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Overview on HHV-6 testing in Clinical and Laboratory Research
Nearly 100% of us have been infected with the HHV-6 virus by early childhood and have antibodies to it, and at least 30% of us have small but detectable levels of latent virus in our blood (Alvarez-Lafuente 2002 & Clark 1996), so the relevant questions are not whether you have the virus, but rather how much virus do you have, and is it active or latent?
PCR DNA tests can detect HHV-6 in the serum during primary roseola infections and in acute transplant reactivation. However, they cannot determine reliably if a patient has a chronic central nervous system (CNS) infection that has reactivated because there is so little virus circulating outside of the tissues. HHV-6A & B viruses (especially HHV-6A) can migrate to the central nervous system and other organs away from the bloodstream. HHV-6A has been found to persist in the spinal fluid long after it has disappeared from the plasma (Caserta 1994).
Researchers at the NINDS have determined from autopsy that bone marrow transplant patients with active or reactivated infections in the CNS tissue have very little HHV-6 in the spinal fluid or serum (Fotheringham 2007). This means that if the HHV-6 is chronically active in the brain tissue, it may be impossible to find any evidence of it in the peripheral blood or even the spinal fluid.
As is the case with pathogens with low viral copy numbers such as HHV-8 and West Nile Virus, indirect evidence of the HHV-6 antibodies are easier to find than the HHV-6 virus itself. Therefore elevated IgG antibody levels (above a threshold) may be the only indication of a reactivated chronic HHV-6 CNS infection.
Another reason that it is so difficult to detect HHV-6 directly by PCR is that there is latent virus in many of our circulating white blood cells. Tests must be done in serum to differentiate active from latent infection. Furthermore, in many viral infections large numbers of virions spill into the plasma when the virus is replicating but this is not the case with HHV-6 since it is spread largely from cell-to-cell or directly through the cells walls. The net result is that very little of it ends up in the serum so PCR tests must be extremely sensitive to detect persistent HHV-6 infections once the acute phase is over.
New assays are desperately needed to identify the kind of low-level chronic activation that has been found in mesial temporal lobe epilepsy and suspected in MS and CFS. Among the assays that hold promise for the future: nested reverse transcriptase testing (nRT-PCR) to look at whether the virus is active in the cells. For a summary chart of all HHV-6 assays, with advantages and disadvantages listed for each, see below:
Elevated IgG antibody levels. Elevated IgG antibody levels can suggest, but not prove active, chronic infection. In a 1996 study of HHV-6 in CFS patients, 89% of the patients with IgG titers of 1:320 and above were found to have active infections by culture. (Wagner, Journal of Chronic Fatigue Syndrome)Stanford infectious disease specialist Jose Montoya believes that the best evidence of smoldering central nervous system (CNS) infection is the IgG antibody to the virus, and not the virus itself. In a pilot study, he found that when patients with high titers of HHV-6 IgG (1:320, 1:640 or higher) and EBV are treated with a potent antiviral; their titers fall substantially along with a significant improvement in symptoms. Elevated IgG Antibodies to HHV-6 cannot indicate with certainty that the infection is active, but high titers support a clinical diagnosis. Similarly, elevated EBV VCA (late antibody) titers also cannot indicate with certainty that an infection is active. However, EBV Early Antigen (EA) antibodies disappear rapidly after an infection is over, so elevated EBV EA antibodies do predict active infection.
Individuals vary in the way they respond to virus: some may not be able to generate antibodies due to a weak immune response. Others may generate large numbers of antibodies to many pathogens, and some healthy individuals have high titers of HHV-6 IgG antibodies. So looking at elevated antibodies to determine active infection is far from a perfect measure. Montoya is currently conducting a placebo controlled trial of Valcyte in symptomatic patients with elevated antibodies to HHV-6 & EBV. If these patients show a dramatic drop in antibody titers and an improvement in symptoms in response to Valcyte treatment, he will demonstrate conclusively that elevated IgG titers can be a sign of active infection and could be a reliable biomarker for monitoring patients.
At Stanford, Montoya uses antibody tests done by immunofluorescence or IFA to determine if a patient might qualify for antiviral treatment. He uses the IFA test at Focus Diagnostics Laboratory and has established a minimum threshold for treatment. He does not use ELISA tests that are offered at many labs (which report a number such as 0-20) because they are difficult to compare to the IFA values established in the literature, and because these assays cannot be used to monitor values over time. Many hospitals also use the IFA test. Other laboratories that use the IFA test include: Focus Diagnostics Laboratory, Specialty Laboratories andMedical Diagnostics Laboratory. IFA kits can be purchased for research purposes as well; BIOCELL Diagnostics, Inc, and a few other companies offer these kits for purchase by investigators.
