Molecular Markers for Detecting Schistosoma Species by Loop-Mediated Isothermal Amplification

Abstract

Schistosomiasis is considered a neglected parasitic disease. Around 280,000 people die from it annually, and more than 779 million people are at risk of getting infected. The schistosome species which infect human beings are Schistosoma mansoni, Schistosoma haematobium, Schistosoma intercalatum, Schistosoma japonicum, Schistosoma guineensis, and Schistosoma mekongi. This disease is also of veterinary significance; the most important species being Schistosoma bovis since it causes the disease in around 160 million livestock in Africa and Asia. This work was aimed at designing and developing a genus-specific loop-mediated isothermal amplification (LAMP) method for detecting the most important schistosome species affecting humans and for the species-specific detection of S. bovis. Bioinformatics tools were used for primer design, and the LAMP method was standardised for detecting the ITS-1 region from S. intercalatum, S. haematobium, S. mansoni, S. japonicum, and S. bovis DNA (generic test) and the NADH 1 gene for specifically detecting S. bovis (at different DNA concentrations). Detection limits achieved were 1 pg DNA for S. mansoni, 0.1 pg for S. haematobium, 1 pg for S. intercalatum, and 10 pg for S. bovis. No amplification for S. japonicum DNA was obtained. The LAMP designed for the amplification of S. bovis NADH-1 worked specifically for this species, and no other DNA from other schistosome species included in the study was amplified. Two highly sensitive LAMP methods for detecting different Schistosoma species important for human and veterinary health were standardised. These methods could be very useful for the diagnosis and surveillance of schistosome infections.

Loop-Mediated Isothermal Amplification in Schistosomiasis

“…A number of LAMP assays have been designed for the species-specific detection of the three main species causing human schistosomiasis ( S. haematobium , S. japonicum and S. mansoni ) and have been applied to schistosomiasis diagnosis, to detect schistosomes-infected snails, and to evaluate efficacy of chemotherapy, both in animal models and human patients. Recently, a LAMP for species-specific detection of the most important veterinary species ( S. bovis ) and a LAMP for simultaneous detection of different Schistosoma species have been also reported [ 31 ]. The selected molecular targets mostly used for LAMP designing for the detection of Schistosoma species are shown in Figure 1 , and different assays features are summarized in Table 1 .…”

Application of a Genus-Specific LAMP Assay for Schistosome Species to Detect Schistosoma haematobium x Schistosoma bovis Hybrids

“…In this work, we tested our previously developed species-specific LAMP assays for S. haematobium [ 44 ], S. mansoni [ 45 ], S. bovis and also the genus-specific LAMP for the simultaneous detection of several Schistosoma species [ 42 ] against both gDNA from pure and, for the first time, hybrid schistosomes. These hybrids were obtained in studies conducted in Côte d’Ivoire [ 26 ] and Corsica, France [ 19 ], and subsequently well characterized by amplification and sequencing of a partial fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and the complete nuclear ribosomal DNA internal transcribed spacer (ITS).…”

“…LAMP assays were accomplished using the reaction mixtures and specific primer sets previously described elsewhere by our group for detection of species-specific S. mansoni based on a mitochondrial minisatellite DNA region [ 45 ], S. haematobium , based on the ribosomal intergenic spacer (IGS) [ 44 ], and S. bovis , based on the mitochondrial NADH subunit 1 [ 42 ]. A genus-specific LAMP assay designed on the internal transcribed spacer 1 (ITS-1) for the simultaneous detection of different species, including S. mansoni , S. haematobium , S. intercalatum and S. bovis , was also applied [ 42 ]. The reactions were carried out using previously described conditions, with the exception of the final reaction volume, which was reduced from 25 μL to 15 μL.…”

“…To date, a number of LAMP approaches have been developed to detect specifically S. haematobium , S. mansoni and S. japonicum in urine, stool, and snails specimens, as recently summarized by Avendaño and Patarroyo [ 41 ]. In addition, a novel species-specific LAMP to detect S. bovis and a genus-specific LAMP to detect different Schistosoma species (including S. haematobium , S. intercalatum , S. mansoni and S. bovis ) have been recently reported by our group [ 42 ]. Due to the schistosome hybridization rapid emergence and spread, and the consequences for disease prevalence, pathological characteristics and treatment, a LAMP test to detect several Schistosoma species (including hybrids forms) would be very useful for the diagnosis and management of schistosomiasis.…”

Loop-Mediated Isothermal Amplification as Point-of-Care Diagnosis for Neglected Parasitic Infections

“…The ITS-1 [149], IGS [153,156] and DraI [163,164] sequences have been studied for S. haematobium diagnosis, a greater detection limit having been obtained with DraI (0.1 fg). Gandasegui et al [153] worked with the IGS region, finding a larger amount of samples to be positive by colorimetry than by turbidimetry (i.e., when adding SYBR Green I to the reaction); however, the effect of the extraction method has been controversial since Gandasegui et al [153] found that sensitivity was greater when DNA was extracted with a commercial kit.…”