Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis

Abstract

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/ micro 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.

Figures

FIG. 1.

Sensitivity of P. falciparum, P. ovale, and P. vivax real-time assay. (A) Amplification (as duplicate) of P. falciparum sample dilutions containing 704 (Toto), 70 (1), 7 (2), and 0.7 (3) parasites/μl. (B) Plot of mean CT values from four replicates tested against the P. falciparum DNA inputs. The plot of CT values and DNA input fits a linear function (R2 = 0.952). (C) Amplification (as duplicate) of P. ovale sample dilutions containing 4620 (Toto), 462 (1), 46 (2), and 4 (3) parasites/μl. (D) Plot of mean CT values from four replicates tested against the P. ovale DNA inputs. The plot of the CT values and DNA input fits a linear function (R2 = 0.952). (E) Amplification (as duplicate) of P. vivax sample dilutions containing 15,000 (Toto), 1,500 (1), 150 (2), 15 (3), and 1.5 (4) parasites/μl. (F) Plot of mean CT values from five replicates tested against P. vivax DNA inputs. The plot of CT values and DNA input fits a linear function (R2 = 0.969).