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Virology

Viral Isolation & Identification

The Virology Section will accept animals for rabies testing from county health departments, county rabies animal control groups, state public health agencies, and veterinarians. Individuals wishing to have animals tested for rabies should contact their local rabies animal control group or their veterinarian. The following information is provided to guide health care professionals in the submission of specimens to the Virology Section for virus isolation and/or identification. It is the policy of the Virology Section to call the submitting agency whenever a viral isolate has been identified.

Specimen Collection, Handling, and Transport

General Considerations

To optimize the ability of the virology laboratory to isolate viral agents from clinical specimens it is very important that the specimens be collected, handled, and transported in a manner that minimizes deleterious effects on any viral agents present. In addition, sufficient information should be provided with a submitted specimen to guide the virology laboratory in the selection of proper inoculation techniques for the viral agents suspected. To maximize the chances for recovery of viral agents all specimens should be transported to the laboratory as soon as possible. Specimens should be sent to the lab on wet ice or ice packs. Specimens that will not be received by the lab within 48 hours should be frozen at -70 centigrade. Do not store viral specimens at freezer temperatures of -10 to -20 centigrade. If a -70 freezer is not available, tightly cap the specimen and freeze, store, and ship the specimen on dry ice.

Viral Transport Media

The medium used for collection and holding of swab specimens should contain protein to stabilize the more labile viruses, and the use of media containing charcoal should be avoided as this may reduce viral recovery rates. For eligible submitters, Hanks Buffered Salt Solution is available from the State Public Health Laboratory Receiving Department (602) 542-1190.

If a medium specifically formulated for collection of viral specimens is not available, a sterile, well-buffered bacteriological broth, such as tryptose phosphate broth, may be used. If no other media is available, sterile water may be used, however, viral recovery will be enhanced by the use of a protein containing media. Cotton-or Dacron-tipped swabs are preferred for collection of specimens; prolonged contact with calcium alginate swabs has been reported to inactivate herpes simplex virus.

Types of Specimens

Specimens that may be tested for the presence of viral agents are the following (click each to learn more):

Autopsy or Biopsy Specimens

Autopsy specimens for virus isolation should be collected within 24 hours after death. Samples (1.0-2.5 cm Tubes of tissue) from probable sites of pathology are collected using separate, sterile instruments and separate sterile containers for each specimen to avoid cross contamination. Tissues are transported to the laboratory on wet ice or cold pack. If they cannot be tested within 48 hours they should be frozen at less than 70 C.

Blood Specimens

Although blood is not the optimal specimen for isolation of most viruses, it may be used for the recovery of some of the vector-borne viruses, Enteroviruses, and CMV. Specimens for virus isolation should be collected as soon as a viral etiology is suspected, otherwise early neutralizing antibody may prevent recovery of virus from the blood. Either serum or leukocyte preparations may be used for viral isolation. For isolation of virus from leukocytes, 8 ml of blood is collected into a tube containing an anticoagulant. Although heparin is often used, it is reported to inactivate herpes simplex virus, and therefore EDTA may be preferable as an anticoagulant. For isolation of virus from the serum or blood clot, 8 ml of blood is collected aseptically without an anticoagulant. Blood specimens should be transported to the laboratory on wet ice or a cold pack, but not frozen.

Cerebrospinal Fluids (CSF)

For virus isolation 3-4 ml of CSF should be collected no later than 7-10 days after onset of illness, placed in a sterile screw-cap tube without collection medium, and transported to the laboratory on wet ice or a cold pack. The specimen should be inoculated as soon as possible after collection, or held at -70 C or lower, since viruses are very labile in this medium.

Cervical Specimens

Specimens should be collected using a speculum. A swab is used to clear the cervix of mucus, and is then discarded. A second swab is inserted about 1 cm into the cervical canal, rotated, and then left in place for a few seconds to absorb secretions. If lesions are seen, they are swabbed. The swab tips are broken off in 3-4 ml of collection medium (Hanks BSS is satisfactory) in a screw-cap container. The specimen is transported to the laboratory on wet ice or a cold pack. If it cannot be tested within 48 hours it should be frozen below -70 C.

Eye Specimens

A swab moistened in sterile saline is used to collect secretions from the palpebral conjunctiva. The swab tip should be broken off into 2-3 ml of viral collection medium (Hanks BSS is satisfactory) and transported to the laboratory on wet ice. Scrapings from the cornea or conjunctiva should be collected by an ophthalmologist or other suitably trained individual, and placed in 2-3 ml of collection medium (Hanks BSS is satisfactory) for isolation attempts. Specimens not tested within 48 hours should be frozen at below -70 C.

