Culture – Viral culture is based upon the inoculation of specimen into specific cell lines and detection of cytopathic effect (CPE) within those cells; specific CPE is representative of a specific virus. This initial observation is then typically confirmed using virus-specific antibody. There are 5 different viral culture orders. The following viruses can be detected using culture: HSV, VZV, Influenza A and B, Parainfluenza 1, 2, and 3, Adenovirus, RSV, CMV, Enterovirus, and Rhinovirus. If SARS is suspected, do not send specimen for culture; page the Nebraska Public Health Laboratory Special Pathogens Laboratory at 888-5588.

a. Herpes simplex virus (HSV) culture (culture is held for 7 days).

b. Respiratory virus culture (panel). Cell culture lines are inoculated to detect influenza A and B, parainfluenza 1, 2, and 3, adenovirus, and respiratory syncitial virus (RSV). Specimen (nasal wash is preferred-see http://info.unmc.edu/redbook/microbiology2.htm for information) is inoculated into tissue culture cell lines and shell vials. The cell lines are selected for their ability to propagate the respiratory viruses listed above. The use of shell vials normally results in faster detection of virus due to the amplification of any existing virus during the centrifugation step of the procedure. After incubation, the shell vials are stained with virus specific monoclonal antibodies for the aforementioned viruses on days 1 and 2. The tissue culture cell lines are examined for 14 days before being reported as negative.

c. Cytomegalovirus (CMV) culture (culture is held for 21 days; although shell vial results [detection of early antigen] are available after 1 day).

d. General virus culture (culture is held for 14 days)

e. Varicella-zoster virus (VZV) culture (culture is held for 14 days).

Direct viral antigen detection – Antigen detection assays are available to detect both influenza A and B as well as RSV. The optimal specimen for both of these assays is a nasal washing (http://info.unmc.edu/redbook/microbiology2.htm) as host cells are needed to isolate the viral antigens. In addition, both HSV and VZV can be detected directly from skin lesions using a direct fluorescent antibody (DFA) test. Call the microbiology laboratory (552-2090) for instructions and the Specimen Receiving Laboratory (559-7616) for test materials if a DFA test for either HSV or VZV is required.

Serology – Both IgM (acute) and IgG antibody titers can be assessed for a number of viral entities including: Epstein Barr Virus (EBV), CMV, HSV, measles, mumps, HIV, and West Nile. Note that EBV, and the measles and mumps virus can not be cultured. Additional testing is available through a reference laboratory. Call the Laboratory Sendout Department at 599-9353 for questions about sendout tests.

Nucleic acid detection – A number of viruses can also be detected using molecular methodologies-these tests are offered by the molecular diagnostics department. Specific questions regarding specimen collection, etc. should be discussed with the molecular diagnostics laboratory (559-7630). The following tests are currently offered: Parvovirus, HHV-6, Enterovirus, HSV, VZV, CMV, EBV, BK, JC, HIV, and Norovirus. Quantitative viral load tests are also available for EBV, CMV, JC, and HIV.

Specimen collection – With a few exceptions (listed below) specimens sent to the laboratory for viral culture should be collected either using a swab containing viral transport medium or within viral transport media. For instance, cultures for HSV or VZV from lesions should be collected using a viral transport medium containing swab. It is important to note that these swabs are distinct from the typical swab for bacterial culture. All tissue specimens should be placed in viral transport medium-it is best not to use a swab when collecting tissue from any source. The three exceptions when viral cultures do not need to be in viral transport medium are: 1) a nasal wash for respiratory virus culture, 2) blood culture for CMV (collect 5 ml blood in a sodium heparin tube and order CMV buffy coat), and 3) sterile fluid (such as CSF) where specimen should not be diluted. Sterile fluids not placed in viral transport media should be kept at 4ºC until transported to the microbiology laboratory.

Guidelines for specimen collection can be found at: http://www.preceptor.com. Then follow links to Laboratory services, section specific policies (Microbiology specimen collection guidelines).