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Principle

Laboratory diagnosis of viral infections requires an understanding of the pathogenesis of the suspected agent and knowledge regarding the stage of infection and the age and immunocompetence of the infected individual. Important factors are the selection of the appropriate specimen, careful collection to optimize recovery of the agent and transport of specimens in a manner that maintains viability and minimizes overgrowth with contaminating organisms.

Selection of Specimens

For best correlation with a particular disease, the specimen should reflect the target organ whenever possible, which is usually based on the clinical symptoms.

Materials

A. Reagents

Viral transport medium (VTM) prevents specimen drying, helps maintain viral viability and retards the growth of microbial contaminants. A number of different VTM formulations are used, either prepared in-house or from a commercial source. At UPMC, M4 is the most commomly used VTM.

VTM typically consists of a protein such as gelatin and antimicrobial agents in a buffered salt solution. VTM tubes usually contain 2 to 3 ml of medium; a few sources provide kits that include VTM tubes and collection swabs. Larger volumes of VTM (5 to 7 ml) are usually prepared for transport of tissue samples; these volumes should not be used for swabs because of the dilution effect.

B. Supplies

1. Sterile, leakproof, screw-cap containers including urine cups, disposable centrifuge tubes (15 and 50 ml), and smaller tubes (e.g., 4-ml Nunc vials, 13x100 tubes), suitable for holding 1 to 2 ml of VTM.
2. Sterile cotton-, Dacron-, or rayon-tipped swabs with plastic or aluminum shafts Small-tip flexible (fine-shafted aluminum) swabs are used for certain samples such as urethral swabs. Do not use calcium alginate swabs, and do not use wooden-shafted swabs.
3. Tuberculin syringe with 26- or 27-gauge needle for aspirating vesicular fluid
4. Blood collection tubes containing anticoagulant (ACD)

C. Equipment

Refrigerator (2 to 8^o C)

Specimen Collection A. Collect specimens as soon after onset of symptoms as possible. The chance of viral recovery is best during the first 3 days after onset and is greatly reduced beyond 5 days with many viruses. Collect autopsy samples as soon after death as possible.

B. Refer to Table for the collection of specimens for viral culture. In general, place swabs into a tube containing a small volume of VTM, and scrapings and small pieces of tissue into a tube containing a small volume of VTM or saline. Place fluid and bulk specimens (e.g., tissue) into a sterile leakproof container; add a volume of VTM sufficient to prevent drying of tissue.

C. Collect specimens as aseptically as possible.

D. Place each specimen into a separate container labeled with the patient's name and identification number, the collection site, the date of collection, and the time of the collection.

E. Obtain a complete patient history, including the date of onset of symptoms, clinical findings, recent exposure history, animal or arthropod contacts or bites, recent travel to areas of endemic infections, and recent vaccines.

Specimen Transport

A. Place the tightly capped specimen container and the appropriate laboratory requisition form into separate compartments of a plastic specimen transport bag.

B. Deliver all specimens to the laboratory as soon after collection as possible, since a loss of infectivity occurs over time; samples containing labile viruses at low titers are those most likely to show loss of infectivity with delayed transport. If immediate delivery is not possible, refrigerate specimens (2 to 8^o C), or place them on wet ice or a cold pack. Loss of viability is slower at refrigeration temperature. Do not freeze samples.

C. Transport specimens for respiratory syncytial virus culture immediately for prompt inoculation into cell culture. Cytomegalovirus and varicella-zoster virus also are relatively labile, and specimens may lose infectivity rapidly.

D. Do not freeze specimens before testing them. Cytomegalovirus and some respiratory viruses are readily inactivated by even one freeze-thaw cycle. If samples must be frozen, freeze them rapidly at -70^oC or lower. Storage at -20^oC is totally unsatisfactory. Store at 4^oC or -70^oC. Survival of labile viruses may be enhanced by using a viral transport medium such as M4 or HBSS with gelatin.