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

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:10.1101/2023.10.31.23297819

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

medRxiv [Preprint]. 2023 Oct 31:2023.10.31.23297819. doi: 10.1101/2023.10.31.23297819.

Authors

Affiliations

¹ Division of Infectious Diseases and Institute for Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States.
² Department of Epidemiology, School of Public Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, Southern Medical University, Guangzhou, China.
³ Department of Epidemiology, Gillings School of Global Public Health, Chapel Hill, NC, United States.
⁴ Kinshasa School of Public Health, Kinshasa, Democratic Republic of the Congo.
⁵ SANRU Asbl, Kinshasa, Democratic Republic of the Congo.
⁶ Muhimbili University of Health and Allied Sciences, Dar Es Salaam, Tanzania.

Abstract

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.

Keywords: Congo; P. ovale curtisi; P. ovale wallikeri; non-falciparum malaria; prevalence; qualitative real-time PCR.

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Preprint

Abstract

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