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J Clin Microbiol. 2020 Feb 26. pii: JCM.01879-19. doi: 10.1128/JCM.01879-19. [Epub ahead of print]

Recombinase polymerase amplification and lateral flow assay for ultrasensitive detection of low-density Plasmodium falciparum infection from controlled human malaria infection studies and naturally acquired infections.

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Institut für Tropenmedizin, Universität Tübingen and German Center for Infection Research, Tübingen, Germany.
Vietnamese - German Center of Excellence in Medical Research, Hanoi, Vietnam.
Centre de Recherches Médicales de Lambaréné and African Partner Institution, German Center for Infection Research, Lambaréné, Gabon.
Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands.
Department of Tropical Medicine, Bernhard Nocht Institute for Tropical Medicine and I. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Sanaria Inc., Rockville, Maryland, USA.
Institut für Tropenmedizin, Universität Tübingen and German Center for Infection Research, Tübingen, Germany


Microscopy and rapid diagnostic tests (RDTs) are the main diagnostic tools for malaria but fail to detect low-density parasitemia that are important for maintaining malaria transmission. To complement existing diagnostic methods, an isothermal reverse transcription recombinase polymerase amplification and lateral flow assay (RT-RPA) was developed. We compared the performance with ultrasensitive reverse transcription qPCR (RT-qPCR) using nucleic acid extracts from blood (n=114) obtained after standardized controlled human malaria infection (CHMI) with Plasmodium falciparum sporozoites. As a preliminary investigation, we also sampled asymptomatic individuals (n=28) in a malaria endemic area (Lambaréné, Gabon) to validate RT-RPA and assess its performance with unprocessed blood samples (dbRT-RPA). In 114 samples analyzed from CHMI-trials, the positive percent agreement to uRT-qPCR was 90% (95% CI: 80-96). The negative percent agreement was 100% (95% CI: 92-100). The lower limit of detection was 64 parasites/ml. In Gabon, RT-RPA was 100% accurate with asymptomatic volunteers (n=28) while, simplified dbRT-RPA showed 89% accuracy. In a subgroup analysis, RT-RPA detected 9/10 of RT-qPCR positive samples, while loop-mediated isothermal amplification (LAMP) detected 2/10. RT-RPA is a reliable diagnostic test for asymptomatic low-density infections. It is particularly useful in settings where uRT-qPCR is difficult to implement.


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