Uses and Pitfalls
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Published: April 2012Print Record of Viewing
The expansion of PCR testing in the microbiology field has improved both the detection of viruses in patient specimens and the turnaround time for testing. It is important to select the best test for detection of specific viruses. Dr. Pritt reviews the various test options and provides guidance on the best tests for identification of viruses in clinical specimens.
Presenter: Bobbi Pritt, MD
- Consultant, Clinical Microbiology Division
- Associate Professor of Laboratory Medicine and Pathology and Microbiology
- Director of the Clinical Virology and Parasitology Laboratory
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Our presenter for this program is Dr. Bobbie Pritt, Director of the Clinical Virology and Parasitology Laboratory in the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minnesota. Dr. Pritt discusses the utility of viral culture — when it is of use and when other tests are suggested for viral identification. Thank you, Dr. Pritt.
Thank you, Sharon, for that introduction. Welcome to a hot topic on Viral Culture: Uses and Pitfalls.
I’d like to begin with a brief overview of viral culture. As many of you probably know, it is a traditional microbiology method that was previously considered to be the “gold standard” for viral diagnosis although this is now changing with wider use of molecular diagnostic tools. Viral culture uses methods such as tube culture and shell vial culture and it is more sensitive than rapid antigen detection methods such as the ubiquitous “card tests” for influenza and RSV.
Traditional tube culture involves the use of a tube containing a monolayer of living cells. The monolayer is infected by virus that is present in the patient specimen and the tubes are then incubated on a roller rack such as that shown here on the right to ensure that cells are bathed in media to keep them alive. The cell monolayers are then periodically observed for damage that is consistent with viral infection. This is referred to as cytopathic effect or CPE.
Multiple Roller Racks Containing Tube Cultures
This next slide shows multiple roller racks containing tube cultures in a walk in incubator.
Technologist Examining the Cell Monolayers
And here you can see a technologist examining the cell monolayers under a traditional light microscope.
Special Stage Attachment
Note that he’s using a special stage attachment that allows the tube to be easily viewed under the microscope.
Negative MRC-5 Cell sheet (human diploid fibroblasts)
This slide shows a nice example of an uninfected cell line as viewed microscopically. In this case, it is a MRC-5 Cell sheet which is made up of human diploid fibroblasts growing in a sheet. You can appreciate here the spindled nature of the cells.
MRC-5 Cell sheet with CMV cytopathic effect (CPE)
When the cell sheet is infected, as shown on this next slide with cytomegalovirus, the cell sheet shows damage or CPE that is characteristic for the infecting virus. The CPE is noted here by the 2 black arrows. Our technologists are highly trained so that they can recognize the different types of CPE caused by each virus.
Shell Vial Culture
Shell vial culture is a modification of the tube culture. It uses a smaller tube containing a coverslip and it is actually the coverslip that contains the cell monolayer. You can see an example of a shell vial and the coverslip it contained on the right. Shell vials are commercially available. Some even contain more than 1 type of cell. When using a shell vial culture, the cell layer is first inoculated with specimen.
Then the tube undergoes centrifugation, followed by a short incubation time which can be as little as 24 hours. This is a marked improvement in turn-around-time for viruses such as CMV which can take up to 14 days to grow in traditional tube culture. Finally, the coverslip is removed and stained with specific monoclonal antibodies for a certain virus. There are also some systems available where a color change indicates the presence of a virus. The advantage of this method is that specific viruses can be isolated in a much shorter period of time than is possible with traditional tube culture. Unfortunately, the shell vials are usually set up so that only 1 virus can be identified per tube.
CMV Shell Vial Results
This next slide shows the coverslips after they have been removed from the shell vials and mounted onto slides. The cell sheet on the coverslip is stained with a monoclonal antibody for a specific virus and then the coverslip is examined under a fluorescent microscope. The image to the right shows a positive result for cytomegalovirus with classic green nuclear staining.
Pitfalls of Culture
Although there are advantages to isolating viruses in culture, there are also some important pitfalls. First, not all clinically important viruses grow in routine culture (and that includes both tube and shell vial cultures). Also, some viruses grow poorly in culture or take an extended period of time to show recognizable CPE. Culture also relies on the presence of viable virus in the specimen. Virus may not be viable due to a number of factors such as prior patient treatment, the type of sample collected, and the conditions under which the specimen is transported to the lab.
