Detection and species determination of malaria parasites by PCR: comparison with microscopy and with ParaSight-F and ICT malaria Pf tests in a clinical environment

Abstract

A rapid procedure for the diagnosis of malaria infections directly from dried blood spots by PCR amplification was evaluated with samples from 52 patients. Plasmodium infections were identified with a genus-specific primer set, and species differentiation between Plasmodium falciparum and Plasmodium vivax was analyzed by multiplex PCR. The PCR test with any of the three primer sets was able to detect as few as four parasites per microliter by gel electrophoresis or by nonisotopic paper hybridization chromatography. The diagnoses obtained by PCR correlated closely with those obtained by Giemsa staining except for two samples observed to have mixed P. falciparum-P. vivax infections. These were initially missed by microscopic analysis. In comparison with antigen-capture assays for P. falciparum, the PCR assays were able to detect three infections that were missed by the ParaSight-F test. The PCR test was negative for nine ParaSight-F-positive samples and one ICT Malaria Pf-positive sample, and these were confirmed to be false-positive results. The PCR thus gave no false-negative or false-positive results. Patients undergoing antimalarial therapy were also monitored by the PCR assay. Four of seven patients who were PCR positive for P. vivax at the time of discharge were later readmitted to the hospital with a recurrence of P. vivax infection. We would like to propose that PCR is a sensitive and easy method that can serve as a useful addition to microscopy for the diagnosis and the clinical monitoring of treatment of malaria.

FIG. 1

Detection of multiplex PCR products with species-specific primers. Random samples of patients’ blood were analyzed. Lanes 1 and 6, blood infected with P. vivax (parasitemias, 1.3 and 0.3%, respectively); lanes 2, 4, 5, and 7, blood infected with P. falciparum (parasitemias, 1.1, 0.8, 0.075, and 0.15%, respectively); lanes 3 and 8, blood from non-malaria-infected patients; lane M, 100-bp marker (Promega). (A) PCR-amplified products were run on a 1% agarose gel in 1× TAE electrophoresis buffer. (B) Paper chromatography hybridization. Oligonucleotide probes specific for P. falciparum (Pf) and P. vivax (Pv) were spotted onto 5-mm-wide nitrocellulose strips. The P. vivax-specific probe was spotted on the top right corner. The P. falciparum-specific probe was spotted on the bottom left corner.

FIG. 2

Assay of sensitivities of genus- and species-specific primer sets for the detection of malaria by PCR. Infected blood was serially diluted with noninfected blood. (A) Genus-specific L1 and L2 primers (initial parasitemia, 0.016%). (B) P. vivax-specific Pv1 and Pv2 primers (initial parasitemia, 0.05%). (C) P. falciparum-specific Pf1 and Pf2 primers (initial parasitemia, 0.075%). The numbers above each lane indicate the number of parasites present per microliter of blood; lane M, 100-bp ladder marker (Promega).

FIG. 3

PCR monitoring of blood samples from patients undergoing treatment for malaria by using the genus-specific primers L1 and L2. Five microliters of blood spotted on filter paper was assayed in each reaction. Blood samples were obtained daily. For patient A (A; initial parasitemia, 0.35%) and patient B (B; initial parasitemia, 0.5%), PCR products could still be detected after day 5. No PCR product was observed after 4 days for patient C (C; initial parasitemia, 0.3%). Lane M, 100-bp ladder (Promega) used as a molecular size marker.