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Journal of Clinical Microbiology, April 2007, p. 1255-1260, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.01975-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Retrospective Species Identification of Microsporidian Spores in Diarrheic Fecal Samples from Human Immunodeficiency Virus/AIDS Patients by Multiplexed Fluorescence In Situ Hybridization{triangledown}

Thaddeus K. Graczyk,1,2* Michael A. Johansson,2 Leena Tamang,1 Govinda S. Visvesvara,3 Laci S. Moura,3 Alexandre J. DaSilva,3 Autumn S. Girouard,2 and Olga Matos4

Department of Environmental Health Sciences, Division of Environmental Health Engineering, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, Maryland 21205,1 Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, Maryland 21205,2 Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Public Services, Atlanta, Georgia 30341,3 Unidade de Protozoarios Oportunistas/VIH e Outras Protozooses, Unidade de Parasitologia e Microbiologia Medicas, Instituto de Higiene e Medicina Tropical, 1495-233 Lisbon, Portugal4

Received 22 September 2006/ Returned for modification 4 December 2006/ Accepted 25 January 2007

In order to assess the applicability of multiplexed fluorescence in situ hybridization (FISH) assay for the clinical setting, we conducted retrospective analysis of 110 formalin-stored diarrheic stool samples from human immunodeficiency virus (HIV)/AIDS patients with intestinal microsporidiosis collected between 1992 and 2003. The multiplexed FISH assay identified microsporidian spores in 94 of 110 (85.5%) samples: 49 (52.1%) were positive for Enterocytozoon bieneusi, 43 (45.8%) were positive for Encephalitozoon intestinalis, 2 (2.1%) were positive for Encephalitozoon hellem, and 9 samples (9.6%) contained both E. bieneusi and E. intestinalis spores. Quantitative spore counts per ml of stool yielded concentration values from 3.5 x 103 to 4.4 x 105 for E. bieneusi (mean, 8.8 x 104/ml), 2.3 x 102 to 7.8 x 104 (mean, 1.5 x 104/ml) for E. intestinalis, and 1.8 x 102 to 3.6 x 102 for E. hellem (mean, 2.7 x 102/ml). Identification of microsporidian spores by multiplex FISH assay was more sensitive than both Chromotrope-2R and CalcoFluor White M2R stains; 85.5% versus 72.7 and 70.9%, respectively. The study demonstrated that microsporidian coinfection in HIV/AIDS patients with intestinal microsporidiosis is not uncommon and that formalin-stored fecal samples older than 10 years may not be suitable for retrospective analysis by techniques targeting rRNA. Multiplexed FISH assay is a reliable, quantitative fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, and E. hellem, as well as Encephalitozoon cuniculi, spores in fecal samples and is a useful tool for assessing spore shedding intensity in intestinal microsporidiosis. The method can be used for epidemiological investigations and applied in clinical settings.

* Corresponding author. Mailing address: Department of Environmental Health Sciences, Division of Environmental Health Engineering, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, MD 21205. Phone: (410) 614-4984. Fax: (410) 955-0105. E-mail: tgraczyk{at}jhsph.edu

{triangledown} Published ahead of print on 7 February 2007.

Journal of Clinical Microbiology, April 2007, p. 1255-1260, Vol. 45, No. 4
0095-1137/07/$08.00+0     doi:10.1128/JCM.01975-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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