Laboratory Evaluation of a Basic Recombinase Polymerase Amplification (RPA) Assay for Early Detection of Schistosoma japonicum

Abstract

Early detection of Schistosoma japonicum (S. japonicum) within its intermediate and definitive hosts is crucial for case finding and disease surveillance, especially in low-endemic areas. Recombinase polymerase amplification (RPA) has many advantages over traditional methods of DNA-amplification, such as polymerase chain reaction (PCR), including high sensitivity and specificity whilst being deployable in resource-poor schistosomiasis-endemic areas. Here, we evaluated the performance of a basic RPA assay targeting the 28srDNA gene fragment of S. japonicum (Sj28srDNA) using schistosome-infected Oncomelania hupensis (O. hupensis) and mouse models, compared to the traditional pathological method and a PCR assay. Overall S. japonicum infection prevalence within O. hupensis hosts by microscopic dissection, PCR and RPA was 9.29% (13/140), 32.14% (45/140) and 51.43% (72/140), respectively, presenting significant differences statistically (χ2 = 58.31, p < 0.001). It was noteworthy that infection prevalence by PCR and RPA performed was 34.44% (31/90) and 53.33% (48/90) in snails within 6 weeks post-infection, while the dissection method detected all samples as negatives. In addition, the basic RPA assay presented positive results from the fourth week post-infection and third day post-infection when detecting fecal DNA and serum DNA, respectively, which were extracted from a pooled sample from mice infected with 20 S. japonicum cercariae. This study suggests that the RPA assay has high potential for early detection of S. japonicum infection within its intermediate and definitive hosts.

Development of real-time and lateral flow recombinase polymerase amplification assays for rapid detection of Schistosoma mansoni

“…To this end, Recombinase Polymerase Amplification (RPA) is an isothermal amplification method that presents great potential. It has been piloted for detection of urogenital schistosomiasis caused by Schistosoma haematobium (Rosser et al, 2015;Rostron et al, 2019;Archer et al, 2020;Frimpong et al, 2021) and intestinal schistosomiasis caused by Schistosoma japonicum (Sun et al, 2016;Xing et al, 2017;Guo et al, 2021;Deng et al, 2022), with promising results for its use in the field at the point-of-need (PON). Only one study has explored the development of a RPA assay to detect Schistosoma mansoni DNA, and this was done using the lateral flow RPA.…”

“…Several LAMP assays have been developed for the detection of S. mansoni in both human and snail hosts over the past years, with favorable results (Abbasi et al, 2010;Hamburger et al, 2013;Fernández-Soto et al, 2014;Gandasegui et al, 2016Gandasegui et al, , 2018Caldeira et al, 2017;Mwangi et al, 2018;García-Bernalt Diego et al, 2019;Price et al, 2019;Mesquita et al, 2021). RPA was described in 2006 (Piepenburg et al, 2006) and since then it has been used mostly for the detection of Schistosoma haematobium (Rosser et al, 2015;Rostron et al, 2019;Archer et al, 2020Archer et al, , 2022Frimpong et al, 2021) and Schistosoma japonicum (Sun et al, 2016;Xing et al, 2017;Guo et al, 2021;Deng et al, 2022), with only one study focused on S. mansoni targeting the ribosomal DNA (rDNA) regions 28S and the internal transcribed spacer (ITS; Poulton and Webster, 2018). Although this work represented a first and important step for the use of RPA for S. mansoni diagnosis, the lateral flow approaches lacked specificity with cross-reactivity with S. haematobium and Schistosoma bovis observed.…”

The Efficiency of Commercial Immunodiagnostic Assays for the Field Detection of Schistosoma japonicum Human Infections: A Meta-Analysis

“…In addition, loop-mediated isothermal amplification (LAMP) assays were developed for accurate, visualized, and early detection of S. japonicum human infections; however, these assays are extremely likely to be contaminated, resulting in false positives [ 71 , 72 , 73 ]. Recently, amplification recombinase-aided isothermal amplification (RAA) and recombinase polymerase amplification (RPA) assays have been developed for early detection of S. japonicum human infections; however, the performance of RAA and RPA assays remains to be investigated for detection of S. japonicum infections in large-scale clinical studies [ 74 , 75 , 76 , 77 , 78 ].…”

Advances in Simple, Rapid, and Contamination-Free Instantaneous Nucleic Acid Devices for Pathogen Detection

“…However, the need for longer primer lengths also limits the detection of short sequences of nucleic acids. Deng et al used the basic RPA method for performance testing on schistosome-infected nail snails and mouse models [178]. RPA was used by Lacharoje et al to detect feline leukemia virus (FeLV) DNA provirus [179].…”