DIAGNOSIS OF STRONGYLOIDES STERCORALIS BY DETECTION OF DNA FRAGMENTS IN URINE
The nematode parasite Strongyloides Stercoralis is difficult to diagnose because the process requires Detection of viable larvae in stool and requires fresh material for examination. Current stool examination procedures for survey work do not meet this need and information about the distribution and extent of this Infection is sparse. Additionally the parasite can cause severe pathology because it is tissue invasive and in immunocompromised individuals, the Infection can disseminate causing fatal disease. It has been reported to affect some tissue transplant recipients. Improved diagnostics do exist and we have shown with other parasites (schistosomes, Malaria) that one can amplify and detect species specific DNA Fragments excreted in the Urine of infected persons. If the Urine (50 ml) is filtered through coarse filter paper, dried and packaged in a plastic sleeve, the DNA is stable. The specimen can be easily transported; cold chain is eliminated and cost of delivery reduced considerably. This is an excellent way to reduce costs in the field. The DNA can be extracted and amplified from the paper (Whatman#3) and is in operation for schistosomiasis in Ghana, Nigeria and Zambia. We plan to apply this technique to detect S. Stercoralis (Ss) in patients. We have obtained pure Ss DNA as a gift, and from GenBank identified a repeat fragment of Ss DNA now used as a target for the proposed diagnostic test. We have a collaborative arrangement with a group in Argentina working in communities afflicted with this and other parasites, and we have received some specimens from infected people and demonstrated the technique. We have shown that live parasites in a host will pass detectable DNA in fluids that are excreted through the Kidney. To demonstrate and expand this Diagnosis we have three aims: 1) we plan to use these and other primers to detect parasite specific Fragments of DNA and test these against a number of field-collected positive and negative filtered Urine specimens. We will sequence all products and ensure they match Ss DNA. 2) We will obtain up to 400 specimens from community based studies in Argentina. These will have been subject to parasitological examination but also to serology and antigen capture in their lab. All specimens sent to Baltimore will be on dry filter paper, similar specimens will be retained in Argentina for transfer of technology. These specimens will be tested, DNA Fragments analyzed to ensure specificity and the results analyzed for specificity and sensitivity. 3) All results from the field and from DN Detection will be subject to Latent Class Modeling. This is a complex statistical procedure to assess the best predictor of true positivity in any one of the tests used. This work will involve collaborators at King's College Hospital in London and will compare efficacy of parasitological, serological (antibody Detection) antigen capture, Detection in stool and DNA specific fragment Detection in Urine. The procedure will be transferred to the field and published.
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