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We describe a novel one-step reverse transcriptase real-time PCR (direct RT-PCR) for Plasmodium falciparum malaria parasites that amplifies RNA targets directly from blood. We developed the assay to identify gametocyte-specific transcripts in parasites from patient blood samples, as a means of monitoring malaria parasite transmission in field settings. To perform the test, blood is added directly to a master mix in PCR tubes and analyzed by real-time PCR. The limit of detection of the assay on both conventional and portable real-time PCR instruments was 100 parasites/mL for 18S rRNA, and 1,000 parasites/mL for asexual (PFE0065W) and gametocyte (PF14_0367, PFGEXP5) mRNA targets. The usefulness of this assay in field studies was explored in samples from individuals living in a high-transmission region in Cameroon. The sensitivity and specificity of the assay compared with a standard two-step RT-PCR was 100% for 18S rRNA on both conventional and portable instruments. For PF14_0367, the sensitivity and specificity were 85.7% and 70.0%, respectively, on the conventional instrument and 78.6% and 90%, respectively, on the portable instrument. The concordance for assays run on the two instruments was 100% for 18S rRNA, and 79.2% for PF14_0367, with most discrepancies resulting from samples with low transcript levels. The results show asexual and sexual stage RNA targets can be detected directly from blood samples in a simple one-step test on a field-friendly instrument. This assay may be useful for monitoring malaria parasite transmission potential in elimination settings, where sensitive diagnostics are needed to evaluate the progress of malaria eradication initiatives.

the american journal of tropical medicine and hygiene

A Direct from Blood Reverse Transcriptase Polymerase Chain Reaction Assay for Monitoring Falciparum Malaria Parasite Transmission in Elimination Settings