Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model

Background Strongyloides stercoralis, the chief causative agent of human strongyloidiasis, is a nematode globally distributed but mainly endemic in tropical and subtropical regions. Chronic infection is often clinically asymptomatic but it can result in severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. There is a great diversity of techniques used in diagnosing the disease, but definitive diagnosis is accomplished by parasitological examination of stool samples for morphological identification of parasite. Until now, no molecular method has been tested in urine samples as an alternative to stool samples for diagnosing strongyloidiasis. This study aimed to evaluate the use of a new molecular LAMP assay in a well-established Wistar rat experimental infection model using both stool and, for the first time, urine samples. The LAMP assay was also clinically evaluated in patients´ stool samples. Methodology/Principal Findings Stool and urine samples were obtained daily during a 28-day period from rats infected subcutaneously with different infective third-stage larvae doses of S. venezuelensis. The dynamics of parasite infection was determined by daily counting the number of eggs per gram of feces from day 1 to 28 post-infection. A set of primers for LAMP assay based on a DNA partial sequence in the 18S rRNA gene from S. venezuelensis was designed. The set up LAMP assay (namely, Strong-LAMP) allowed the sensitive detection of S. venezuelensis DNA in both stool and urine samples obtained from each infection group of rats and was also effective in S. stercoralis DNA amplification in patients´ stool samples with previously confirmed strongyloidiasis by parasitological and real-time PCR tests. Conclusions/Significance Our Strong-LAMP assay is an useful molecular tool in research of a strongyloidiasis experimental infection model in both stool and urine samples. After further validation, the Strong-LAMP could also be potentially applied for effective diagnosis of strongyloidiasis in a clinical setting.
Author Summary Human strongyloidiasis, a soil-transmitted infection mainly caused by Strongyloides stercoralis, is one of the most neglected among the so-called neglected tropical diseases (NTDs). The difficult diagnosis lead to an underreporting of infection rates. Strongyloidiasis can easily be misdiagnosed because many infections remain asymptomatic and the lack of sensitivity of the conventional fecal-based techniques for morphologically identification of infective larvae in feces. Although serologic tests are useful, a limitation in standardization to avoid cross-reactions still remains. There is an urgent need to improve more sensitive and specific diagnostic tests, particularly in immunocompromised patients or candidates to immunosuppressive treatments. Several molecular approaches for Strongyloides spp. DNA detection have already been assayed, but they have a very limited use in routine diagnostic, particularly in endemic areas. In addition, all molecular approaches for Strongyloides spp. DNA detection have always been mainly assayed for stool samples and no other more advantageous biological samples, such as urine, have been investigated for molecular purposes. In this study we have developed, for the first time, a molecular assay using LAMP methodology as a simple, sensible and robust method for the detection of S. venezuelensis DNA in a well-established Wistar rats experimental infection in both stool and urine samples. The LAMP assay was also successfully evaluated in patients´ stool samples. Our LAMP assay (Strong-LAMP) is an useful molecular tool in a strongyloidiasis experimental infection model and could be a potential field-friendly diagnostic test in a clinical setting, following further validation.