Abstract
BackgroundHuman schistosomiasis, mainly due to Schistosoma mansoni species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting S. mansoni infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. A promising approach is the loop-mediated isothermal amplification (LAMP) technology. Compared to PCR-based assays, LAMP has the advantages of reaction simplicity, rapidity, specificity, cost-effectiveness and higher amplification efficiency. Additionally, as results can be inspected by the naked eye, the technique has great potential for use in low-income countries.Methodology/Principal findingsA sequence corresponding to a mitochondrial S. mansoni minisatellite DNA region was selected as a target for designing a LAMP-based method to detect S. mansoni DNA in stool samples. We used a S. mansoni murine model to obtain well defined stool and sera samples from infected mice with S. mansoni cercariae. Samples were taken weekly from week 0 to 8 post-infection and the Kato-Katz and ELISA techniques were used for monitoring the infection. Primer set designed were tested using a commercial reaction mixture for LAMP assay and an in house mixture to compare results. Specificity of LAMP was tested using 16 DNA samples from different parasites, including several Schistosoma species, and no cross-reactions were found. The detection limit of our LAMP assay (SmMIT-LAMP) was 1 fg of S. mansoni DNA. When testing stool samples from infected mice the SmMIT-LAMP detected S. mansoni DNA as soon as 1 week post-infection.Conclusions/SignificanceWe have developed, for the first time, a cost-effective, easy to perform, specific and sensitive LAMP assay for early detection of S. mansoni in stool samples. The method is potentially and readily adaptable for field diagnosis and disease surveillance in schistosomiasis-endemic areas.
Highlights
Schistosomiasis, a disease caused by parasitic worms of several species of genus Schistosoma, is one of the 17 neglected tropical diseases (NTDs) recognized by World Health Organization (WHO) [1]
Specificity and sensitivity of polymerase chain reaction (PCR) using outer primers To make sure that the expected target was amplified, a conventional PCR reaction was performed using outer primers F3 and B3 to amplify S. mansoni DNA
When in silico comparisons of the expected 206 bp sequence were carried out using BLASTn searches with the currently available genomes of S. mansoni, S. haematobium and S. intercalatum at Wellcome Trust Sanger Institute web site and S. japonicum at GenDB web site, respectively, the higher homology in alignment length, percentage of identities and Evalue were obtained for S. mansoni
Summary
Schistosomiasis, a disease caused by parasitic worms of several species of genus Schistosoma, is one of the 17 neglected tropical diseases (NTDs) recognized by World Health Organization (WHO) [1]. Human schistosomiasis, mainly caused by Schistosoma mansoni species, is one of the most widespread of all human parasitic diseases, ranking second only to malaria in terms of its socioeconomic and public health importance in developing countries in tropical and subtropical areas, especially in SubSaharan Africa. Traditional parasitological methods, such as Kato-Katz assay for counting eggs in feces, are relatively inexpensive and easy to perform providing basic information on prevalence and infection intensity. Mainly due to Schistosoma mansoni species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting S. mansoni infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. As results can be inspected by the naked eye, the technique has great potential for use in low-income countries
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