I. Introduction
The following viruses may be detected from various tissue or biopsy sources:
Lung: Adenovirus/CMV/enterovirus/respiratory viruses
Liver: CMV/non-culturable viral hepatitis
Brain: HSV/VZV/enterovirus/measles
Kidney: CMV
Heart: Enterovirus
Postpartum: CMV
Bone Marrow: CMV
For other viruses requested, refer to Appendix XV (Virus isolation and identification) to ensure the appropriate media is inoculated.
http://bahankuliahkesehatan.blogspot.com/
II. Collection and Transport
The sample should be collected directly adjacent to the affected tissue and placed in viral transport medium. If a delay in transport or processing is anticipated, keep the specimen at 4oC.
III. Procedure
A. Processing of Specimens:
a) If the tissue is received in a dry container, add 1-3 ml of maintenance medium.
b) Macerate the tissue using the small tissue grinder.
c) If a large amount of tissue/cellular material is present, transfer suspension to a sterile 15 ml centrifuge tube and spin at 700 x g for 10 minutes
c) Use the supernate as inoculum.
If Fetal / Post-partum tissue is received and Parvovirus PCR is requested, forward specimen to the Virology PCR section.
- Direct Examination:
Not usually performed.
- Isolation and Identification:
Specimens | Methods | Cell Linesa | Incubation at 36oC | Stain used/Read |
| LungbLiver/Bone Marrow/Kidney/ FetalTissue | Shell Vial | MRC-5 R-Mixb MRC-5 (if requested) MRC-5 (if requested) MRC-5 (if requested) | 2 days 2 days 1 day 1 day 2 days | CMV-IE RS* HSV1 HSV2 VZV |
| Shell Vial for CPE | E-Mix MRC-5 (summer**) | 5 days 5 days | Read daily*** Read daily*** | |
| Brain | Shell Vial | MRC-5 MRC-5 MRC-5 | 2 days 1 day 2 days | CMV-IE HSV-bivalent VZV |
| Shell Vial for CPE | E-Mix MRC-5 (summer**) | 5 days 5 days | Read daily*** Read daily*** |
aMRC-5 = Human Fibroblast cells
b E-Mix or MRC-5 for Shell Vials for CPE inoculated with Lung tissue should be read for daily and if CPE is observed, stain a double Cytoprep with SimulFluor Respiratory virus Screen and HSV bivalent monoclonal antibody stains.
R-Mixb Shell Vial inoculated with Lung tissue should be stained with SimulFluor Respiratory virus Screen.
** Summer= May to October
*** If CPE is detected, the technologist will pass the infected supernatant to a new shell vial and prepare a cell suspension of the infected culture so that multiple cell spots (on slide) can be stained with the enterovirus D3, Coxsackie B and ECHO stains to identify the virus.
See Appendix II for detailed shell vial procedure.
See Appendix III for detailed tube culture (shell vials for CPE) procedure.
IV. Reporting Results
Shell Vial: Negative Report: “Negative for ______virus.”
Positive Report*: “POSITIVE for _______virus.”
Shell Vials for CPE: Negative Report: “No virus isolated”
Positive Report*: “POSITIVE for _______virus.”
Toxicity Report: "Virology Culture: Specimen toxic to cell culture.
Contaminated Report: "Virology Culture: Specimen is heavily contaminated with bacteria and/or fungus. Unable to perform Virology Culture.
V. References
1. Leland D. 1996. Clinical Virology. W.B. Saunders Company.
2. Gleaves, Curt A. et al. Cumitech 15A “Lab Diagnosis of Viral Infections”.
American Society for Microbiology, August 1994.
Tidak ada komentar:
Posting Komentar