Reverse transcription quantitative PCR to detect low density malaria infections

<jats:title>Abstract</jats:title><jats:p>Targeted malaria elimination strategies require highly sensitive tests to detect low density malaria infections (LDMI). Commonly used methods for malaria diagnosis such as light microscopy and antigen-based rapid diagnostic tests (RDTs) are not sensitive enough for reliable identification of infections with parasitaemia below 200 parasites per milliliter of blood. While targeted malaria elimination efforts on the Thailand-Myanmar border have successfully used high sample volume ultrasensitive quantitative PCR (uPCR) to determine malaria prevalence, the necessity for venous collection and processing of large quantities of patient blood limits the widespread tractability of this method. Here we evaluated a real-time quantitative reverse transcription PCR (qRT-PCR) method that significantly reduces the required sample volume compared to uPCR. To do this, 304 samples collected from an active case detection program in Kayin state, Myanmar were compared using uPCR and qRT-PCR. <jats:italic>Plasmodium</jats:italic> spp. qRT-PCR confirmed 18 of 21 uPCR <jats:italic>Plasmodium falciparum</jats:italic> positives, while <jats:italic>P. falciparum</jats:italic> specific qRT-PCR confirmed 17 of the 21 uPCR <jats:italic>P. falciparum</jats:italic> positives. Combining both qRT-PCR results increased the sensitivity to 100% and specificity was 95.1%. These results show that malaria detection in areas of low transmission and LDMI, can benefit from the increased sensitivity of qRT-PCR especially where sample volume is limited.</jats:p>