Kamis, 25 Agustus 2011

OCULAR SPECIMENS

ocular spesimen I. Introduction

Viral infections of the eye (conjunctivitis, corneal ulcers, etc) are usually due to herpes simplex virus (HSV), varicella-zoster virus (VZV) and adenoviruses. Ulcerative lesions are usually due to HSV and VZV. For vitreous fluid, the two most common viruses isolated are cytomegalovirus (CMV) and varicella-zoster (VZV). Other viruses which may cause conjunctivitis, such as enteroviruses, will be looked for only if specifically requested.

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II. Collection and Transport

Specimens should be collected using a clean, sterile swab and gently swabbing the conjunctiva or ulcerative lesion. Place the swab in viral transport medium and send to the laboratory as soon as possible. Vitreous fluid should be collected in a clean, sterile container. If a delay in transport or processing is anticipated, the specimen should be kept at 4oC until processed. If a delay of more than 72 hours is anticipated, the specimen should be frozen at –70oC. Avoid repeated freeze-thaw cycles.

 

III. Procedure

A. Processing of Specimens:

Specimens should be set up immediately or stored at -70°C. Vortex patient sample in transport medium for 30 seconds. Remove excess fluid from the swab and discard the swab. Refer to Appendices II and III for Shell Vial and Tube culture inoculation, respectively.

B. Direct Examination:

If requested, prepare one double-well cytospin. Stain one well with HSV bivalent 1/2 antibody and the other with VZV monoclonal antibody.

Refer to Appendix V for immunofluorescent staining technique.

C. Isolation and Identification:

Specimen

Method

Cell Linea

Incubation at 36oC

Stainb used/Read

Conjunctiva,

cornea

Shell Vial

MRC-5

MRC-5

R-MixToo*

1 day

2 days

2 days

HSV-bivalent

VZV

P123/Ad*

Shell Vial for CPE

E-Mix (if enterovirus is requested)

MRC-5 (summer, if enterovirus is requested)

5 days

5 days

Read daily for 5 days

Read daily for 5 days

Vitreous fluid

Shell Vial

MRC-5

MRC-5

MRC-5

R-MixToo*

1 day

2 days

2 days

2 days

HSV-bivalent

VZV

CMV-IE

P123/Ad*

Shell Vial for CPE

E-Mix (if enterovirus is requested)

MRC-5 (summer**, if enterovirus is requested)

5 days

5 days

Read daily for 5 days

Read daily for 5 days

aMRC-5 = Human Fibroblast cells; R-Mix= R-Mix Fresh cellsTM from Diagnostic Hybrids Inc.

bHSVbivalent= Monoclonal DFA stain for HSV1 and HSV2

bVZV= Monoclonal DFA stain for Varicella zoster virus

bCMV-IE= Monoclonal IFA stain for Cytomagalovirus Immediate Early antigen

bP123/Ad = Monoclonal IFA stain for parainfluenza virus 1, 2 & 3

P123/Ad* = if R-MixToo* is positive for virus, inoculate another R-MixToo shell vial so that the isolate can be frozen

** summer= May - October

D. Interpretation and Processing of Cultures:

a. For shell vial procedure:

If CMV is requested, fix and stain after 2 days (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

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, issue a report indicating contamination.

I. Reporting Results

Direct: Negative report: “Negative for _________virus.”

Positive report*: “POSITIVE for ________virus.”

Shell Vial: Negative report: “Negative for _________virus.”

Positive report*: “POSITIVE for ________virus.”

Shell Vial for CPE: Negative Report: “No virus isolated”

Positive Report*: “POSITIVE for _________ virus.”

 

V. Reference

1. Gleaves, Curt A. et al. Cumitech 15A “Lab Diagnosis of Viral Infections”.

American Society for Microbiology, August 1994.

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