A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of Schistosoma mansoni in Stool Samples: A Diagnostic Approach in a Murine Model

Abstract

Background

Human 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 findings

A 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/Significance

We 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.

Author Summary

Schistosomiasis is one of the most widespread of all human parasitic diseases, Schistosoma mansoni being the most important species causing human intestinal schistosomiasis. The diagnosis of the disease is mainly based on parasitological and serological methods, but they are not effective in detecting S. mansoni infections in the acute stage of the disease. New diagnostic tools to detect the disease during the first weeks would be desirable, permitting early treatment and preventing the pathology associated with chronic infections. An approach is the loop-mediated isothermal amplification (LAMP) technique, which can amplify DNA with high specificity and sensitivity under isothermal conditions. DNA amplification and reading of results require minimum equipment, thus the technique has great potential for use in diagnosis of neglected tropical diseases. In our study, we developed and evaluated a LAMP assay for the early detection of S. mansoni DNA in stool samples from mice experimentally infected with the parasite. The results indicated that our LAMP assay is specific, sensitive and cost-effective in detecting S. mansoni DNA in stool samples as soon as one week post-infection, when parasitological and serological methods are not effective. The assay has the potential to be developed further as a field diagnostic tool for use in schistosomiasis-endemic areas.