Development of a single tube hemi-nested PCR for genus-specific detection of Plasmodium in oligoparasitemic patients.

Primers targeting the Plasmodium small-subunit (SSU) rDNA were designed to amplify DNA from P. vivax, P. falciparum, P. malariae, and P. ovale, using conventional PCR, two-step nested PCR (HNPCR), and single tube hemi-nested PCR (STHNPCR). The limit of detection of parasite DNA for the conventional PCR, HNPCR, and STHNPCR were 10 pg, 0.01 pg, and 0.1 pg, respectively, indicating that the STHNPCR is 100-fold more sensitive than conventional PCR, and only 10 times less sensitive than HNPCR. In addition, the detection limit was also defined using blood from a patient infected with P. falciparum. Using the saponin method, the detection limit of the conventional PCR, HNPCR, and STHNPCR were 70, 0.7, and 0.07 parasites/microl, respectively. Finally, the three techniques were evaluated using blood from 30 patients receiving antimalarial treatment, and negative by microscopy and conventional PCR. The HNPCR could still detect specific DNA in 16/30 patients, whereas STHNPCR detected parasite DNA in 10/30 patients, but the difference was not statistically significant. No significant correlation was found between presence of clinical manifestations and presence of parasite DNA, detected by either HNPCR or STHNPCR. We conclude that these sensitive molecular diagnostic systems can be used for the diagnosis of asymptomatic oligoparasitemic patients.