Herpes Simplex and Varicella Zoster Viruses
Fast, Efficient and Accurate Processing for
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Published: December 2009Print Record of Viewing
Dr. Pritt provides an overview of testing for Varicella-Zoster and Herpes Simplex Virus by Rapid PCR and explains the recent changes to specimen requirements for these assays.
Presenter: Dr. Bobbi Pritt
- Assistant Professor of Laboratory Medicine and Pathology
- Director of the Clinical Virology and Parasitology Laboratories in the Division of Clinical Microbiology at Mayo Clinic
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Our presenter for this program is Dr. Bobbi Pritt, Assistant Professor of Laboratory Medicine and Pathology and Director of the Clinical Virology and Parasitology Laboratories in the Division of Clinical Microbiology at Mayo Clinic. Dr. Pritt will provide an overview of testing for Varicella-Zoster and Herpes Simplex Virus by Rapid PCR and explain the recent changes to specimen requirements for these assays.
To begin, herpes simplex virus, otherwise known as HSV, and varicella zoster virus, or VZV, are 2 common viruses in the herpesviridae family. They both can cause lesions of the skin and mucosal membranes: 2 common manifestations of HSV infection are oral herpes or cold sores, and genital herpes, while VZV causes chicken pox and shingles. Rarely, HSV and VZV can also cause systemic disease, but dermal and anogenital disease are much more common. The classic presentations of these diseases can usually be diagnosed without laboratory testing, but there are also situations in which the viruses have overlapping clinical manifestations, and laboratory testing may be indicated in these instances.
There are several laboratory testing options for HSV and VZV. A quick and simple method is the Tzanck smear, in which scrapings of cells from the base of an unroofed blister are placed on a glass slide and stained with Giemsa or papanicolaou stain. The microscopist then looks for the presence of virally- infected cells, which classically, as shown here, are multinucleated, and have glassy nuclear contents, marginalized nuclear chromatin, and nuclear molding. This presentation is diagnostic for HSV and VZV herpes viruses, but it cannot distinguish between the 2 of them.
In order to actually differentiate HSV and VZV, alternative methods such as viral culture or PCR must be used. PCR includes both conventional and real-time methods, and it is the fastest, most sensitive method for detection of both viruses. So, given these advantages, real-time PCR is the only method used at Mayo Clinic for diagnosis of HSV or VZV from anogenital and dermal specimens, which are the most common specimens that we receive for these viruses.
HSV/VZV Real-time PCR Sample Workflow
Until recently, samples for HSV and VZV PCR were processed according to the following workflow. First, samples from characteristic lesions, as shown here, were collected on a swab by the clinician and then placed in a transport tube for the journey to the lab. The transport tubes that we previously accepted could contain M5 viral culture transport media, or could simply be a culturette tube with a moistened sponge inside to keep the sample hydrated.
Once the sample arrived in our laboratory, the swab was removed and placed in M5 media, if it wasn’t in this media already. DNA from the sample was then extracted on the Roche MagNA pure automated extraction instrument, and then pipetted by the virology laboratory technologists into an individual glass cuvette, along with the appropriate PCR mastermix. The cuvettes were finally placed on a carousel with other samples as shown in the upper right hand corner of this slide, and the carousel was placed on the Roche Light Cycler 2.0 instrument for PCR amplification.
Although we strive for rapid turnaround time for reporting test results, and we perform HSV and VZV testing several times a day, we are limited by the time it takes to extract and amplify the samples. Specifically, it takes 2 hours to extract DNA from the swab samples using the MagNA pure instrument.
Therefore, in an effort to increase the efficiency and speed of our assay, we queried whether it would be possible to bypass the extraction step altogether by using an alternative processing method.
Is Extraction Required for Dermal and Anogenital Specimens?
First, we needed to examine why extraction was done for HSV and VZV PCR testing. We decided to focus specifically on dermal and anogenital samples, since these comprise the bulk of the samples that we receive for HSV and VZV PCR testing.
