Real-time PCR detection of mixedPlasmodium ovale curtisiandwallikerispecies infections in human and mosquito hosts
Authors: Potlapalli Varun , Muller Meredith S. , Ngasala Billy , Ali Innocent Mbulli , Na Yu Bin , Williams Danielle R. , Kharabora Oksana , Chhetri Srijana , Liu Mei S. , Carey-Ewend Kelly , Lin Feng-Chang , Mathias Derrick , Tarimo Brian B. , Juliano Jonathan J. , Parr Jonathan , Lin Jessica T. 
Published: April 2023

ABSTRACTPlasmodium ovale curtisi(Poc) andPlasmodium 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 detectPocandPowwere modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/μL (95% CI 0.4-1.6) forPocand 4.5 plasmid copies/μL (95% CI 2.7-18) forPow, or 0.1 and 0.8 parasites/μL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103plasmid copies/μL (roughly 200 parasites/μL). Mock mixtures were used to establish criteria for classifying mixedPoc/Powinfections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 100copies/μL (<1 parasite/μL). 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 mixedPoc/Powinfections where nested PCR detected a singlePospecies. When real-time PCR was applied to 14 oocyst-positiveAnophelesmidguts saved from mosquitoes fed onP. ovale-infected persons, mixedPoc/Powinfections were detected in 11 (79%). Based on these results, 8/9P. ovalecarriers transmitted bothP. ovalespecies to mosquitoes, though bothPospecies 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 mixedPoc/Powinfections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated.AUTHOR SUMMARYPlasmodium ovale, one of five species of malaria known to infect humans, in fact represents two distinct species,P. ovale curtisi(Poc) andwallikeri(Pow), that can only be distinguished using molecular diagnostics. ThoughPocandPowcirculate in the same regions in Africa and Asia, mixed infections, where both are found in the same human host, have rarely been described. In this study, we modified existing real-time PCR assays targeting 18S rRNA and developed an algorithm to detect mixedPoc/Powinfections. We then applied these assays to field-collected samples from Tanzania and Cameroon, including blood samples fromP. ovale-infected persons andP. ovale-positive mosquito midguts saved from mosquito feeding assays. We detected bothPocandPowin roughly 10% of humanP. ovaleblood-stage infections, and surprisingly, in a majority of blood-fed mosquitoes. This suggests thatPocandPowco-infect the same hosts more frequently than previously realized.