Real-time PCR detection of mixedPlasmodium ovale curtisiandwallikerispecies infections in human and mosquito hosts

Abstract

Plasmodium ovalecurtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining malaria species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/uL (95% CI 0.4-1.6) for Poc and 4.5 plasmid copies/uL (95% CI 2.7- 18) for Pow, or 0.1 and 0.8 parasites/uL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/uL (roughly 200 parasites/uL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 100 copies/uL (<1 parasite/uL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to 14 oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovale-infected persons, mixed Poc/Pow infections were detected in 11 (79%). Based on these results, 8/9 P. ovale carriers transmitted both P. ovale species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.

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

“…DRC samples were collected as part of a 2017 study investigating malaria diagnostic test performance in three provinces, Kinshasa, Bas-Uele, and Sud-Kivu[25]. Tanzania samples were collected from participants enrolled in a malaria transmission study in rural Bagamoyo district from 2018-2019 [9, 35, 36]. Enrolled subjects provided informed consent or assent.…”

“…as previously described [2]. Poc versus Pow species was determined using two singleplex real-time PCR assays as previously described [9].…”

“…P. ovale comprises two distinct non-recombining species, P. ovale curtisi ( Poc ) and P. ovale wallikeri ( Pow ) [7]. Poc and Pow have potential differences in clinical symptomatology and latency [8], but existing diagnostic assays have limited ability to detect and distinguish them, and require multiple steps or prolonged cycling time that increases risk of false-positive results [9].…”

“…A nested PCR assay was developed in 2013 [16], and while it can detect samples with 2-10 parasites/µl, this assay requires multiple steps and long turnaround time. Available single-target quantitative real-time PCR assays require separate runs to distinguish Poc and Pow [9, 17, 18]. Because of the limitations of the existing assays, most studies of P. ovale spp.…”