Primary infections & IgM antibody levels. In a primary infection, which typically occurs before the age of two, IgG antibody levels increase four-fold within several weeks. Most pediatricians test for IgM antibodies to confirm a case of HHV-6 associated roseola or febrile seizures. The IgM antibodies appear within a few weeks and can be detected for several months. A fourfold rise in IgG titers or the presence of IgM antibodies are considered proof of active infection. The HHV-6 IgM antibodies are typically produced only with the primary infection, and not in subsequent reactivations. For this reason, the HHV-6 IgM test is not very useful for adults. Many physicians believe incorrectly that an adult with no IgM antibodies means that the infection must not be active.
Reactivated HHV-6B in Transplant Patients. Transplant patients who are given drugs to suppress their immune systems after a transplant often get reactivated viral infections. The virus typically reactivates in the lungs or organ tissue and sometimes in the brain tissue causing CNS dysfunction including depression and cognitive problems. In these acute reactivations, HHV-6B DNA can often be detected transiently by PCR in the whole blood and less frequently in the serum. Transplant physicians use qualitative PCR in the serum or quantitative PCR in whole blood to look for reactivation in these patients (Gautheret-Dejean 2002). The variant detected is almost always variant B (>97%), which is different from other conditions such as MS where the variant detected is primarily variant A (Alvarez-Lafuente 2004). Antibody tests are not as useful because they may take weeks to show a change in titer.
Quantitative PCR testing. Since so many healthy individuals have detectable levels of latent virus in their white blood cells, PCR DNA tests of whole blood are not useful unless the test is quantitative, and the absolute level of virus can be compared to a healthy population (Gautheret-Dejean 2002). When the virus is found in the serum or plasma it is considered a sign of active infection. However, HHV-6 and EBV are very “cell associated” which means there is very little free virus spilled in the serum, even in an active infection. This is because the virus is spread from cell-to-cell through the cell walls (Zhen 2005). This is in contrast to other infections where free virus leaves the cell, enters the plasma and then infects another cell. Labs that do quantitative PCR for HHV-6 include Viracor and Focus Diagnostics Laboratory. Viracor also includes variant typing.
Qualitative PCR testing. Qualitative PCR tests are useful if done on serum or plasma and the detection of DNA in serum or plasma is considered evidence of active infection (Secchierio 1995). Tests done on whole blood are not useful for detecting active infection because there is so much latent virus in the cells of healthy individuals. This makes it impossible to differentiate latent from active virus in cells using whole blood.
Variant Typing. HHV-6B has been associated with febrile seizures, status epileptics and mesial temporal lobe epilepsy. HHV-6A has been associated with MS and chronic fatigue syndrome, and may be more prevalent in patients with HIV. Both variants have been associated with encephalitis. The labs that are able to type variant A vs. variant B are: Viracor, Focus Diagnostics, Wisconsin Viral Research and Redlabs.
Nested PCR. Nested PCR is a technique that “amplifies” a traditional PCR. It is not available at any of the large commercial laboratories due to its expense and the difficulty with false positives. Unless extreme measures are taken, false positive results due to contamination can be a problem. Two specialty commercial labs in the US currently offer nested PCR on serum. A positive result in serum or plasma is considered an active infection. However repeat testing or a procedure called “sequencing the band” is advised to rule out a false positive. A nested PCR on whole blood can be useful to differentiate A variant vs. B variant, but it cannot differentiate between latent and active virus because so many healthy persons have latent virus in their white blood cells.Wisconsin Viral Research and Redlabs do nested PCR on serum; Redlabs also does nested PCR on whole blood for typing the variant. It is easier to find the virus in whole blood than in serum, so this is useful for variant typing, but a positive nested PCR result in whole blood does not indicate active infection since many healthy individuals have latent virus in the cells that is detectable by nested PCR.
Rapid Culture. HHV-6 is notoriously difficult to culture and will not replicate unless the virus is stimulated with chemicals. It is also time consuming and expensive. For this reason, no hospitals or large commercial laboratories offer a culture assay. There is one commercial lab in the US, Wisconsin Viral Research, that offers a rapid culture test. A positive result demonstrates that the patient has virus in the cells, and it can determine the variant, but this test cannot tell a patient whether that virus was active or latent before it was stimulated in culture. Many healthy individuals have latent virus in their cells that will activate when stimulated with chemicals.
