Accurate and sensitive TaqMan real-time PCR assays to detect and quantify simian malaria parasites Plasmodium cynomolgi, Plasmodium inui and Plasmodium coatneyi.

Abstract

Background: Human infections with simian parasites such as Plasmodium inui, P. cynomolgi, and P. coatneyi have been reported in several countries within Southeast Asia. Though infections caused by these parasites are mostly asymptomatic and mild, this could hamper the efforts to eradicate malaria in this region. In this report, we outlined the development and optimisation of new TaqMan-based real-time PCR assays to accurately identify and quantify P. inui, P. cynomolgi, and P. coatneyi.

Methods: Species-specific hydrolysis probes were designed for real-time PCR assays targeting variable regions within the 18S rRNA genes of P. inui, P. cynomolgi and P. coatneyi. The assays were tested against plasmid DNA containing the target 18S rRNA gene as well as plasmid DNA harbouring 18S rRNA gene fragments of other human and simian Plasmodium species. The assays were further tested using genomic DNA samples from macaques and Anopheles mosquitoes that were previously confirmed to be positive for simian Plasmodium by nested PCR assays.

Results: The developed assays demonstrated high specificity, amplifying only the target 18S rRNA gene of their respective Plasmodium species. The assays were also able to detect and quantify as few as 10 copies of template DNA per PCR reaction, which is equivalent to approximately five parasites, with amplification efficiencies of > 95%. The generated standard curves for each assay showed linear relationship between the copy numbers (10-10⁶ copies) and the cycle threshold (Ct) values. The accuracy of the assays was further supported by their ability to produce results equivalent to those obtained using conventional nested PCR assays.

Conclusions: The newly developed assays can accurately detect and quantify P. inui, P. cynomolgi and P. coatneyi, with limit of detection of approximately five parasites. Given the streamlined nature of real-time PCR, these assays could be implemented for routine malaria surveillance in areas where zoonotic malaria is prevalent. In addition, these assays are suitable for the detection of submicroscopic infections and in mosquito samples, which often have low parasite densities.