Development of a real-time PCR Assay for Detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine Clinical Diagnosis.

A TaqMan-based real-time PCR qualitative assay for the detection of three species of malaria parasites—
Plasmodium falciparum, P. ovale, and P. vivax—was devised and evaluated using 122 whole-blood samples from
patients who had traveled to areas where malaria is endemic and who presented with malaria-like symptoms
and fever. The assay was compared to conventional microscopy and to an established nested-PCR assay. The
specificity of the new assay was confirmed by sequencing the PCR products from all the positive samples and
by the lack of cross-reactivity with Toxoplasma gondii and Leishmania infantum DNA. Real-time PCR assay
showed a detection limit (analytical sensitivity) of 0.7, 4, and 1.5 parasites/l for P. falciparum, P. vivax, and
P. ovale, respectively. Real-time PCR, like nested PCR, brought to light errors in the species identification by
microscopic examination and revealed the presence of mixed infections (P. falciparum plus P. ovale). Real-time
PCR can yield results within 2 h, does not require post-PCR processing, reduces sample handling, and
minimizes the risks of contamination. The assay can therefore be easily implemented in routine diagnostic
malaria tests. Future studies are warranted to investigate the clinical value of this technique.