Development of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of Schistosoma haematobium

Abstract

BackgroundAccurate diagnosis of urogenital schistosomiasis is vital for surveillance and control programmes. While a number of diagnostic techniques are available there is a need for simple, rapid and highly sensitive point-of-need (PON) tests in areas where infection prevalence and intensity are low. Recombinase Polymerase Amplification (RPA) is a sensitive isothermal molecular diagnostic technology that is rapid, portable and has been used at the PON for several pathogens.ResultsA real time fluorescence RPA assay (RT-ShDra1-RPA) targeting the Schistosoma haematobium Dra1 genomic repeat region was developed and was able to detect 1 fg of S. haematobium gDNA. Results were obtained within 10 minutes using a small portable battery powered tube scanner device that incubated reactions at 40 °C, whilst detecting DNA amplification and fluorescence over time. The assay’s performance was evaluated using 20 urine samples, with varying S. haematobium egg counts, from school children from Pemba Island, Zanzibar Archipelago, Tanzania. Prior to RPA analysis, samples were prepared using a quick crude field DNA extraction method, the Speed Extract Kit (Qiagen, Manchester, UK). Positive assay results were obtained from urine samples with egg counts of 1–926 eggs/10 ml, except for two samples, which had inconclusive results. These two samples had egg counts of two and three eggs/10 ml of urine.ConclusionsThe RT-ShDra1-RPA assay proved robust for S. haematobium gDNA detection and was able to amplify and detect S. haematobium DNA in urine samples from infected patients. The assay’s speed and portability, together with the use of crude sample preparation methods, could advance the rapid molecular diagnosis of urogenital schistosomiasis at the PON within endemic countries.

Analytical and Clinical Assessment of a Portable, Isothermal Recombinase Polymerase Amplification (RPA) Assay for the Molecular Diagnosis of Urogenital Schistosomiasis

“…RT-ShDra1-RPA assay design (primers/probes and preliminary testing) has been described in detail elsewhere [ 46 ]. RPA reactions were performed using TwistDx (Cambridge, UK) RPA Exo Kits according to the manufacturer’s instructions.…”

“…PCR assays, and crucial preliminary steps needed to isolate DNA from urine samples, however, require expensive and fragile technical equipment, suitable laboratory infrastructure and trained laboratory personnel—all rarely available within schistosomiasis-endemic areas, thus impeding the use of these methods at the point-of-care [ 40 , 41 , 42 ]. For these reasons, a variety of alternative and portable DNA amplification technologies have been developed for diagnostic purposes, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), the latter of which has been used to detect trace levels of S. haematobium ova-derived DNA within laboratory-prepared samples as well as in clinical urine samples [ 43 , 44 , 45 , 46 ]. Unlike PCR, the RPA assay uses a low-temperature isothermal reaction, requires only minimal equipment and takes comparatively far less time to carry out [ 34 , 40 ].…”

“…Unlike PCR, the RPA assay uses a low-temperature isothermal reaction, requires only minimal equipment and takes comparatively far less time to carry out [ 34 , 40 ]. In addition, the RPA can be performed using lyophilised reagents that do not require a cold chain and also by using a crude DNA extraction process that can be easily prepared under field conditions, rendering the RPA well suited for point-of-care use within schistosomiasis-endemic areas [ 46 ].…”

“…Here, we assessed the analytical and clinical diagnostic performance of a previously developed and field-deployable RPA assay (RT-ShDra1-RPA) that targets the S. haematobium Dra1 repeat genomic region [ 46 ]. This was carried out using varying concentrations of commercially synthesised S. haematobium Dra1 copies as well as laboratory-prepared ddH 2 O samples spiked with differing numbers of S. haematobium eggs.…”

An update on non-invasive urine diagnostics for human-infecting parasitic helminths: what more could be done and how?

“…To overcome many of the logistical and methodological challenges presented by PCR-based diagnostics, the point-of-care recombinase DNA-polymerase amplification (RPA) assay has also recently been developed and has been used to successfully detect and amplify S. japonicum and F. hepatica cfDNA in human stool samples, as well as S. haematobium DNA within (Shiff et al, 2011;Ibironke et al, 2012;Lodh et al, 2014) RPA (Rostron et al, 2019) rDNA internal transcribed spacer (ITS) region PCR (Sandoval et al, 2006) 77 S. mansoni rDNA internal transcribed spacer (ITS) region PCR (Sandoval et al, 2006) 28S rDNA region PCR (Sandoval et al, 2006) 110 bp region from highly repeated 121 bp fragment (Hamburger et al, 1991) PCR (Enk et al, 2012;Lodh et al, 2013Lodh et al, , 2014 Oligonucleotides: F3; B3; FIP; BIP (Hamburger et al, 2013) LAMP (Lodh et al, 2017) Unnamed 650 Lymphatic system (adult stage/L3 larval stage) and/or circulatory system (microfilariae larval stage)…”