Rectal Swabs

The specimen should be collected no later than 7-10 days after onset of illness. A cotton or Dacron tipped swab is moistened with collection medium free from serum, inserted 4-6 cm into the rectum and rubbed against the mucosa until visible fecal material adheres to the swab. Two swabs should be collected in this manner and the tips broken off in 3 - 4 ml of collection medium. The specimen should be transported to the laboratory on wet ice or cold pack. If the specimen cannot be tested within 48 hours, it should be frozen at <= -70 C.

Saliva

A swab should be rubbed over the buccal mucosa opposite the upper molars in the proximity of the Stensen's ducts, then over the floor of the mouth anterior to the tongue. The swab tip should be broken off into 3-4 ml of collection medium (Hanks BSS is satisfactory), then transported to the laboratory. Specimens which cannot be tested within 48 hours should be frozen at below -70 C. Saliva collected by aspiration or expectoration into a sterile container also may be used for virus isolation.

Semen Specimens

Semen specimens collected into sterile screw-cap jars should be sent to the laboratory on wet ice or cold pack.

Stool Specimens

Stool specimens for viral isolation attempts should be collected as soon as possible, and usually no later than 7-10 days after onset of illness. Enteroviruses may be excreted for weeks so if infection with these viruses is suspected, stool specimens collected later than 10 days post onset may be collected. Three to four grams of the specimen (2-3 tsp.) should be placed in a sterile, screw-cap glass or plastic bottle. The specimen should be transported to the laboratory on wet ice or cold pack. If the specimen cannot be tested within 48 hours after collection, it should be frozen at -20 C to -70 C. A temperature of -20 C is satisfactory for specimens being tested only for Enteroviruses.

Throat and Nasopharyngeal Specimens

Virus isolation is most successful if respiratory specimens are taken within the first 3 days of illness, and they should be collected no later than 5 days after onset. For virus isolation, swabs from both the throat and nasal passage should be collected. The pharynx is swabbed vigorously with a cotton swab moistened with collection medium free of serum. (Hanks BSS is a good collection medium). A dry swab is inserted into the nostril parallel with the palate and gently rotated. The applicator sticks are broken off, leaving both of the swab tips in a vial containing 2- 3 ml of collection medium. Ensure that the swabs are broken off short enough so as not to interfere with proper closing of the vial and causing leakage.

Specimens should be transported to the laboratory on wet ice and inoculated as soon as possible. If the specimens cannot be inoculated into cell culture within 48 hours, specimens should be frozen at -70 C. Note, respiratory specimens should not be frozen at -20 C temperatures as this will markedly reduce chances of isolating respiratory viruses. In general, freezing should be avoided if possible.

Urine Specimens

Urine specimens are generally tested only for Cytomegalovirus, although Mumps virus, Adenovirus, and some other viruses can sometimes be found in urine. The specimen should be collected at the onset of illness or as soon as possible if congenital disease is suspected. Chances of recovering virus may be enhanced by examining 2-3 consecutive urine specimens. Clean voided specimens (10-20ml) are collected in sterile containers and transported to the laboratory on wet ice or cold pack. It is particularly important that specimens for CMV be transported to the laboratory as soon as possible after collection. When submitting urine specimens it is very important to inform the laboratory what viral agent is suspected.

Vesicular Lesion Specimens

Vesicular fluids and cellular material from the base of lesions should be collected for virus isolation during the first 3 days of the eruption. Vesicles are washed gently with sterile saline and the vesicular fluids are aspirated with a 26-gauge or 27-gauge needle attached to a tuberculin syringe, or with a capillary pipette. The fluids should be diluted in 2-3 ml of collection medium (Hanks BSS is satisfactory) to prevent clotting. Alternatively, the cleansed vesicles may be opened and the fluids collected from several vesicles may be collected onto a swab which is then broken off into 2-3 ml of collection medium (e.g., Hanks BSS). Cellular material from the base of the vesicles may be added to the vesicular fluids for virus isolation attempts. The base of the opened vesicle is scraped with a scalpel blade, without producing bleeding, and the cellular material is placed in collection medium together with the vesicular fluid. Specimens should be transported to the laboratory on wet ice or with a cold pack or frozen at <=70 C if they cannot be inoculated within 48 hours.