Kamis, 25 Agustus 2011

CEREBRAL SPINAL FLUID

I. Introduction

Cerebral Spinal Fluid (CSF) will be routinely cultured for cytomegalovirus (CMV), herpes simplex virus (HSV), varicella-zoster virus (VZV) and enteroviruses (coxsackie, echo and polio virus). PCR for these viruses will be performed if specifically requested. Other viruses that may be isolated from CSF include mumps virus and adenovirus. Requests for Rubella virus, JC virus, BK virus and arbovirus should be referred to the Public Health Laboratory (PHL).

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II. Collection and Transport

Specimens should be collected in a clean, sterile container and sent to the laboratory as soon as possible. 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 as soon as possible after arriving in virology laboratory. After processing, an aliquot of up to 2 mL of the left-over specimen should be stored at -70°C in a cryovial.

a. If the specimen is requested for PCR and viral culture and

i. The amount of specimen is <0.5 mL, perform PCR only.

ii. The amount of specimen is between 0.5-1.0 mL, perform PCR and tube cultures.

iii. The amount of specimen is >1.0 mL, perform PCR, tube culture and shell vial assay.

 

b. If specimen is requested for viral culture only and

iv. The amount of specimen is <0.5mL, perform the culture only.

v. If the amount of specimen is >0.5mL, perform tube culture and shell vial assay.

 

c. For PCR testing, aliquot 0.2-1 mL first (freeze aliquot unless PCR can be performed immediately) before proceeding. A minimum of 0.2 mL is needed for each of PCR and RT-PCR test.

 

d. CSF specimens will be inoculated directly into shell vials without further processing.

B. Direct Examination:

Method

Virus(es)

Location

PCR

HSV / CMV / EBV/VZV/ Parvo

In-house

RT-PCR*

Enterovirus

West Nile virus

In-house

PCR

Adenovirus

Research Lab

PCR

HHV6,7

Research Lab

*RT-PCR = Reverse Transcription PCR using Qiagen Isolation Kit, RealArt reagents and Roche LightCycler. CMV= cytomegalovirus; EBV= Epstein-Barr virus; HSV= Herpes simplex virus; VZV= Varicella-zoster virus; HHV6,7= Human herpes virus types 6,7

Note: HSV PCR is routinely performed on all CSF requesting virus testing, other PCR and RT-PCR tests are performed only upon request and with approval by a microbiologist.

 

C. Isolation and Identification:

Method

Cell Linea

Incubation at 36oC

Stain used/Read

Shell Vial

MRC-5 (if requested)

MRC-5 (if requested)

2 days

4 days

CMV-IE

VZV

Shell Vial for CPE

E-Mix

MRC-5 (summer b)

5 days

5 days

Read daily

Read daily

aMRC-5 = Human Fibroblast cells

b summer = from May to October.

 

D. Interpretation and Processing of Cultures:

a) Shell vial procedure:

i) For CMV, fix and stain 1 shell vial after 2 days (or next working day).

ii) If VZV is requested, fix and stain 1 shell vial after 4 days (or next working day).

See Appendix II for detailed shell vial procedure.

b) Shell Vial for CPE should be examined daily for Cytopathic effect (CPE). Any culture demonstrating 2+ or more CPE should be confirmed using appropriate monoclonal antibodies 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 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 uninterpretable due to bacterial/fungal contamination, replant the specimen.

 

IV. Reporting Results

PCR: Negative Report: “Negative for ________virus. This is a research test”

Positive Report*: “POSITIVE for ________ virus. This is a research test.”

Indeterminate Report: “Indeterminate by PCR. This is a research test”

Shell vial: Negative Report: “Negative for ______________ virus.”

Positive Report*: “POSITIVE for _______________ virus.”

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

Positive Report*: “______________ virus isolated”

Toxicity Report: "Specimen toxic to cell culture.”

Contaminated Report: "Specimen is heavily contaminated with bacteria and/or fungus. Unable to perform Virology Culture.”

 

V. References

1. Gleaves, Curt A. et al. Cumitech 15A “Lab Diagnosis of Viral Infections”. American Society for Microbiology, August 1994.

2. Collier L, Balows A, Sussman M. Topley’s and Wilson’s Microbiology and Microbial Infections. Volume 1, Ninth Ed. 1998

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