title: Retrospective species identification of microsporidian spores in diarrheic fecal samples from human immunodeficiency virus/AIDS patients by multiplexed fluorescence in situ hybridization.
abstract: 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 10(3) to 4.4 x 10(5) for E. bieneusi (mean, 8.8 x 10(4)/ml), 2.3 x 10(2) to 7.8 x 10(4) (mean, 1.5 x 10(4)/ml) for E. intestinalis, and 1.8 x 10(2) to 3.6 x 10(2) for E. hellem (mean, 2.7 x 10(2)/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.
authors.affiliation: Department of Environmental Health Sciences, Division of Environmental Health Engineering, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, MD 21205, USA. tgraczyk@jhsph.edu
authors.forename: Thaddeus K
authors.initials: TK
authors.lastname: Graczyk
authors.forename: Michael A
authors.initials: MA
authors.lastname: Johansson
authors.forename: Leena
authors.initials: L
authors.lastname: Tamang
authors.forename: Govinda S
authors.initials: GS
authors.lastname: Visvesvara
authors.forename: Laci S
authors.initials: LS
authors.lastname: Moura
authors.forename: Alexandre J
authors.initials: AJ
authors.lastname: DaSilva
authors.forename: Autumn S
authors.initials: AS
authors.lastname: Girouard
authors.forename: Olga
authors.initials: O
authors.lastname: Matos
mesh_terms: D017088:AIDS-Related Opportunistic Infections / Q000175:diagnosis* / Q000382:microbiology; D015169:Colony Count, Microbial; D003967:Diarrhea / Q000382:microbiology*; D016819:Encephalitozoon / Q000145:classification / Q000302:isolation & purification*; D016890:Encephalitozoonosis / Q000175:diagnosis / Q000382:microbiology; D021862:Enterocytozoon / Q000145:classification / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008856:Microscopy, Fluorescence; D016881:Microsporidiosis / Q000175:diagnosis* / Q000382:microbiology; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D013172:Spores, Fungal / Q000302:isolation & purification