Multiplexed real-time PCR assay for discrimination of Plasmodium species with improved sensitivity for mixed infections

Abstract

The implementation of real-time PCR for the diagnosis of malaria has been hampered by poor sensitivity for the detection of mixed infections. We have optimized a method that enhances the sensitivity of detection of minor species in mixed infections within a single multiplex reaction. Our assay uses species-specific forward primers in combination with a conserved reverse primer and largely overcomes primer competition for the minor species DNA. With a blind panel of clinical samples, we successfully identified the species in 13/16 mixed infections. This assay was further validated with 91 blood samples and demonstrated a specificity and sensitivity for single infections of 100% compared with nested PCR as the "gold standard." This test has been implemented for routine confirmation of malaria species in Alberta, Canada. In comparison with species identification by microscopy, the real-time PCR test demonstrated greater sensitivity for the identification of species causing low-level and mixed infections and for the discrimination of Plasmodium species other than Plasmodium falciparum. Our experience supports a role for real-time PCR in the identification of malarial species in conjunction with microscopy.

FIG. 1.

Simultaneous detection of four Plasmodium species. A mixture of genomic DNAs of four Plasmodium species served as the template in the species identification real-time PCR. Rn, normalized reporter.

FIG. 2.

Manual baseline correction improves sensitivity for mixed infections. In this specimen, the minor species P. vivax was undetected with the Auto C_T baseline parameter (A) but was clearly identified with the Manual C_T baseline (B) (cycles 3 to 15). Rn, normalized reporter.