Development of a loop-mediated isothermal amplification (LAMP) and a polymerase chain reaction (PCR) assays for diagnosis of Fasciola hepatica in animal faeces and comparison with traditional diagnostic methods

Abstract: The liver fluke Fasciola hepatica is a parasitic trematode prevalent in mammals, primarily in sheep and cattle. There is a wide range of methods for diagnosis of F. hepatica infections, such as coproscopy, coproantigen ELISA, serum ELISA, PCR and Loop-mediated isothermal amplification (LAMP). As with all diagnostic methods, each presents benefits and disadvantages. Coproscopy requires no sophisticated equipment, but its robustness is limited due to difficulty of species identification and inability to detect early F. hepatica infections. Coproantigen ELISA can detect infections during the pre-patent period, yet its sensitivity in field applications is still debated. Serum ELISA is a good method for large herd screening, although it provides less insight to the infection status. PCR can differentiate between species using primers targeting the ITS2 region of F. hepatica genome. LAMP is a molecular method based on rapid amplification of target DNA under isothermal conditions. Both PCR and LAMP have only recently been attempted for F. hepatica identification in faeces. The aim of the study was to develop and set up LAMP and PCR methods for diagnosis of F. hepatica in ruminant faeces and to compare these molecular techniques with coproscopy, coproantigen detection and serology. A total of 64 faecal and blood samples were collected from 64 sheep and cattle from four farms in Sweden. Faecal samples were examined by faecal egg counts (FEC) with a sedimentation method and coproantigen ELISA using the Bio-X Bovine Fasciola hepatica Antigen ELISA Kit (Bio-X Diagnostics, Belgium). Serologic testing with an in-house ELISA was conducted on all serum samples. PCR and LAMP were performed with DNA extracted directly using PowerFecal® DNA isolation kit (MO BIO, USA) from faecal samples. F. hepatica eggs were present in 28 animals, while coproantigen and antibodies were detected in 36 and 53 animals respectively. PCR and LAMP managed to amplify only 3 and 6 samples respectively. Based on a composite reference standard, results showed that LAMP and PCR had a sensitivity of 14% and 8% respectively, which was much lower compared to the 78% sensitivity of FEC and 100% sensitivity of both coproantigen and serum ELISA. FEC, coproantigen ELISA and PCR all had 100% specificity, while LAMP and serum ELISA had 96% and 39% specificity respectively. In conclusion, FEC and coproantigen ELISA were good diagnostic tools for detection of patent F. hepatica infections. PCR and LAMP results could possibly improve with further development of faecal DNA extraction techniques.