In these cases, the main purpose of extraction is to purify viral DNA remove inhibiting substances. Since HSV and VZV DNA is typically abundant in anogenital and dermal samples, and samples collected on a swab contain very few, if any, inhibiting substances, we hypothesized that extraction served little purpose, other than removing the phenol red pH indicator from the M5 media. We know that the phenol red can interfere with the detection of the PCR product, but if samples are not submitted in M5, then extraction may not be necessary.
Direct Processing in Neutralization Buffer: An Alternative to MagNA Pure Extraction
Based on work with other PCR assays that we perform in our lab, such as those for group A strep and Bordatella pertussis, we had experience using a direct processing method in neutralization buffer that allows us to bypass nucleic acid extraction. With this method, swab samples that arrive in the microbiology laboratory can be placed directly into a conical tube containing neutralization buffer. Then after a brief heating and vortexing step, the sample can then be transferred to the PCR cuvette and undergo amplification. DNA extraction is not necessary.
This would require all samples to arrive as swabs without M5 media, like the swab in the culturette transport tube shown here. Again, if the swab was place in M5 media, then extraction would still be required to remove the phenol red.
Match Up Identifying Information
However, if only a swab in a culturette tube is received, the following steps can then be taken to process the sample. First, the identifying information on the swab container is matched up with the printed labels to ensure that the correct patient sample is being processed. This image shows a technologist in initial processing performing this important check.
Remove Swab from the Culturette Tube
Next, the swab is removed from the culturette tube and placed into the tube with neutralization buffer. The shaft of the swab is clipped off as shown here, and the lid is then placed on the new tube. The clippers used to cut the swab shaft are cleaned thoroughly in bleach and ethanol between each patient sample, to prevent cross contamination between samples.
Heat Swab in Neutralization Buffer
The swab in neutralization buffer is then heated for six minutes in order to render it noninfectious, and is then subjected to a brief vortexing step to dislodge viral material from the swab into the neutralization buffer.
Sample is Ready for PCR
Now, the sample is ready for PCR, and can be pipetted directly into the PCR cuvette. This new process takes only 35 minutes, compared to the normal 2 hour extraction process. PCR time remains the same.
A New Workflow for Dermal and Anogenital Swabs
So, going back to our workflow diagram, the new procedure looks as follows: The swab sample would still be obtained in the clinical setting and placed in a transport device. But in this case, only a transport device without M5 media, such as a culturette tube, can be used. Note the presence of moist sponges inside the culturette transport tube, as shown with the blue arrow. These sponges keep the specimen hydrated during transport. When the swab is received by the microbiology lab, and it is then placed in neutralization buffer, which is heated, vortexed, and amplified as described previously. Again, our processing time before PCR has now decreased from two hours to only 35 minutes.
Direct Processing in Neutralization Buffer: Comparable to DNA Extraction?
Well, this appears to be a great process, which could benefit the patient by potentially decreasing turnaround time. The question is whether or not it produces comparable results compared to the method using M5 media and MagNA Pure extraction. In order to determine this, we performed a simple study, where known HSV and VZV PCR positive samples from anogenital and dermal sources that had been previously sent in using a culturette tube were collected in the lab. The culturette transport tubes from these samples still contained the sponges which had surrounded the original swabs during transport, and we know that these sponges retain a significant amount of viral DNA. Therefore, we were able to obtain this DNA by placing two swabs inside of each tube and compressing the sponge around the swabs, thus inoculating the swabs with viral DNA. Obviously, these swabs would not be expected to yield as much viral DNA as the original swab that was sent in, but it provided sufficient viral DNA from a clinical source to allow us to perform a comparison of methods.
To do this, one of these two new swabs was placed in 3mL of M5 media as would normally occur when a swab in a culturette tube is received in the lab. The sample then underwent routine extraction. The other swab was place into a tube containing neutralization buffer (which is abbreviated here as an NB tube). Then, 5 microliters of both the extracted and NB samples were amplified by HSV and VZV PCR on the LightCycler instrument.
