Field evaluation of a recombinase polymerase amplification assay for the diagnosis of Schistosoma japonicum infection in Hunan province of China

Abstract

Background: Current diagnostic methods for Schistosoma japonicum infection are insensitive for low-density infections. Therefore, a new diagnostic assay based on recombinase polymerase amplification (RPA) technology was established and assessed for field applification.

Methods: The S.japonicum RPA assay was developed to target highly repetitive retrotransposon SjR2 gene of S japonicum, and its sensitivity and specificity were assessed by serial dilution of S. japonicum genomic DNA and other related worm genomic DNA respectively. The RPA diagnostic validity was first evaluated in 60 fecal samples from healthy people and patients, and then compared with other diagnostic tests in 200 high-risk individuals living in endemic areas.

Results: The real time RPA assay could detect 0.9 fg S. japonicum DNA within 15 min and distinguish S. japonicum from other worms. The validity analysis of RPA for the detection of S. japonicum in stool samples from 30 S. japonicum-infected patients and 30 healthy persons indicated 100% sensitivity and specificity. When testing 200 fecal or serum samples from a high-risk population, the percentage sensitivity of RPA was 100%, whereas that of indirect hemagglutination assay (IHA) and enzyme-linked immunosorbent assay (ELISA) were 80.3% and 85.2% respectively. In addition, the RPA presented better consistency with the stool-based tests than IHA and ELISA. Overall, the RPA was superior to other detection methods with respect to detection time, sensitivity, and convenience.

Conclusions: This is the first time we applied the RPA technology to the field evaluation of S. japonicum infection. And the results suggest that RPA-based assays can be used as a promising point-of-care test for the diagnosis of schistosomiasis.

Keywords: Diagnosis; Field evaluation; Recombinase polymerase amplification; Schistosoma japonicum.

Fig 1

Analytical sensitivity of real-time RPA and real-time PCR for Schistosoma japonicum detection. Fluorescence development via real-time detection using a dilution range of 9 pg/μL–0.9 fg/μL of the S. japonicum genomic DNA. a Real-time RPA: 9 pg/μL, represented by the red line; 900 fg/μL, green; 90 fg/μL, pink; 9 fg/μL, cyan; 0.9 fg/μL, blue; negative control, black. b Real-time PCR: 9 pg/μL represented by the red line; 900 fg/μL, orange; 90 fg/μL, light green; 9 fg/μL, green; 0.9 fg/μL, cyan; negative control, pink

Fig 2

The specificity of Schistosoma japonicum RPA. S. japonicum is represented by the blue line; S. mansoni, black; S. sinensium, green; S. haematobium, pink

Fig 3

Agreement between RPA, Kato-Katz thick smear and MHT for stool-based diagnosis of Schistosoma japonicum infections. Values indicate the number of positive samples in each group