A Sensitive Species-specific Reverse Transcription Real-time PCR Method for Detection of and

As the global burden of malaria decreases and countries strive towards disease elimination, there is a greater demand for sensitive diagnostics to target the submicroscopic reservoir of infection. We describe here a sensitive species-specific RT-qPCR method to differentiate between and infections at the submicroscopic level. With amplification of the 18S rRNA genes from total nucleic acids (both DNA and RNA), we discern and with a limit of detection of 10 parasites/mL and 18 copies/μL, respectively. This assay was validated with 519 blood samples, negative by thick-smear, from febrile and asymptomatic cohorts from Colombia. These results were directly compared to a qPCR-based method (DNA only) as the gold standard. Of the samples from patients who presented with fever ( = 274), 34 infections were identified by RT-qPCR (16 , 15 , and 3 mixed), of which only 10 infections were identified at the species level by qPCR. Within the asymptomatic cohort ( = 245), 13 infections were identified by RT-qPCR (3 , 3 , and 7 mixed), whereas the species for only one infection was determined by qPCR. We conclude that this species-specific RT-qPCR method provides a more sensitive tool for species identification compared to DNA based qPCR methods.