Kamis, 25 Agustus 2011


bronkus I. Introduction

Viral pneumonia in immunocompromised patients is commonly due to cytomegalovirus (CMV) or respiratory syncytial virus (RSV). Other viruses that may be detected include influenza, parainfluenza, adenovirus, enterovirus and measles viruses. Requests for viruses other than the above may require the use of additional media. Refer to Appendix XV (Virus Isolation and Identification).




II. Collection and Transport

Sputum, bronchial washes and aspirates are to be submitted in a clean, sterile container. If a delay in transport or processing is anticipated, keep the specimen at 4oC. Multiple bronchial washes and/or BAL samples received from the same patient at the same time should be pooled and processed as a single BAL sample.


III. Procedure


A. Processing of Specimens:

i) Bronchial washes and aspirates:

a) Approximately 2-3 mL of centrifuged sediment is to be received from the specimen receiving area (planting).

b) Prepare direct smears (if required) from the sediment. Very thick/mucoid specimens may require dilution of a portion of the sediment with sputolysin prior to direct smear preparation to avoid excessively thick smears. (See Appendix XXII)

ii) Sputum:

a) Combine equal amounts of sputum and a 1:10 dilution of sputolysin and vortex gently.

b) Prepare direct smears, if required, from the above mixture.


B. Direct Examination:

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.

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 hrs, processing and smear preparation should be completed, however staining, reading and reporting results may be completed the next day (except on Fridays, consult Charge technologist).


C. Isolation and Identification:


Cell Linea

Incubation at 36oC

Stainb used/Read

Shell Via l



2 days

2 days



Shell Vial for CPE

If enterovirus is requested:


MRC-5 (summer**)

5 days

5 days

Read daily***

Read daily***

aMRC-5 = Human Fibroblast cells

b CMV-IE = Monoclonal IFA stain for Cytomegalovirus Immediate Early antigen

b RS= SimulFluor Respiratory virus Screen DFA staining

*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

*** 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.


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 CMV requested, fix and stain on day 2 (or next working day).

See Appendix II for detailed shell vial procedure.

b) Shell Vial Cultures for CPE should be examined a 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 supernate (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, send out report indicating contamination.


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: "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.


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.


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