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

et al. (2014) A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of Schistosoma mansoni in Stool Samples: A Diagnostic Approach in a Murine Model. PLoS Negl Trop Dis 8(9): e3126. doi:10.1371/journal.pntd.0003126 A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of Schistosoma mansoni in Stool Samples: A Diagnostic Approach in a Murine Model Pedro Ferna ndez-Soto 0 Javier Gandasegui Arahuetes 0 Alicia Sa nchez Herna ndez 0 Julio Lo pez Aba n 0 Aaron R. Jex, University of Melbourne, Australia 0 Bele n Vicente Santiago , Antonio Muro 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. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Real Federacio n Espan ola de Fu tbol-Sociedad Espan ola de Medicina Tropical y Salud Internacional, 2013 (RFEF-SEMTSI, 2013) and by Junta de Castilla y Leo n (Ref. no. SA342U13). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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]. Presently, 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. The disease is endemic in 74 countries infecting more than 200 million people worldwide, with 732 million people at risk of infection in known transmission areas [2], [3], [4]. On a global scale, one of thirty individuals has schistosomiasis [5]. It is also noted that the prevalence of imported schistosomiasis is increasingly a problem in non-endemic areas due to the growing number of international travelers to endemic areas, expatriates and immigrants from endemic countries [6], [7], [8]. Over time, several diagnostic techniques including parasitological and immunological methods have been tested for diagnosis of schistosome infection. As is well known, 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. However, a 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. major limitation of these methods is their lack in sensitivity, especially in low-grade infections, as occurs in areas of low prevalence or in individuals with recent infections [9]. In addition, they are cannot be carried out in the acute phase of schistosomiasis since parasite has not started yet to lay eggs. When parasites cannot be directly detected, the immunological methods are usually applied to patients with schistosomiasis clinical signs. However, serology-based analyses currently continue to present problems, such us obtaining schistosome antigens, inability to discriminate between current or previous infection, high level of cross reactivity as well as persistence of antigens and antibodies after chemotherapy usually causing false positive results [10]. To overcome the shortcomings of both parasitological and immunological diagnostic methods, the development of new, more sensitive and specific molecular diagnostic tools for the dia (...truncated)