Development of real-time and lateral flow recombinase polymerase amplification assays for rapid detection of Schistosoma mansoni

Abstract

BackgroundAccurate diagnosis followed by timely treatment is an effective strategy for the prevention of complications together with reducing schistosomiasis transmission. Recombinase Polymerase Amplification (RPA) is a simple, rapid, sensitive, and specific isothermal method with low resource needs. This research aimed at the development and optimisation of a real-time (RT) and a lateral flow (LF) RPA assay for the detection of Schistosoma mansoni.MethodologyRecombinase Polymerase Amplification reactions were performed at full- (as recommended) and half-volumes (to reduce costs), with RT or LF detection systems targeting the S. mansoni mitochondrial minisatellite region. The specificity was assessed using gDNA from other Schistosoma species, helminths co-endemic with S. mansoni, human stool, and urine, and Biomphalaria snail hosts. The analytical sensitivity was evaluated using serial dilutions of gDNA, synthetic copies of the target, and single eggs. The ability of both assays to detect the S. mansoni DNA in human urine and stool samples was also tested. The long-term stability of the RT-RPA reagents was evaluated by storing the reaction components in different temperature conditions for up to 3 weeks.ResultsThe RT- and the LF-RPA (SmMIT- and SmMIT-LF-RPA, respectively) presented similar results when used full- and half-volumes, thus the latter was followed in all experiments. The SmMIT-RPA was 100% specific to S. mansoni, able to detect a single egg, with a limit of detection (LOD) of down to 1 fg of gDNA and one synthetic copy of the target. The assay was able to detect S. mansoni DNA from stool containing 1 egg/g and in spiked urine at a concentration of 10 fg/μl. SmMIT-RPA reagents were stable for up to 3 weeks when kept at 19°C, and 2 weeks when stored at 27°C. The SmMIT-LF-RPA cross-reacted with Clinostomidae, presented the LOD of 10 fg and one synthetic copy of the target, being able to detect a single egg and 1 egg/g in a stool sample. The LOD in spiked urine samples was 10 pg/μl.ConclusionThe half-volume SmMIT-RPA is a promising method to be used in the field. It is specific, sensitive, robust, and tolerant to inhibitors, with a long-term stability of the reaction components and the real-time visualisation of results.

Development of CRISPR/Cas13a-based assays for the diagnosis of Schistosomiasis

“…Potential target genes include the mitochondrial NADH dehydrogenase 1 ( nad1 ) and the SjR2 retrotransposon for S. japonicum , 60 and the mitochondrial minisatellite DNA region (SmMIT) for S. mansoni . 61 The SHERLOCK assay procedure could be streamlined by combining the RPA reaction with Cas13a detection (Cas13a protein, crRNA and reporter) in one single reaction (one-pot detection). This would simplify the protocol, improving ease-of-use and reducing processing time, as well as reducing the risk of introducing contaminants.…”

“…Potential target genes include the mitochondrial NADH dehydrogenase 1 ( nad1 ) and the SjR2 retrotransposon for S. japonicum , 60 and the mitochondrial minisatellite DNA region (SmMIT) for S. mansoni . 61 …”

Tropical infectious diseases and the skin: Diagnostic and treatment updates since the WHO's integrated campaign against neglected tropical skin diseases

“…4 These methods have also been applied to Leishmaniasis and Schistosoma detection. 5,6 Monitoring treatment efficacy is another area that requires better POCT. Early or low-grade parasitic infections are frequently undetected by stool or urine microscopy.…”

“…Their sensitivities for detecting disease are promising; the estimated LAMP sensitivity for M. ulcerans is between 73.75% and 91.49% 4 . These methods have also been applied to Leishmaniasis and Schistosoma detection 5,6 …”

Development and assessment of a novel gold immunochromatographic assay for the diagnosis of schistosomiasis japonica

“…Improved coprological tests, such as the saline gradient method (9), formalin-ethyl acetate sedimentationdigestion (FEA-SD) (10) and Helmintex method (i.e., isolates eggs from fecal samples with the use of paramagnetic particles in a magnetic field) (11) showed an increased diagnostic sensitivity compared with the traditional techniques; yet are usually labour-intensive and have a lengthy processing time. In addition, polymerase chain reaction (PCR) technology-based molecular diagnostics, including real time quantitative (q)PCR- (12)(13)(14)(15), droplet digital (dd) PCR-based assays (16)(17)(18), loop-mediated isothermal amplification (LAMP) (19)(20)(21), and recombinase polymerase amplification (RPA) (22)(23)(24)(25) are alternative tools for schistosomiasis diagnosis due to their outstanding accuracy; however, these tests require experienced human resources and are expensive (e.g. the relatively high cost of DNA extraction, qPCR reagents and/or equipment), limiting their application in remote areas with limited resources.…”