the DRC. and prevalence among symptomatic individuals sampled across three provinces of the DRC were estimated.

Results: The best-performing and 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 and , respectively. Species was determined in 80% of all spp.-positive field samples and 100% of those with >10 parasites/μl. Most spp. field samples from the DRC were found to be infections.

Conclusions: We identified promising multi-copy targets for molecular detection and differentiation of and and used them to develop a new duplex real-time PCR assay that performed well when applied to diverse field samples. Though low-density infections are not reliably detected, the assay is highly specific and can be used for high-throughput studies of spp. epidemiology among symptomatic cases in malaria-endemic countries like the DRC.