Viral bronchitis, bronchiolitis and pneumonia are commonly caused by RSV or Parainfluenza in infants and young children. CMV pneumomitis may be seen in newborn/premature infants.
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II. Collection and Transport
Endotracheal tube (ETT) aspirates or auger suctions are generally collected into a sterile container. If a delay in transport or processing is anticipated, keep the specimen at 40C.
III. Procedure
A. Processing of Specimens:
a) If sample is for virology only, flush approximately 2 mL of viral transport media through tubing and use for inoculation. If sample is to be split for other tests (i.e. C&S) use sterile saline.
b) Refer to Appendix II and III for Shell Vial and Tube Culture inoculation, respectively.
B. Direct Examination:
(a) Prepare one double-well cytospin slide for immunofluorescent staining. Stain one well with RSV/Para3 SimulFluor monoclonal antibody and one with SimulFluor Respiratory virus Screen monoclonal antibody. If a specific virus is requested, prepare appropriate number of additional slides and stain using specific individual monoclonal antibodies (Refer to Appendix VI Direct antigen Detection from specimens – SimulFluor Respiratory Screen Protocol Scheme 2)
(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.
Complete direct smear results the same day for specimens received in the virology section by 14:00 hours. For specimens received between 14:00 and 15:30
a) to Appendices IV and V). If positive, record in freezer program and freeze the cells and supernate (Refer to Appendix X and XII).
b) 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.
c) 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, issue a report indicating contamination.
Reporting Results
Direct: Negative Report: “Negative for respiratory virus”
PositiveReport*: “POSITIVE for ______virus.”
Shell Vial: Negative Report: “Negative for Cytomegalovirus.”
Positive Report*: “POSITIVE for _______virus.”
Toxicity Report: "Virology Tube Culture: Specimen toxic to cell culture.
Contaminated Report: "Virology Tube Culture: Specimen is heavily contaminated with bacteria and/or fungus. Unable to perform Virology Shell Vial Assay.”
*Notifiy Infection Control of all positive respiratory virus results and document.
*Notify Medical Officer of Health of all positive influenza virus results and document.
V. Reference
- Greenberg, S. et al. Cumitech 21 “Lab Diagnosis of Viral Respiratory Disease”.
American Society for Microbiology, March 1986.
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