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 primers-dimers. Both the P. malariae and P. ovale cooperative primer-based assays had at least 10-fold lower detection limit compared with the corresponding conventional primer-based assays. More important, the cooperative primer-based assays were evaluated in a cross-sectional study using 560 samples obtained from two health facilities in Ghana. The prevalence rates 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 Plasmodium falciparum. The study demonstrates the public health importance of including detection tools with lower detection limits in routine diagnosis and surveillance of nonfalciparum species. This will be necessary for comprehensively assessing the effectiveness of malaria interventions and control measures aimed toward global malaria elimination.

Figure 1

The specificity and detection limits of Plasmodium malariae and Plasmodium ovale assays. Assays were performed using Plasmodium falciparum (Pf), P. malariae (Pm), P. ovale curtisi (Po curtisi), P. ovale wallikeri (Po wallikeri), and Plasmodium vivax (Pv) genomic DNA. A: Melt curve for P. malariae (blue). B: Melt curves for P. ovale curtisi (green) and P. ovale wallikeri (orange) assays. Other colors represent the P. falciparum, P. vivax, and nontemplate control (NC). C: Separation of the resulting P. malariae real-time quantitative PCR (qPCR) amplicons on 1.5% agarose gel. D: Separation of the resulting qPCR amplicons for P. ovale subspecies on 1.5% agarose gel. Expected amplicon sizes for P. ovale curtisi and P. ovale wallikeri were 528 and 521 bp, respectively. E: The detection limits for P. malariae (blue curve) and P. ovale (red curve) assays were determined using a 10-fold serial dilution of plasmids. Plasmid concentrations were log-transformed and then analyzed using probit analysis. The probability of detection was plotted against the plasmid copy numbers/μL of the DNA. Molecular weight marker (MM) shown in bp.

Figure 2

Comparison of cooperative and conventional real-time quantitative PCR (qPCR) assays. Assays were performed using serially diluted Plasmodium malariae (MRA-179) and Plasmodium ovale (MRA-180) plasmids. The qPCR amplicons were separated on 1.5% agarose gel. Molecular weight marker (MM) shown in bp.

Figure 3

Prevalence of Plasmodium species among study participants. A: The prevalence of genus Plasmodium determined by microscopy and real-time quantitative PCR (qPCR) in Ewim and Sogakope. B: The prevalence of Plasmodium falciparum (Pf), Plasmodium malariae (Pm), and Plasmodium ovale (Po) determined by SYBR Green–based qPCR assays and microscopy among the combined study population. C: The proportion of Plasmodium species mono and mixed infections among study participants in the two study sites. D: The prevalence of Plasmodium species positive cases by age group (in years). The prevalence was determined by expressing the total number of positive cases as a percentage of the total sample size for each of the stratified age groups. n = 560 study participants (A); n = 178 in Ewin (A); n = 382 in Sogakope (A).

Figure 4

Quantification of Plasmodium species copy numbers. Data have been presented as box plot, where the boxes represent the interquartile range, the line through the box is the median, and the whiskers indicate the 1^st and the 99^th percentiles. The geometric mean of the parasite load is denoted by the plus symbol (+), whereas the individual dots represent outliers. Parasite copy numbers were compared using U-test. Statistical significance has been represented. A: The overall parasite load for Plasmodium falciparum, Plasmodium malariae, and Plasmodium ovale infections. B:Plasmodium species parasite load by age group (years) stratification. C: Correlation of parasite load determined by microscopy and real-time quantitative PCR (qPCR) C_T values. n = 479 P. falciparum infections (A); n = 104 P. malariae infections (A); n = 31 P. ovale infections (A). ∗P < 0.05, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.