A novel duplex qualitative real-time PCR assay for the detection and differentiation of Plasmodium ovale curtisi and Plasmodium ovale wallikeri malaria
Authors: He Wenqiao , Sendor Rachel , Potlapalli Varun R. , Kashamuka Melchior M. , Tshefu Antoinette K. , Phanzu Fernandine , Kalonji Albert , Ngasala Billy , Thwai Kyaw Lay , Juliano Jonathan J. , Lin Jessica T. , Parr Jonathan B. 
Published: November 2023

Background

P. ovalespp. infections are endemic across multiple African countries and are caused by two distinct non-recombining species,P. ovale curtisi(Poc) andP. 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 ofPocandPowthat can be used to improve our understanding of these parasites.

Methods

Repetitive sequence motifs were identified in availablePocandPowgenomes 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 55P. ovalespp. samples and 40 non-ovalePlasmodiumsamples from the DRC.PocandPowprevalence among symptomatic individuals sampled across three provinces of the DRC were estimated.

Results

The best-performingPocandPowtargets 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 forPocandPow, respectively. Species was determined in 80% of allP. ovalespp.-positive field samples and 100% of those with >10 parasites/µl. MostP. ovalespp. field samples from the DRC were found to bePocinfections.

Conclusions

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

Author Summary

Non-falciparum malaria is gaining attention, especially in settings whereP. falciparumtransmission is declining.Plasmodium ovale curtisi(Poc) andwallikeri(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 distinguishPocandPowand are not well-suited for high-throughput use, hindering our understanding ofP. ovalespp. epidemiology. Mining recently availablePocandPowreference 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-densityPowinfections, this new assay can be used for high-throughput studies of symptomaticP. ovalespp. infections in malaria-endemic countries. We apply this tool to samples collected during a large study conducted in the DRC and investigateP. ovalespp. epidemiology across health centers in three provinces.