RESEARCH ARTICLE Reverse transcription PCR to detect low density malaria infections [version 2; peer review: 2 approved with reservations, 1 not approved] Peter Christensen 1,2 , Zbynek Bozdech 3 , Wanitda Watthanaworawit 1 , Laurent Rénia 4,5 , Benoît Malleret 4,6 , Clare Ling 1,7 , François Nosten 1,7 1 Shoklo Malaria Research Unit, Mahidol University, Maesot, Tak, 63110, Thailand 2 Microbiology and Immunology, University of Otago, Dunedin, Otago, 9016, New Zealand 3 School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Singapore 4 Singapore Immunology Network, A*STAR, Singapore, 138648, Singapore 5 A*STAR ID Labs, A*STAR, Singapore, 138648, Singapore 6 Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117545, Singapore 7 Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, UK First published: 22 Feb 2021, 6:39 https://doi.org/10.12688/wellcomeopenres.16564.1 Latest published: 04 Aug 2021, 6:39 https://doi.org/10.12688/wellcomeopenres.16564.2 v2 Abstract Background: 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. Methods: Here we evaluated a real-time reverse transcription PCR (RT-qPCR) method that 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 RT-qPCR. Results: Plasmodium spp. RT-qPCR confirmed 18 of 21 uPCR Plasmodium falciparum positives, while P. falciparum specific RT-qPCR confirmed 17 of the 21 uPCR P. falciparum positives. Combining both RT-qPCR results increased the sensitivity to 100% and specificity was 95.1%. Conclusion: Malaria detection in areas of low transmission and LDMI can benefit from the increased sensitivity of ribosomal RNA detection by RT-PCR, especially where sample volume is limited. Isolation of high quality RNA also allows for downstream analysis of malaria Open Peer Review Reviewer Status Invited Reviewers 1 2 3 version 2 (revision) 04 Aug 2021 report version 1 22 Feb 2021 report report report Gisely Melo, Instituto de Pesquisa Clínica Carlos Borborema, Manaus, Brazil 1. Muneaki Hashimoto, National Institute of Advanced Industrial Science and Technology (AIST), Hayashi-cho, Japan 2. Julian Tanner , The University of Hong Kong, Pokfulam, Hong Kong 3. Any reports and responses or comments on the article can be found at the end of the article. Page 1 of 17 Wellcome Open Research 2021, 6:39 Last updated: 04 OCT 2021