|Thurston Enriquez, Jeanette|
Submitted to: African Journal of Microbiology Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 11, 2008
Publication Date: January 30, 2008
Citation: Dowd, S.E., Thurston Enriquez, J.A. 2008. Evaluation and guidelines for use of polymerase chain reaction-computer database homology comparison (PCR-CDHC) for detection and species determination of human pathogenic microsporidia. African Journal of Microbiology Research. 2(1):001-007. Interpretive Summary: Microsporidia are new and emerging pathogens that have been found in a variety of livestock and are thought to be zoonotic and waterborne. Methods for detection of these new and emerging pathogens have been developed utilizing the Polymerase Chain Reaction and molecular sequencing technology. There are a variety of human pathogenic varieties of these microsporidia and the ability to determine the species of these human pathogenic species from among non-pathogenic varieties is of great import. Methods are described and validated in this manuscript for the use of PCR and sequencing methods combined with Computer Database Homology Comparison (CDHC) for determining the species of microsporidia isolated from livestock waste. The development of this methodology and its successful validation provides the first step toward needed epidemiology evaluation of their role as zoonotic pathogens.
Technical Abstract: The potential for waterborne disease and zoonotic transmission of at least two species of human pathogenic microsporidia has heightened interest in clinical and environmental detection methods for these organisms. Detection using the polymerase chain reaction (PCR) followed by computer database homology comparison (CDHC) (PCR-CDHC) was reported previously by this research group. As a result, PCR-CDHC has been employed by many research groups around the world for species determination of human pathogenic microsporidia. To validate the CDHC speciation approach, a phylogenetic tree was generated using the small subunit ribosomal DNA sequences (SSU-rDNA) of a large number of microsporidia. An index of similarity was created and used as an assessment of CDHC's ability to differentiate between closely related species. Polymerase chain reaction followed by dye termination PCR sequencing and subsequent CDHC of the sequences was performed on 8 species of microsporidia including four human pathogenic strains. The four non-human pathogenic microsporidia tested by this approach were those shown by the phylogenetic analyses to be very closely related to the other human pathogenic species as determined by branch length. In all cases, the CDHC approach was able to correctly identify all eight species of microsporidia. To provide an example of PCR-CDHC, a "universal" and two previously published pathogen-specific microsporidia PCR protocols followed by PCR-CDHC was conducted to assess their ability to detect naturally occurring microsporidia species in swine wastewater. Only one primer set resulted in a PCR-CDHC analysis where presumptive human pathogenic microsporidia was detected. Subsequent CDHC showed these presumptive positive PCR results were actually false positives. With the appropriate primer set, PCR-CDHC proves to be a reliable method that can be used for specific species determination of human pathogenic microsporidia in samples where non-pathogenic species may be present.