Development of Cooperative Primer-based Real-time PCR Assays for the Detection of Plasmodium malariae and Plasmodium ovale.

Abstract

Plasmodium malariae and Plasmodium ovale are increasingly gaining public health attention as the global transmission of falciparum malaria is decreasing. However, the absence of reliable Plasmodium species-specific detection tools has hampered accurate diagnosis of these minor Plasmodium species. In this study, SYBR Green-based real-time PCR assays were developed for the detection of P. malariae and P. ovale using cooperative primers that significantly limit the formation and propagation of primer-dimers. Both the P. malariae and P. ovale cooperative primer-based assays had at least 10-fold lower detection limit compared to the corresponding conventional primers. More importantly, the cooperative primer-based assays were evaluated in a cross-sectional study using 560 samples obtained from two health facilities in Ghana. The prevalence of P. malariae and P. ovale among the combined study population were 18.6% (104/560) and 5.5% (31/560), respectively. Among the Plasmodium positive cases, P. malariae and P. ovale mono-infections were 3.6% (18/499) and 1.0% (5/499), respectively, with the remaining being co-infections with P. falciparum. The study demonstrates the public health importance of including detection tools with lower detection limits in routine diagnosis and surveillance of non-falciparum species. This will be necessary for comprehensively assessing the effectiveness of malaria interventions and control measures aimed towards global malaria elimination.