Development and evaluation of recombinase-aided amplification (RAA) assay for detecting Schistosoma haematobium gene in urine samples

Abstract Background: A sensitive assay to diagnose S. haematobium infection (which causes urogenital schistosomiasis) is needed to improve control and elimination interventions. However, the sensitivity of immunological diagnostic methods and other methods (such as reagent strip testing to detect microhematuria and microscopy-based egg counting) for detecting S. haematobium infection is low. Therefore, we aimed to develop a rapid and specific assay based on recombinase-aided amplification (RAA) for surveillance and subsequent control of S. haematobium infection in low-endemic areas, evaluate its sensitivity and specificity, and validate its diagnostic performance using urine samples from infected individuals. Methods: Using a mitochondrial COX1 gene fragment of S. haematobium , we designed a fluorescent probe according to the principle of RAA, and then established and optimized a reaction system for use in an RAA assay. We also assessed the sensitivity and specificity of the RAA assay and validated its diagnostic performance (compared to microscopy-based egg counting) using urine samples from imported patients with light (<50 eggs/10mL urine) and heavy (≥50 eggs/10mL urine) S. haematobium infection loads and negative controls. Results: The established RAA assay (at 39°C in 20 minutes) could detect as little as 10 copies/μL of S. haematobium recombinant plasmid and had no cross-reaction with Schistosoma mansoni , Schistosoma japonicum, Ancylostoma duodenale , Clonorchis sinensis , Echinococcus granulosus , or Ascaris lumbricoides . The validation of the RAA assay showed that it had 100% consistency with microscopy-based egg counting.Conclusion: As it is simple to use and has high sensitivity and specificity, the RAA assay for S. haematobium can be used in field diagnostics to support urogenital schistosomiasis control and elimination in Africa and regions with imported cases.