BACKGROUND: Clostridium difficile is known as the major cause of nosocomially acquired diarrhea. Various phenotypic and genotypic methods have been used to subtype C. difficile strains. The purpose of the present study is to evaluate several typing methods which can be used as tools for subtyping C. difficile isolates for epidemiological studies. METHODS: In two Korean tertiary care hospitals, a total of 81 C. difficile isolates were collected from symptomatic, hospitalized patients in 1998. All isolates were examined for the release of toxin A and toxin B by PCR assay and cell culture assay. Also arbitrarily primed-PCR and PCR-ribotyping profiles were determined for the typing of C. difficile strains on a genetic level. RESULTS: The toxin B gene was detected in 65.4% (54/81) of isolates by both PCR assay and cell cultureassay. Nine types were identified with T-7 primer, and 13 types were identified with PG-05 primer in AP- PCR. Sixteen types were identified in PCR-ribotyping. When two typing methods were compared, reproducibility by PCR-ribotyping was 100%, while it was only 83% and 33% AP-PCR with primer T-7, and PG-05, respectively. The discrimination index was 0.88 for PCR-ribotyping, 0.82 for AP-PCR with primer T-7 and 0.81 with primer PG-05. CONCLUSION: These data suggest that PCR-ribotyping provides a reproducible, discriminatory, and simple alternative to conventional molecular approaches for typing strains of C. difficile.
Cell Culture Techniques ; Clostridium difficile* ; Clostridium* ; Diarrhea ; Discrimination (Psychology) ; Epidemiologic Studies ; Humans ; Polymerase Chain Reaction ; Tertiary Healthcare

PURPOSE: The death of retinal pigment epithelial(RPE) cells plays an important role in the development of age-related macular degeneration. We studied whether ultraviolet could induce apoptotic cell death and the increase of reactive oxygen intermediates (ROI) in the RPE cells. METHODS: Cultured human RPE cells were exposed to doses of between 0 and 80 mJ/cm2 UVB as an attached monolayer. The number of viable cells was assessed by trypan blue assay. The apoptosis was determined by flow cytometry using annexin V and propidium iodide (PI). Intracellular ROI was measured using DCFH-DA. RESULTS: UVB-exposed cells showed a dose-dependent cell death. The number of cells decreased in 20~30% at the dose of 40 mJ/cm2 UVB 24 hours after UVB exposure. The proportion of annexin V positive cells and intracellular ROI increased after UVB exposure. CONCLUSIONS: These results suggest that UV-induced oxidative damage may be one of the important mechanisms of RPE cell death.
Annexin A5 ; Apoptosis ; Cell Death ; Epithelial Cells* ; Flow Cytometry ; Humans ; Macular Degeneration ; Oxygen ; Propidium ; Retinal Pigment Epithelium ; Retinaldehyde* ; Trypan Blue