Collect specimens as outlined in the Viral Specimen Collection Table, as soon after onset as possible. The best specimens for viral isolation are those collected during the first 3 days after onset of symptoms.

Collect all specimens as aseptically as possible. Swabs used to obtain specimens for viral isolation should be made of sterile cotton or Dacron with plastic or aluminum shafts. Avoid swabs with wooden shafts. DO NOT USE CALCIUM ALGINATE SWABS FOR VIRAL ISOLATION SPECIMENS.

All swabs and small tissue specimens for viral isolation should be placed in viral transport media (VTM). Typically, viral transport media consists of protein such as bovine serum albumin or gelatin to stabilize the virus and antimicrobial agent in a buffered salt solution. Transport medium can be purchased commercially. Swabs should be placed in 2-3 ml of medium, larger volumes of medium should not be used because of the dilution effect.

Place each specimen into a separate, leakproof container labeled with the patient’s name, the collection site, and the date of collection. For additional information on the packaging of specimens, see The TDSHS Manual of Reference Services.

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Specimen Submission Form

Complete a G-1a specimen submission form for each specimen submitted to the Viral Isolation Section. Specimens will not be tested without a completed G-1a form. Follow the Guidelines for Specimen Shipping and Mailing for shipping specimens to the laboratory. Forms may be obtained from the Laboratory Office.

Specimen Transport and Handling

Specimens for viral isolation should be transported to the laboratory as quickly as possible after collection since the chance of isolating a virus declines with increased time in transit. Samples containing labile viruses (i.e. respiratory syncytial virus, cytomegalovirus and varicella zoster virus) at low titers are those most likely to show loss of infectivity with delayed transport. For short-term transit or storage, specimens should be kept at refrigerator temperatures on wet ice or cold packs. If a longer transit time (greater than 2-3 days) is anticipated, the specimen should be frozen at -70° C and transported to the laboratory on dry ice.

Specimens for chlamydia isolation should be put in chlamydia transport media and kept at refrigerator temperatures on wet ice or cold packs if they can be processed within 48 hr after collection. If not, they should be frozen at -70° C.

Specimens for direct detection (i.e. EM, PCR) should be kept at refrigerator temperatures on wet ice or cold packs. Freezing specimens for direct detection is not optimal.

Specimens must be transported in accordance with all safety and labeling regulations. The TDSHS Manual of Reference Services includes detailed instructions for packaging and shipping specimens and isolates.

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Testing Methods

Viral Isolation

Viruses cause a wide range of clinical symptoms and are shed from various anatomical sites during the infection. The site selected for specimen collection should be based on the clinical symptoms (see Viral Specimen Collection Table) and the seasonal and/or local epidemiological trends. Once a specimen is received in the laboratory, it is processed and inoculated into living host systems (i.e. cell cultures). Inoculated host systems are incubated to allow any virus present in the specimen to grow and replicate. During the incubation period, if observation indicates the presence of a virus, additional laboratory tests are performed to confirm the virus’s presence and identity.

Reference Culture: Viral Isolation

Laboratories who would like final identification and/or confirmation of their results may send isolates to the TDSHS Laboratory.

Isolates can be sent either frozen on dry ice or at ambient temperatures in cell culture monolayers that are shipped when CPE is first evident in the monolayers. Fill the tube or vial with media to prevent drying of the monolayer during transit if the tube or vial gets turned during transit.

Although the Viral Isolation laboratory will confirm an adenovirus identification, prior approval is required for typing of adenovirus isolates since they are ubiquitous. Typing of adenoviruses is limited to situations when the virus is the suspect cause of death, is involved in a significant outbreak, or under other unusual circumstances. Contact the Medical Virology Branch of the Laboratory at (512) 458-7515 to discuss the possibility of typing an adenovirus isolate.

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Electron Microscopy

The electron microscope has been an invaluable tool for the basic understanding of viruses. Clinical application of the electron microscope has yielded another use: the rapid and unequivocal recognition of viruses. The results of studies with the electron microscope during the past have ranged from describing a new virus for the first time to the rapid recognition of viruses in clinical specimens.

The use of the electron microscope in the detection and identification of viruses offers many advantages. Using a negative stain technique, results are generally available in hours, not days. The infectivity of the virus does not need to be maintained, and viruses which are difficult or impossible to culture can be identified. The approach is open-ended which allows a wide variety of agents to be visualized, and since the actual agent is visualized, there are no false positive results.

Specimens for electron microscopy should not be placed into transport medium or fixative. Package specimens on wet ice or cold packs and transport as quickly as possible. Do not freeze specimens for electron microscopy.

See sample electron micrographs

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Dengue Isolation

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Dengue is an acute febrile illness resulting from infection with 1 of 4 closely related arboviruses (dengue serotypes 1,2,3, and 4).

A laboratory diagnosis of dengue is obtained by a rise in antibody titer between acute and convalescent sera (drawn at least 14 days apart), the presence of IgM, the isolation of dengue virus from an acute phase serum specimen, or the detection of RNA in the serum by PCR.

Submission of specimens for dengue isolation should be coordinated with Infectious Disease Epidemiology and the Laboratory.

Serum is the optimal specimen for isolation of dengue virus. Isolation of dengue virus from serum is most successful when the blood is collected within 3 days of onset of symptoms, although virus has been recovered from sera collected up to 5 days after onset. Ship the serum cold, on ice packs, by overnight carrier to the TDSHS Laboratory if the specimens will arrive at the laboratory within 2-3 days of collection. If the specimen will be delayed more than 2-3 days, ship the specimens frozen, on dry ice. However, if the specimen is being shipped as whole blood rather than serum, send it only on cold packs. DO NOT FREEZE WHOLE BLOOD OR SEND WHOLE BLOOD ON DRY ICE. This will lyse the red blood cells and be toxic to the cell cultures, compromising the ability to isolate the virus.

Specimens will be rejected for isolation if the specimen is not received in a sterile container, there is excessive delay in transport, the specimen is not refrigerated or frozen during transport, or if the date of collection is not within 5 days of onset of symptoms.

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Last Updated August 15, 2005