Development of a Plasmodium PCR for monitoring efficacy of antimalarial treatment

Abstract

We report in this work a highly sensitive and nonradioactive PCR method for the detection of the four species of parasite causing human malaria. Plasmodium-specific primers corresponding to the small-subunit rRNA genes of the malaria parasite were used, and a 291-bp fragment was amplified. Our results showed a high specificity for the four human Plasmodium species, and we were able to detect one parasite in 50 microl of whole blood. The responses of 12 patients infected with Plasmodium falciparum to antimalarial therapy were monitored by PCR diagnosis and examination of thick blood film for at least 20 min by an experienced microscopist. For one patient this study allowed early diagnosis of therapeutic failure, confirmed 7 days later by examination of the thick blood film. A total of 134 samples were examined; 94 were positive by PCR, and among these 68 were positive by thick blood film examination. The sensitivity of the thick blood film was 72.3% compared to PCR and 60.7% compared to dot blot hybridization.

FIG. 1

Determination of the genus specificity of PCR. (A) Analysis by agarose gel electrophoresis and ethidium bromide staining. (B) Southern blot and hybridization with a digoxigenin-11–dUTP-labeled probe. Lane 1, φX174 DNA cleaved with HaeIII as a molecular size marker, lane 2, L. infantum (10⁶ organisms); lane 3, T. gondii (10⁶ organisms); lane 4, T. gondii DNA (1 μg); lane 5, human DNA (1 μg); lane 6, T. cruzi (10⁶ organisms); lane 7, P. vivax (0.3%); lane 8, P. ovale (0.1%); lane 9, P. malariae (1%); lane 10, pBR322 DNA cleaved with HaeIII as a molecular size marker; lane 11, P. falciparum (0.5%). Arrows at the right indicate the 291-bp PCR product.

FIG. 2

Determination of the absolute sensitivity of PCR. (A) Analysis by agarose gel electrophoresis. (B) Southern blot hybridization with a digoxigenin-11–dUTP-labeled probe. Lane 1, pBR322 DNA cleaved with HaeIII as a molecular size marker; lanes 2 to 10, 10-fold dilutions of a single isolate with uninfected erythrocytes (1 to 10⁻⁸%). Arrows at the right indicate the 291-bp PCR product.