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

Abstract

Fasciola hepatica is the most widely distributed trematode infection in the world. Control efforts may be hindered by the lack of diagnostic capacity especially in remote endemic areas. Polymerase chain reaction (PCR)-based methods offer high sensitivity and specificity but require expensive technology. However, the recombinase polymerase amplification (RPA) is an efficient isothermal method that eliminates the need for a thermal cycler and has a high deployment potential to resource-limited settings. We report on the characterization of RPA and PCR tests to detect Fasciola infection in clinical stool samples with low egg burdens. The sensitivity of the RPA and PCR were 87% and 66%, respectively. Both tests were 100% specific showing no cross-reactivity with trematode, cestode, or nematode parasites. In addition, RPA and PCR were able to detect 47% and 26% of infections not detected by microscopy, respectively. The RPA adapted to a lateral flow platform was more sensitive than gel-based detection of the reaction products. In conclusion, the Fasciola RPA is a highly sensitive and specific test to diagnose chronic infection using stool samples. The Fasciola RPA lateral flow has the potential for deployment to endemic areas after further characterization.

Figure 1.

Recombinase polymerase amplification test for Fasciola hepatica (FAS-RPA) characterization. Agarose gel electrophoresis of FAS-RPA reactions. (A) FAS-RPA limit of detection showing ∼300-bp products using F. hepatica DNA at concentration between 10 ng/μL and 1.6 pg/μL. Additional unspecific bands are observed at the 600-bp level. (B) Evaluation of FAS-RPA specificity showing amplification of F. hepatica DNA but not DNA extracted from trematode, cestode, and nematode parasites. Fh = F. hepatica; Scm = Schistosoma mansoni; Ts = Taenia solium; Al = Ascaris lumbricoides; Hkw = hookworm; Tt = Trichuris trichiura; Hn = Hymenolepis nana; Ss = Strongyloides stercoralis; Hs = Homo sapiens; NTC = no-template control; 100 bp = DNA ladder.

Figure 2.

Real-time polymerase chain reaction for Fasciola hepatica (FAS-PCR) characterization: amplification and melt curves. (A) FAS-PCR reactions using F. hepatica DNA at concentrations between 10 ng/μL and 1.6 pg/μL. (B) Evaluation of FAS-PCR specificity showing amplification of F. hepatica DNA, but no amplification with trematode, cestode, and nematode parasites' DNA (no amplification curves appear in the figure). Fh = F. hepatica; Scm = Schistosoma mansoni; Ts = Taenia solium; Al = Ascaris lumbricoides; Hkw = hookworm; Tt = Trichuris trichiura; Hn = Hymenolepis nana; Ss = Strongyloides stercoralis; Hs = Homo sapiens; NTC = no-template control.

Figure 3.

Recombinase polymerase amplification test for Fasciola hepatica (FAS-RPA) lateral flow. (A) Limit of detection for FAS-RPA products detected by lateral flow using serial 10-fold dilutions of F. hepatica DNA. The lowest DNA concentration detected was 1 pg/μL. (B) FAS-RPA products detected by lateral flow using clinical samples with F. hepatica eggs. (C) FAS-RPA products detected by lateral flow using clinical samples without F. hepatica eggs. NTC= no-template control; PC = positive control.