Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE credits
Strongyloides stercoralis (S. stercoralis) is an intestinal nematode mainly present in tropical areas of the world. Most infections with this parasite are asymptomatic, but in immunosuppressed patients S. stercoralis may disseminate throughout the body and cause gastrointestinal symptoms as well as shock, neurological malfunctions and septicemia. When patients are treated with immunosuppressive therapy, chronic infections may be reactivated. In some instances infection with S. stercoralis may be fatal.
Entamoeba histolytica (E. histolytica) is a pathogenic amoeba that can induce both intestinal and extraintestinal infections. Infections with E. histolytica most commonly occur in developing countries. Differentiation between the pathogenic E. histolytica and the related apathogenic amoeba Entamoeba dispar (E.dispar) is not possible by microscopic examination.
This master thesis, performed at the Swedish Institute for Communicable Disease Control (SMI), aimed at establishing a qualitative method for the detection of S. stercoralis with real-time PCR as it is a fast, accurate and highly sensitive technique for qualitative or quantitative genomic analysis. This would provide an improved preparedness for the diagnostic unit when physicians suspect infection with S. stercoralis.
Another aim of the thesis was to establish a qualitative real-time PCR method for differentiation between E. dispar and E. histolytica. The current method for differentiation between these two species at SMI is traditional gel-based PCR. A transfer from PCR to real-time PCR would contribute to a less time consuming and more efficient diagnostic procedure. A TaqMan probe and primers specific for the gene encoding 18S ribosomal RNA (18S rRNA) for S. stercoralis were acquired and tested for use with the same PCR program and reagents as other real-time PCR methods performed at SMI. Primer and probe concentrations were optimized. The test of analytical specificity indicated that the primer/probe system was specific for S. stercoralis. The amplification efficiency and analytical sensitivity was also determined. Two systems specific for E. histolytica and E. dispar respectively targeting the gene encoding 18S rRNA were acquired and tested in the same manner.
All optimized systems of the real-time PCR methods proved to be compatible with the desired instrument, PCR program and reagents. Weak signals were obtained with the E. dispar specific system when template of E. histolytica was added. With the knowledge of how to correctly interpret the result data, accurate diagnosis could still be obtained despite this issue. If the result from an investigation of accuracy is satisfactory, validation and subsequent implementation of the method for differentiation between E. histolytica and E. dispar is recommended. The optimization of the S. stercoralis specific method indicates that validation can be initiated.
2013. , 41 p.