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PCR amplification and species determination of microsporidia in formalin-fixed feces after immunomagnetic separation.
Appl Environ Microbiol. 1998 Jan; 64(1):333-6.AE

Abstract

The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens.

Authors+Show Affiliations

Department of Microbiology, University of Arizona, Tucson 85721, USA. MS2virus@aol.comNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article

Language

eng

PubMed ID

9435086

Citation

Dowd, S E., et al. "PCR Amplification and Species Determination of Microsporidia in Formalin-fixed Feces After Immunomagnetic Separation." Applied and Environmental Microbiology, vol. 64, no. 1, 1998, pp. 333-6.
Dowd SE, Gerba CP, Enriquez FJ, et al. PCR amplification and species determination of microsporidia in formalin-fixed feces after immunomagnetic separation. Appl Environ Microbiol. 1998;64(1):333-6.
Dowd, S. E., Gerba, C. P., Enriquez, F. J., & Pepper, I. L. (1998). PCR amplification and species determination of microsporidia in formalin-fixed feces after immunomagnetic separation. Applied and Environmental Microbiology, 64(1), 333-6.
Dowd SE, et al. PCR Amplification and Species Determination of Microsporidia in Formalin-fixed Feces After Immunomagnetic Separation. Appl Environ Microbiol. 1998;64(1):333-6. PubMed PMID: 9435086.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - PCR amplification and species determination of microsporidia in formalin-fixed feces after immunomagnetic separation. AU - Dowd,S E, AU - Gerba,C P, AU - Enriquez,F J, AU - Pepper,I L, PY - 1998/1/22/pubmed PY - 1998/1/22/medline PY - 1998/1/22/entrez SP - 333 EP - 6 JF - Applied and environmental microbiology JO - Appl Environ Microbiol VL - 64 IS - 1 N2 - The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens. SN - 0099-2240 UR - https://www.unboundmedicine.com/medline/citation/9435086/PCR_amplification_and_species_determination_of_microsporidia_in_formalin_fixed_feces_after_immunomagnetic_separation_ L2 - http://aem.asm.org/cgi/pmidlookup?view=long&pmid=9435086 DB - PRIME DP - Unbound Medicine ER -