Application of real-time polymerase chain reaction (PCR) analysis for detection and discrimination of malaria parasite species in Thai patients.

ABSTRACT

A TaqMan real-time PCR system was used to detect and discriminate the 4 species of human malaria parasites in clinical blood samples. A 150-base pair (bp) region of the small subunit ribosomal RNA (SSU rRNA) gene of each malaria parasite, including species-specific sequences to be detected by TaqMan probe, was used as a target for PCR analysis. The PCR method used universal primers and species-specific TaqMan probes for Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. The detection threshold for the method, as determined with serial dilution of cultured P. falciparum-infected erythrocytes, was 5 parasite-infected erythrocytes per reaction. Fifty blood samples of falciparum malaria and a second set of 50 samples of vivax malaria, diagnosed by microscopic examination at the Hospital for Tropical Diseases, Mahidol University, Thailand, were analyzed by real-time PCR. In the 50 samples of microscopically-diagnosed falciparum malaria, 40 were regarded as P. falciparum single infection, 7 were P. falciparum and P. vivax mixed infections, and 3 were P. vivax single infection by real-time PCR. In the second set of 50 samples of microscopically diagnosed vivax malaria, all were considered P. vivax single infection by PCR. Neither P. ovale nor P. malariae infection was identified in the 100 blood samples. Real-time PCR analysis was shown to be more sensitive and accurate than routine diagnostic methods. Application and extension of the PCR method reported here will provide a powerful tool for further studies of malaria.