The most performing techniques enabling early diagnosis of infectious diseases rely on nucleic acid detection. Today, because of their high technicality and cost, nucleic acid amplification tests (NAAT) are of benefit only to a small fraction of developing countries population. By reducing costs, simplifying procedures and enabling multiplexing, paper microfluidics has the potential to considerably facilitate their accessibility. However, most of the studies performed in this area have not quit the lab. This letter brings NAAT on paper closer to the field, by using clinical samples and operating in a resource-limited setting. We first performed isothermal reverse transcription and Recombinase Polymerase Amplification (RT-RPA) of synthetic Ribonucleic Acid (RNA) of Ebola virus using paper microfluidics devices. We further applied this method in Guinea to detect the presence of Ebola virus in human sample RNA extracts, with minimal facilities (carry-on detection device and freeze-dried reagents on paper). RT-RPA results were available in few minutes and demonstrate a sensitivity of 90.0% compared to the gold-standard RT-PCR on a set of 43 patient samples. Furthermore, the realization of a nine-spot multilayered device achieving the parallel detection of three distinct RNA sequences opens a route toward the detection of multiple viral strains or pathogens.

The diagnosis of infectious diseases is entering a new and interesting phase. Technologies based on paper microfluidics, coupled to developments in isothermal amplification of Nucleic Acids (NAs) raise opportunities for bringing the methods of molecular biology in the field, in a low setting environment. A lot of work has been performed in the domain over the last few years and the landscape of contributions is rich and diverse. Most often, the level of sample preparation differs, along with the sample nature, the amplification and detection methods, and the design of the device, among other features. In this review, we attempt to offer a structured description of the state of the art. The domain is not mature and there exist bottlenecks that hamper the realization of Nucleic Acid Amplification Tests (NAATs) complying with the constraints of the field in low and middle income countries. In this domain however, the pace of progress is impressively fast. This review is written for a broad Lab on a Chip audience.