The results of the two methods were then compared both qualitatively and semi-quantitatively.
Comparison of Detection - HSV
Here are the results of 300 HSV samples in which the NB and extraction methods are compared. Interestingly, 87 positive samples were detected by both methods, but an additional 20 samples were detected by the NB method. These samples had been previously confirmed as positive by PCR, so we were confident in saying that the NB method detected a substantial number of additional positive samples.
Results: Comparison of Amplification Cycle Threshold
We also examined the cycle thresholds or crossing points of the PCR amplification curves. The lower the crossing point, the higher the amount of viral DNA in the sample. Shown here are just the results from the first eight samples, but it is enough to show that the crossing points are consistently lower using the NB method compared to the M5 extraction method. And this trend continued throughout all 300 samples, demonstrating that the NB method resulted in a great yield of amplified HSV DNA.
Comparison of Detection - VZV
We also assayed 100 known positive VZV samples using the two methods. As you see here, 12 positive samples were detected by both methods, and a single additional positive sample was detected by the NB method. This demonstrates that the NB method is at least equivalent to the M5 extraction method.
Results: Comparison of Amplification Crossing Points
The crossing points with the NB method were also equal or lower than those using the M5 extraction methods, again showing that the NB method is at least equal to the M5 extraction method yielding amplifiable VZV DNA.
Questions and Answers
So, the question may be asked, “If I’m submitting a dermal or anogenital swab for HSV and/or VZV PCR, should the swab be placed M5 viral transport media or just in a culturette tube?” As you have probably guessed from my previous slides, the swab should just be submitted in the culturette tube without M5 media. Without M5 media, there is no need to submit the sample for extraction. A commonly used culturette tube is shown here. Be sure that the culturette tube that is chosen contains a moist sponge to keep the sample hydrated and maintain the nucleic acid during transport.
Another question that you may have already thought of is whether or not there is an advantage to using M5 media for maintaining detectable DNA during transport. Clearly, we wouldn’t want to recommend the culturette tube for transport if the M5 media provided a significant advantage.
Well, it turns out that M5, while an excellent transport media for viral culture, is not actually beneficial for PCR detection of HSV and VZV from dermal and anogenital swabs. In fact, it is better to submit the sample in a culturette tube, because the virus does not need to be viable in order for DNA to be detected, and the addition of M5 media may dilute the sample, and decrease the sensitivity of detection.
M5 Media vs Culturette Tubes: Experiment
We proved this using another simple experiment. We took a positive HSV culture and make three 10-fold dilutions of the culture material. We then placed 10 swabs into the 3 dilutions, for a total of 30 swabs. Five swabs from each dilution went into individual containers with M5 media, while another five swabs from each dilution went into individual culturette tubes. One M5 tube and one culturette tube were then processed daily for 5 days, according to the appropriate extraction or neutralization buffer procedures. The design of this study took the whole process into account, including transport and pre-PCR processing and the results strongly favored the combination of the culturette tube with the NB.
M5 Media vs Culturette Tubes: Results
As shown on this table, the cycle threshold or crossing point was consistently lower for swabs that underwent culturette storage and neutralization buffer processing compared to the crossing points of swabs stored and processed by the conventional M5 method. As mentioned previously, a lower crossing point indicates that more viral DNA was recovered from the sample.
Therefore, we can draw the following conclusions from this work:
First, swab specimens can be sent directly to the laboratory in a culturette tube, without need for M5 transport media. In fact, the culturette is the preferred transport method.
Second, traditional DNA extraction is not necessary for real-time PCR detection of HSV and VZV from anogenital and dermal specimens, since culturette transport followed by direct processing in NB provides equal or greater yield of nucleic acid.
So, this simple change in processing will yield significant time savings, may decrease turnaround time, and for HSV, may increase the yield of viral DNA. We have begun using this process in our lab and are no asking our clients to submit anogenital and dermal swabs for HSV and VZV testing in a culterette tube and not in M5.