Advances in the recombinase polymerase amplification platform for urogenital schistosomiasis diagnosis (ShDraI-RPA) towards implementation at the point-of-care

Accurate diagnosis of schistosomiasis is crucial to achieve disease elimination as a public health problem. Rapid and highly sensitive diagnostic tools that can be used in decentralized environments and/or at the point-of-care are needed. This work optimises and simplifies an existing isothermal molecular diagnostic platform (recombinase polymerase amplification, RPA) for urogenital schistosomiasis, the ShDraI-RPA, with a focus on delivering a more accurate diagnosis in endemic settings. The standard ShDraI-RPA oligonucleotides were modified, incorporating a phosphorothioate backbone into the reverse primer and inverting the probe fluorophore and quencher, to prevent false positive results due to secondary structure formation. The sensitivity and specificity of the modified assay were evaluated on a donor urine spiked with one S. haematobium egg and an array of other schistosomes and human urinary tract pathogens. The stability of RPA reagents was assessed by storing them at ambient temperature (± 27 °C) in a dark environment for up to 90 days. Sample preparations were explored to develop a simple, rapid and low resource methodology that would complement the ShDraI-RPA platform when used in remote settings. The modified ShDraI-RPA assay was robust, sensitive and specific to S. haematobium group species, detecting down to 10 fg of gDNA and ten synthetic Dra I copies. DNA amplification was achieved at 42 °C within 20 min and results could easily be visualized using a portable fluorometer or under blue light. RPA reagents remained stable when stored in the absence of light at ± 27 °C for up to 30 days. A two-step DNA extraction method proved optimal for extracting DNA from single S. haematobium eggs in spiked urine. The optimized ShDraI-RPA platform shows improved specificity and sensitivity and has now reached several of the target product profile requirements set out by the WHO for the ideal diagnostic test for schistosomiasis.