Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
3397706 | Clinical Microbiology and Infection | 2011 | 7 Pages |
Although microscopy remains the reference standard for malaria diagnosis, molecular tools are attracting increasing interest. To improve the detection of mixed infections, we developed a four-primer real-time PCR with four Plasmodium species-specific forward primers, based on the pan-primer design with universal Plasmodium primers as described previously. 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 (Pf), 54 Plasmodium vivax (Pv), 52 Plasmodium ovale (Po) and 13 Plasmodium malariae (Pm) single infections, 27 mixed infections (14 Pf + Pm; 12 Pf + Po; one Pv + Pm) and 17 negative specimens. We found lower cycle threshold values than with the pan-primer PCR, with a mean difference of 2.23, a higher analytical sensitivity (in asexual parasites/µL: Pf/Pv, 0.02; Po, 0.004; Pm, 0.006) and 15 extra mixed infections. As compared with microscopy, 17 extra mixed infections were detected and Plasmodium species were identified in four microscopy-positive samples in which species identification was not possible. Additionally, the PCR corrected 13 species mismatches between Po and Pv, and in 11 cases detected Pf as a second species that was not identified by microscopy and in five of them was not detected by rapid diagnostic tests (RDTs). PCR confirmed the presence of Pf in 30/46 histidine-rich protein-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.