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

Penelope Rostron,Stefanie Knopp,Shaali M Ame,David Rollinson,Bonnie L Webster,Fatma Kabole,Said M Ali,Faki Bakar,Fiona Allan,Tom Pennance

doi:10.1186/s13071-019-3755-6

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.

Highlights

Accurate diagnosis of urogenital schistosomiasis is vital for surveillance and control programmes
Schistosoma haematobium template DNA For the assay development S. haematobium adult worm genomic DNA originating from the Zanzibar island Unguja was provided by the Schistosomiasis Collection at the Natural History Museum (SCAN) [32]
The reactions were run at 40 °C for 20 minutes in an Axxin T-16 isothermal device (T-16 ISO), which measures the increase in fluorescence, due to DNA amplification, over time

Summary

Introduction

Accurate diagnosis of urogenital schistosomiasis is vital for surveillance and control programmes. As a control programme achieves success, low infection intensity is common within the population, with a high proportion of those infected excreting low numbers of schistosome eggs that may escape detection by routine methods, namely urine filtration and haematuria detection strips [16, 17]. This increases the need for diagnostic sensitivity and specificity to prevent false negative diagnoses [7, 17]. The recently developed, and very promising, circulating anodic antigen (CAA) based test offers high sensitivity and is currently being optimized and evaluated for PON testing [18, 19]

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Discussion

Conclusion