Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri infections in human and mosquito hosts

Varun R. Potlapalli, Meredith S. Muller, Billy Ngasala, Innocent Mbulli Ali, Yu Bin Na, Danielle R. Williams, Oksana Kharabora, Srijana Chhetri, Mei S. Liu, Kelly Careyewend, Feng Chang Lin, Derrick Mathias, Brian B. Tarimo, Jonathan J. Juliano, Jonathan B. Parr, Jessica T. Lin

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Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct nonrecombining Plasmodium 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/μL (95% CI 0.4–1.6) for Poc and 4.5 plasmid copies/ μL (95% CI 2.7–18) for Pow, 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 103 plasmid copies/μL (roughly 200 parasites/μL). 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/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 in 19 samples, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovale-infected persons, mixed Poc/Pow infections were detected in 11/14 (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.

Original languageEnglish (US)
Article numbere0011274
JournalPLoS neglected tropical diseases
StatePublished - Dec 2023
Externally publishedYes

ASJC Scopus subject areas

  • Public Health, Environmental and Occupational Health
  • Infectious Diseases

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