Recombinase Polymerase Amplification Compared to Real-Time Polymerase Chain Reaction Test for the Detection of Fasciola hepatica in Human Stool

Fasciola hepatica is the most widely distributed trematode with millions of people infected worldwide.1 The highlands of Bolivia and Peru are hyperendemic areas with a prevalence of up to 70% determined by stool microscopy. Furthermore, the areas that are endemic and hyperendemic for fascioliasis are expanding as better data are emerging from diverse countries. In northwest Ethiopia where only sporadic fascioliasis had previously been described, 3.3% of children were found infected in 2013.2 In some provinces of Vietnam, a prevalence between 2% and 11% have been reported.3 Similarly, new endemic areas have been described in Iran and Kyrgystan.4,5 Nonetheless, accurate data on the distribution and burden of fascioliasis are still limited. Difficulties in assessing the epidemiology of Fasciola stem from the focal distribution of the infection even in endemic areas and the poor performance of currently available diagnostic methods.6 Fascioliasis surveillance by stool microscopy is highly dependent on personnel training and motivation. In addition, microscopy suffers from poor sensitivity.6 Although egg concentration techniques may achieve higher sensitivity, they still require testing of multiple specimens and most are not standardize to provide quantitative results.7–9 The KatoKatz test is a quantitative technique recommended for diagnosis, but it is insensitive in low-burden infections.10 Therefore, as endemic areas of Fasciola expand, improved diagnostic capacity is needed. Coproantigen detection in the stool by enzyme-linked immunosorbent assay (ELISA) is highly sensitive and specific.11 A version using the MM3 monoclonal antibody has been successfully used to test human fecal samples, but no commercial version is available and the potential for point-of-care use has not been tested.12 The ideal diagnostic method should require minimal personnel training, be deployable to resource-limited settings, provide semiquantitative or quantitative results, and be highly sensitive to detect low-burden infections. Isothermal DNA amplification methods have been touted as alternative diagnostic techniques that might be more widely applicable than polymerase chain reaction (PCR)– based tests that use expensive thermal cycling equipment and require extensive personnel training. In particular, recombinase polymerase amplification (RPA) uses a highly efficient displacement polymerase that amplifies detectable products at a constant temperature of 37°C in as little as 20 minutes.13 Several tests using RPA have demonstrated high sensitivity and specificity for detecting small amounts of viral, bacterial, and parasite DNA.14–16 The objective of this study was to perform the characterization of an RPA and real-time PCR tests to detect Fasciola DNA in stools and compare their sensitivity and specificity using microscopytested human samples as the comparator. This study provides the first proof of concept for the future adaptation of the Fasciola RPA to semiquantitative point-of-care diagnosis. METHODS Ethics statement. Samples were collected as part of an ongoing epidemiologic study of fascioliasis in the Peruvian highlands. In this study, three stool samples per subject were tested by microscopy including the Kato-Katz and Lumbreras rapid sedimentation tests for the presence of helminth eggs and larvae.17,18 Participants in the epidemiologic study were also tested for F. hepatica serum antibodies using FAS2 ELISA (Bionoma, Lima, Peru). The study used de-identified stool samples of those consenting to the use of their discarded specimens for future studies. Samples were banked at the Universidad Peruana Cayetano Heredia and University of Texas Medical Branch Collaborative Research Center in Cusco, Peru. The study protocol was reviewed by the Universidad Peruana Cayetano Heredia and University of Texas Medical Branch Institutional Review Boards. Specimen procurement. Adult parasites and eggs of F. hepatica were obtained from naturally infected cattle livers *Address correspondence to Miguel M. Cabada, Infectious Diseases Division, Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas and Universidad Peruana Cayetano Heredia-University of Texas Medical Branch Collaborative Research Center-Cusco, Cusco, Peru. E-mail: 341 and gallbladders that had been condemned at a local slaughterhouse in Cusco. Echinococcus granulosus, Diphyllobothrium spp., Strongyloides stercoralis, Taenia solium, and