Isolation of the Herpes Simplex Virus by Shell Vial Culture / 대한진단검사의학회지

BACKGROUND: Cell culture is the golden standard method for Herpes simplex virus (HSV) isolation. However, some specimens require many days to develop any cytopathic effect (CPE). We developeda rapid sensitive culture technique for HSV isolations. METHODS: This study included a total of 133 patients with suspected HSV infection. Specimens were centrifuged onto a Vero cell monolayer in a shell vial. The CPE was observed daily during the5-day incubation by inverted-phase microscope. The direct immunofluorescence (DIF) stain with aHSV specific antibody was performed 2 days after sample inoculation. The negative samples in theDIF stain were reinoculated in the new shell vials after extraction of the monolayer. Polymerase chainreaction for HSV detection was performed using the original samples. RESULTS: The CPE was observed 30 (64%), 39 (83%), 43 (92%), 44 (94%), and 46 (98%) cases at1, 2, 3, 4, and 5 days incubation, respectively. The DIF stain detected 46 cases (98%) at 2 days incubation. The CPE was observed in another 7 cases at 1-day incubation after the reinoculation of negative samples. The PCR detected 47 (100%) of 133 cases. CONCLUSIONS: The reinoculation of negative sample in a shell vial culture is a rapid sensitive methodfor HSV isolation.

Genotyping of Vibrio parahaemolyticus by Infrequent Restriction Site Polymerase Chain Reaction / 대한임상미생물학회지

BACKGROUND: Infrequent restriction site PCR (IRS-PCR) is a recently described DNA fingerprinting technique based on selective amplification of restriction endonuclease-cleaved fragments. We applied of IRS-PCR to clinical isolates of Vibrio parahaemolyticus associated with diarrhea. METHODS: IRS-PCR assay was performed with adaptors for XbaI and HhaI restriction sites. A total of 35 strains of V. parahaemolyticus which were isolated from clinical specimens of patients with diarrhea were analyzed. The isolates were collected from different geographic areas of Seoul (n=12), Incheon (n=21) and Gwangju (n=2) during 1998-2000 in Korea. RESULTS: In IRS-PCR, amplifed DNA fragments between 50 and 400 bp were found to be the most reproducible in this study. When V. parahaemolyticus isolates were amplified with AH1 and PX-G as primers, 35 isolates could be grouped into five IRS-PCR patterns: A (n=16), B (n=4), C (n=6), D (n=5) and E (n=4). The patterns were subdivided into 15 subtypes: A1, A2, B1, B2, B3, B4, C1, C2, C3, D1, D2, D3, E1, E2 and E3. The IRS-PCR patterns of V. parahaemolyticus did not show any relationship with serotype or geographic origin, but the isolates from same outbreak produced a same pattern(A1). CONCLUSION: The results provide evidence of the discriminatory power of the IRS-PCR method as it applies to V. parahaemolyticus.