Comparison of three real-time PCR methods with blood smears and rapid diagnostic test in Plasmodium sp. infection

In cases of malaria, rapid and accurate diagnosis of Plasmodium sp. is essential. In this study three different quantitative, real-time PCR methods were compared with routine methods used for malaria diagnosis. A comparative study was conducted prospectively in the laboratories of Montpellier and Nîmes University Hospitals. The methods used for routine diagnostic malaria testing consisted of microscopic examination of Giemsa-stained blood smears and rapid diagnostic tests. Three quantitative real-time PCR methods (qRT-PCR) were tested: qRT-PCR1 amplified a specific sequence on the P. falciparum Cox1 gene, qRT-PCR2 amplified a species-specific region of the multicopy 18S rDNA, and qRT-PCR3 amplified a mitochondrial DNA sequence. Among the 196 blood samples collected, 73 samples were positive in at least one of the five tests. Compared with the routine method, there were no false negatives for P. falciparum diagnosis in either qRT-PCR1 or qRT-PCR3. In all P. ovale, P. vivax and P. malariae infections diagnosed from blood smears, qRT-PCR1 was negative, as expected, whereas qRT-PCR2 and qRT-PCR3 were positive and concordant (simple kappa coefficient = 1). One negative sample from microscopy was positive with both qRT-PCR2 and qRT-PCR3. Together, qRT-PCR3 and the combined qRT-PCR1 and qRT-PCR2 were concordant with routine methods for malaria diagnosis (99% and 99.5%, respectively). These three rapid, molecular qRT-PCR methods, used alone or in association, showed excellent results, with high concordance, accuracy and reliability in malaria diagnosis.