Viruses That Grow In Culture and Incubation Time
This chart on the next slide shows the viruses that will grow in culture and note that it is a somewhat short list. I’d like to just call attention to 3 common viruses that take a very long time to grow in culture. These are VZV, CMV, and RSV. VZV and RSV, in particular, often fail to grow in culture at all.
Viruses That Do NOT Grow in Routine Cell Culture
There are also a number of relatively common viruses that do NOT grow in culture at all, such as Epstein-Barr virus (EBV), JC virus, BK virus, Arboviruses and that includes West Nile and Dengue viruses and most agents of viral gastroenteritis.
Best Tests for Identification of Cultivable Viruses
Given these limitations of culture, it is not surprising that it has been replaced in most instances by more rapid, sensitive, and specific tests. This chart here shows the viruses that are commonly sought in patient specimens ant the optimal method for detecting them in the laboratory. Notice that the primary test for almost all viruses listed is PCR.
Suggested Tests for Viral Identification
So let’s now go over some common scenarios where viruses are suspected and the suggested tests for detecting them. One such scenario is influenza like illness in a patient for whom treatment will be given. In this setting, the test of choice is a multiplex PCR assay test for influenza A, influenza B, and RSV. In immunocompromised hosts, a larger panel of viruses may be desired, in which case additional PCR tests for Adenovirus, CMV, HSV, Parainfluenza and Rhinovirus may be desired. In this setting, viral culture may also be useful because it is a relatively affordable test that will detect most of these viruses. Again, the important limitation of viral culture in this setting is the long turn-around-time of 2 weeks or more.
Another common scenario is when a patient presents with a dermal or genital lesion that is suspicious for infection with HSV or VZV. Because VZV grows poorly in culture, the optimal tests in this setting are PCR for both HSV and VZV. An important caveat is that viral culture would be required is HSV resistance to acyclovir is suspected. In this case, HSV would need to be grown in cell culture prior to undergoing susceptibility testing. Another unique setting is the neonate with potential exposure to HSV during delivery. In this case, HSV PCR or viral culture would be appropriate, and some experts prefer culture over PCR because this would indicate that the virus is viable and not just consist of residual viral DNA from the mother.
CSF is another commonly submitted specimen for viral detection, particularly in patients with meningitis or encephalitis. In this setting, the tests of choice would include enterovirus PCR, HSV and VZV PCR if indicated by the clinical picture, and arbovirus serology as indicated. It is important to emphasize again that arboviruses do NOT grow in routine cell culture, so this is not something that culture should be used for. Also, virus is rarely grown in culture from CSF, which is why I’ve listed PCR tests rather than culture-based tests in this setting.
Some physicians will make the point that culture is useful when novel or emerging viruses are suspected such as in samples from patients with: avian influenza, SARS, small pox, monkey pox, emerging pathogens, or even viral agents of bioterrorism. In these settings, however, viral culture in the routine clinical laboratory would not be recommended due to the risk of infection to laboratory personnel. Therefore, the physician should notify the laboratory if one of these agents is suspected so that the specimen can be sent to a specialized laboratory such as the CDC or other public health lab.
I’d like to now say a word about some other types of specimens. Specifically, bone marrow, blood, and lymph node are specimens from which viruses are rarely identified by culture, and PCR is almost always the test of choice for viral identification. This may include quantitative PCR on whole blood or plasma for viruses such as EBV, CMV, BKV, HHV-6, and adenovirus. Urine is another specimen that rarely yields virus in culture and PCR is typically the test of choice. It is used for detection of BK virus, adenovirus, CMV, and other viruses. In some situations, culture might also be used for detection of mumps virus and adenovirus. And of course, in all of these settings, tests for other organisms such as bacteria and fungi should be considered as appropriate.
So in summary, although viral culture is considered a “gold standard,” it is rarely the test of choice. This is due to multiple reasons, including the long turnaround time, the limited number of viruses that will grow in routine culture, unlike bacterial culture which will support the growth of numerous common bacteria and the fact that many viruses don’t grow well or grow very slowly in culture. Therefore, I encourage physicians to be familiar with the different viruses associated with various clinical presentations so that the appropriate tests can be ordered.
Thank you again for joining us today for this brief review of viral culture.