Validation of a four-primer real-time PCR as a diagnostic tool for single and mixed Plasmodium infections

While microscopy remains the gold standard for malaria diagnosis, molecular tools gain more interest. To improve the detection of mixed-infections, we developed a four-primer real-time PCR with 4 Plasmodium species-specific forward primers, based on the pan-primer design with universal Plasmodium primers as described before. After validation for analytical sensitivity, specificity and reproducibility, the four-primer-PCR was evaluated on 351 blood samples from patients presenting at the outpatient clinic of the Institute of Tropical Medicine (Belgium). With the four-primer-PCR, we identified 188 Plasmodium falciparum, 54 P. vivax, 52 P. ovale and 13 P. malariae single infections, 27 mixed-infections (14 Pf+Pm; 12 Pf+Po; 1 Pv+Pm) and 17 negative specimens. Compared to the pan-primer-PCR, we observed lower Ct-values with a mean difference of 2.23, a higher analytical sensitivity (Pf/Pv: 0.02; Po 0.004; Pm: 0.006 asexual parasites/mul) and 15 extra mixed-infections. Compared to microscopy, we detected 17 extra mixed-infections and identified Plasmodium species in 4 microscopic-positive samples in which species identification was not possible. Additionally, the PCR corrected 13 species mismatches between P. ovale and P. vivax and detected 11 P. falciparum as a second species that was not identified by microscopy and in five of them not picked up by RDT. PCR confirmed the presence of P. falciparum in 30/46 HRP-2 positive samples that were microscopy negative. We conclude that the presently developed four-primer real-time PCR is complementary to standard malaria diagnostic tests in clinical laboratories with an added value for simultaneous identification of the four Plasmodium species and the detection of mixed-infections