- Reverse Transcriptase (messenger RNA) PCR. This technique of examining samples for messenger RNA is generally accepted as a gold standard for detecting active, vs. latent infection. It has been used at several academic and research laboratories, but no commercial laboratory, currently offers the test for diagnostic purposes Sensitivity can be an issue because messenger RNA can be degraded quickly if the RNA is not stabilized with a stabilizing solution immediately after the blood sample is drawn. However, recent studies show potential (Pradeau 2006).
- Antigenemia. Detection of viral protein in the peripheral blood or in biopsy tissue by putting infected cells on slides and then staining them with specific monoclonal antibodies. This technique can determine the variant type and is semi-quantitative. It has been used in Finland to monitor transplant patients undergoing immunosuppression (Loginov 2006). Kits for conducting HHV-6 Immunofluorescence Assay (IFA) are available commercially through BIOCELL Diagnostics, Inc, and others.
- Immunohistochemistry (IHC). This technique is useful in analyzing biopsy or autopsy sections. Cells from the tissue are stained with HHV-6 monoclonal antibodies to determine which proteins were expressed at the time the biopsy was taken. Pathologists can also differentiate A variant from B variant by using variant specific antibodies.
- Fluorescent In situ hybridization (FISH). Detection of PCR-amplified DNA in an intact cell by hybridization after PCR by using a labeled probe. This technique has been used in the study of MS and tissues; quantitative analysis of viral mRNA expression indicates that both normal white matter and lesions in patients had significantly higher HHV-6 expression suggesting a role in pathogenesis (Opsahl 2005).
- Early Antigen Assay. The most sensitive antibody test for active infection was available briefly to researchers in the late 90’s and has never made it into commercial production due to the difficulties in production; it was based on antibodies to the early antigen (EA) or a protein produced by the virus in the early stages of infection. Since the EA antibodies disappear rapidly after active infection is over (unlike the “late antigen” antibodies that persist for much longer), they may be a better predictor of active infection. Studies of HHV-6 in CFS and MS that looked for antibodies to IgM and IgG early antigen (the antibody to a protein produced during active infection) 50-70% of these patient groups had active infections. The HHV-6 Foundation is collaborating with a team of scientists to try to get this test on the market and available to researchers and diagnostic labs.
- Loop-mediated isothermal amplification (LAMP). This is a novel technique developed in Japan that allows rapid detection of HHV-6 DNA using simple and relatively inexpensive equipment already owned by most hospitals, making it suitable for rapid diagnosis of roseola (Ihira 2007).
- Avidity. Antibodies that are recently produced have low avidity while those produced in the past have high avidity. Thus the avidity of antibodies can be used to determine the stage of infection. Katherine Ward in the U.K has used avidity analysis of HHV-6 antibodies in the identification of primary vs. latent infection in several studies (Ward 2005).
- Western Blot. This technique can be used to identify early and late viral antibodies and is quite sensitive. However, it is not practical for routine testing as it is labor intensive.
- Antibody & Antigen Capture with Electrochemiluminescence (ECL). Steve Jacobson at the NINDS has developed a novel method to differentiate active from latent virus using this new technology. He has been able to improve sensitivity significantly over that of the nested PCR.
- PCR-EIA. Yi-Wei Tang at Vanderbilt University has developed a method to improve sensitivity by using a two step PCR procedure that would combine a magnetic based nucleic acid extraction method and PCR amplification with a colormetric microliter plate identification that allows him to reach an analytical sensitivity of <50 copies/ml for detection of HHV-6. This assay can also differentiate A from B variant. Dr. Tang is in the process of developing this assay for clinical use.
NOTE: This page has been reviewed by Dharam Ablashi, Scientific Director of the HHV-6 Foundation and co-discoverer of HHV-6, as well as the following scientists: Louis Flamand, Ph.D., Rheumatology and Immunology Research Center, Quebec, Canada, Gary Pearson, Ph.D., Georgetown University School of Medicine (Retired), Washington, DC
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Direct detection of human herpesvirus 6 DNA in serum by the loop-mediated isothermal amplification method.
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The natural history and laboratory diagnosis of human herpesviruses-6 and -7 infections in the immunocompetent.
Learn more about the different tests available:
IgG & IgM Antibody -IgG titres at 1:320 or above may mean an active infection)
-normal IgM does not rule out a chronic reactivation
• Quantitative PCR on serum (can’t detect most low-grade CNS infections)
• Nested PCR on serum (more sensitive, but still can’t pick up many CNS infections)