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REAL-TIME PCR DETECTION OF THREE HUMAN-PATHOGENIC SPECIES FROM THE MICROSPORIDIAL GENUS ENCEPHALITOZOON
Citation:
Hester, J. D., M Varma, A. M. Bobst, M W. Ware, H.D A. Lindquist, AND F W. Schaefer III. REAL-TIME PCR DETECTION OF THREE HUMAN-PATHOGENIC SPECIES FROM THE MICROSPORIDIAL GENUS ENCEPHALITOZOON. Presented at VII International Workshops on Opportunistic Protists (IWOP), Cincinnati, OH, June 13-16, 2001.
Impact/Purpose:
Develop a method to detect microsporidians in water.
Description:
Three microsporidial species from the genus Encephalitozoon, E. hellem, E. cuniculi and E. intestinalis, have emerged as important opportunistic pathogens of humans affecting organ transplant recipients, AIDS patients, and other immunocompromised patients. Even though these three microsporidial species have the potential to be waterborne via body secretions (i.e., feces, urine, or respiratory secretions), little is known about the occurrence of these protozoan parasites in water matrices. Methods capable of detecting these microsporidial species in surface waters have been severely limited because of complex matrices and low organism density.
We report here the development of a 5'-nuclease assay for the quantitative detection of E. hellem, E. cuniculi, and E. intestinalis. Species-specific oligonucleotide primer sets and a genus-specific, fluorescent-labeled Taqman probe were designed to target the rDNA gene for all three Encephalitozoon species. DNA isolation from microsporidial spores that were quantitated using both flow cytometric and hemacytometer counts permitted an extremely accurate assessment of this assay's level of sensitivity. After optimization of the assay conditions, we were able to specifically detect a single infectious spore as well as obtain a standard curve with a linear range across 5 logs of microsporidial spore counts for all three human-pathogenic microsporidia in surface waters and may serve as a sensitive and specific diagnostic tool in clinical analysis.