These specimens are set up for routine isolation of respiratory viruses (Influenza A and B, Parainfluenza 1, 2, and 3, Respiratory Syncytial Virus, and Adenovirus). Direct antigen detection will be performed during the winter months (November to April, inclusive) and upon request only from May to October. Additional cell lines or shell vials may be required if specific viruses are requested. For other viruses requested, refer to Appendix XV (Isolation and Identification) to ensure that the appropriate media is inoculated. http://bahankuliahkesehatan.blogspot.com/
II. Collection And Transport
Specimens should be obtained as early in the patient’s illness as possible. Throat, nasopharyngeal or nasal swabs should be collected using a clean, sterile swab and placed into viral transport medium and transported to the laboratory as soon as possible. If a delay in transport or processing for up to 72 hours is anticipated, keep the specimen at 4oC.
III. Procedure
A. Processing of Specimen:
Specimens can be kept refrigerated for up to 72 hours. Vortex patient sample in transport medium for 30 seconds. Remove excess fluid from the swab and discard the swab. Specimen in the transport medium can be transferred to a 2 mL cryovial. After preparation of slide (if needed) and inoculation of cultures, store cryovial at -70°C for 6 months. The original specimen container should be kept at 4°C for 1 week.
B. Direct Examination:
Specimens from infants and those requesting RSV:
Prepare one double-well cytospin slide for immunofluorescent staining using the SimulFluor RSV/P3 monoclonal antibody for one well and SimulFluor respiratory virus screen monoclonal antibody for the second well. Refer to Appendix VI for SimulFluor Screen Protocol.
Specimens not from infants and not requesting RSV:
Prepare one double-well cytospin slide for immunofluorescent staining using the SimulFluor influenza A/B monoclonal antibody for one well and SimulFluor respiratory virus screen monoclonal antibody for the second well. Refer to Appendix VI for SimulFluor Screen Protocol.
NB: (a) If a specific respiratory virus(es) is/are requested, prepare appropriate number of additional slides and stain using available SimulFluor stain panels and specific individual monoclonal antibodies.
(b) If direct smear is positive, the technologist will mark the inoculated R-Mix shell vial so that the coverslip will not be stained. Instead, a cell suspension of the R-Mix monolayer is prepared so that multiple cell spots (on slide) can be stained with SimulFluor stain panels and/or specific individual monoclonal antibodies.
Specimens (non stat) received in the virology lab by 14:00 hours: Complete direct smear testing the same day.
Specimens (non stat) received between 14:00 – 15:30 hours: Process specimen and prepare smear, however, staining, reading and reporting results may be completed the next day.
C. Isolation and Identification:
Specimens | Method
| Cell Linea | Incubation at 36oC | Stainb used/Read |
| Specimens from infants in NICU | Shell Via l | MRC-5 R-Mix* | 2 days 2 days | CMV-IE RS* If direct smear is positive, stain slide with corresponding SimulFluor panels |
| Shell Via l for CPE (if enterovirus is requested) | E-Mix MRC-5 (summer**) | 5 days 5 days | Read daily If CPE is present, pass &/or stain with enterovirus (DHI D3), Coxsackie B and ECHO stains | |
| Other specimens | Shell Via l | R-Mix* | 2 days | RS* If direct smear is positive, stain slide with corresponding SimulFluor panels |
aMRC-5 = Human Fibroblast cells
b CMV-IE = Monoclonal IFA stain for Cytomegalovirus Immediate Early antigen
b RS= SimulFluor Respiratory virus Screen DFA staining
b HSVbivalent= Monoclonal DFA stain for HSV1 and HSV2
*If direct smear is positive, the technologist will mark the R-Mix shell vial so that the coverslip will not be stained. Instead, prepare a cell suspension of the R-Mix monolayer so that multiple cell spots (on slide) can be stained with SimulFluor stain panels and/or specific individual monoclonal antibodies.to identify the virus.
** summer= May to October
D. Interpretation and Processing of Cultures:
a) For shell vial procedure:
i) Fix and stain R-Mix shell vial on day 2 (or next working day).
ii) If HSV requested, fix and stain on day 1 (or next working day).
iii) If CMV required, fix and stain on day 2 (or next working day).
See Appendix II for detailed shell vial procedure.
b) Shell Vials for CPE should be examined daily for Cytopathic effect (CPE). Any culture demonstrating 2+ or more CPE should be confirmed using appropriate monoclonal antibodies and immunofluorescent staining (Refer to Appendices IV and V). If positive, record in freezer program and freeze the cells and supernatant (Refer to Appendix X and XII).
c) Any culture demonstrating CPE for which a virus cannot be detected using monoclonal antibodies or other in-house methods and toxicity has been ruled out (see below) should be referred to the Public Health Laboratory (PHL) for electron microscopy and further work-up. Consult the charge/senior technologist or medical microbiologist.
d) Culture Toxicity: If toxicity is suspected in a tube culture (rounding of cells, sloughing of cells, granular cytoplasm of cells or unusual CPE), the cells should be scraped and appropriate monoclonal antibody staining performed. Negative stain results indicate the need for a passage. Scrape cells and add 0.2 ml of these scraped cells to a fresh tube containing 2 ml of media (1:10 dilution) and proceed again with tube culture method. (Appendix III). If toxicity or CPE persists, refer to the charge/senior technologist for review.
e) Contaminated Culture: If the tube culture is visibly contaminated and uninterpretable, replant the specimen.
IV. Reporting Results
Direct: Negative Report: “Negative for respiratory viruses.”
Positive* Report: “POSITIVE for _________virus.”
Shell Vial: Negative Report: “Negative for ________ virus”
Positive* Report: “POSITIVE for _________virus.”
Toxicity Report: "Specimen toxic to cell culture.”
Contaminated Report: "Specimen is heavily contaminated with bacteria and/or fungus. Unable to perform Virology Shell Vial
V. References
1. Gleaves, Curt A. et al. Cumitech 15A “Lab Diagnosis of Viral Infections”.
American Society for Microbiology, August 1994.
2. Greenberg, S. et al. Cumitech 21 “Lab Diagnosis of Viral Respiratory Disease”.
American Society for Microbiology, March 1986.
Tidak ada komentar:
Posting Komentar