LAMP Assay Developed for Threadworms in Stool and Urine

Strongyloides stercoralis, commonly known as threadworm, is a nematode globally distributed but mainly endemic in tropical and subtropical regions and is the chief causative agent of human strongyloidiasis.

The diagnosis of strongyloidiasis is suspected when clinical signs and symptoms, eosinophilia or serologic findings are observed, but definitive diagnosis is accomplished by parasitological examination of stool samples allowing the morphological identification of S. stercoralis, including direct smear in saline, the spontaneous sedimentation method, and centrifugation.

Scientists at the University of Salamanca (Salamanca, Spain) and their colleagues developed, a molecular assay using loop mediated isothermal amplification (LAMP) methodology as a simple, sensible and robust method for the detection of S. venezuelensis DNA in a well-established rodent model infection in both stool and urine samples. The LAMP assay was also successfully evaluated in patients´ stool samples. The LAMP assay (Strong-LAMP) is a useful molecular tool in a strongyloidiasis infection model and could be a potential field-friendly diagnostic test in a clinical setting, following further validation.

Stool samples were collected from 12 patients who showed significant levels of immunoglobulin E (IgE), eosinophilia or other symptoms suggestive of disease. Stool samples were examined after arrival by qualified laboratory technicians. Eleven of these 12 stool samples were subjected to different parasitological methods as screening tests for strongyloidiasis, including microscopic examination (MOE) for the presence of rabditiform larvae in direct fecal smears, agar plate culture (APC) or Harada-Mori´s filter paper culture method (HMM).

DNA was extracted from the human stool samples and tested by an optimized real-time polymerase chain reaction. To evaluate the ability of the LAMP assay designed to amplify S. venezuelensis DNA in real samples, the team used DNA extracted from the pooled feces and urine samples taken daily from each experimentally infected group of rats with different doses. To check whether LAMP assay designed was also able to amplify DNA from S. stercoralis in clinical samples, they used the patients´ stool samples included in the study.

All patients´ stool samples were tested by RT-PCR and LAMP to compare results. The RT-PCR resulted positive in 6/7 patients´ stool samples with confirmed strongyloidiasis by parasitological tests previously applied. In addition, a positive result was obtained in a sample to which no parasitological test could be performed; negative results were obtained in negative parasitological samples for S. stercoralis. All patients´ stool samples with confirmed strongyloidiasis by parasitological tests could be detected by LAMP, including the sample, which resulted negative in previous RT-PCR analyses.

The authors concluded that the Strong-LAMP assay can be applied effectively for the detection of S. stercoralis DNA in patients´ stool samples. The successfully amplification of Strongyloides spp. DNA in infected urine samples by LAMP assay as well as the advantages that urine would have in collection, storage and processing in comparison to patients´ stool samples, should make us consider the possibility of starting to use urine specimens in diagnosing human strongyloidiasis. The study was published on July 14, 2016, in the journal Public Library of Science Neglected Tropical Diseases.

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