Development and optimization of polymerase chain reaction-based malaria diagnostic methods and their comparison with quantitative buffy coat assay

Polymerase chain reaction (PCR)-based assays targeting the small-subunit rR NA were developed and evaluated, allowing for the simultaneous diagnosis of Plasmodium falciparum and Plasmodium vivax DNA in human blood samples. The PCR methods and quantitative buffy coat (QBC) were compared in 402 patient s. The heminested PCR method showed a sensitivity of 97.4%, which was super ior to the sensitivity of the QBC method (91.7%, P < 0.05), to simple PCR ( 84.6%, P < 0.001), and to PCR with digoxigenin labeling (PCR-DIG) (88.5%, P < 0.001). The PCR-DIG and QBC analyses were more sensitive than simple PCR (P < 0.003 and P < 0.05, respectively). There was no significant differenc e between the sensitivities of the QBC assay and the PCR-DIG assay. The spe cificity for the 3 PCR-based methods was 100%, superior to the specificity calculated for the QBC assay (88.95%, P < 0.009). The frequency of a positi ve result in groups from endemic areas but without detectable parasitemia i ncreased, in order, from simple PCR, QBC test, PCR-DIG, to heminested PCR. An association between a positive PCR result and a history of malaria was a lso found. Taken together, these data suggest that this technology could be further developed to screen people with oligoparasitemia and to monitor ma laria treatment.