Post by ArgyrosfeniX on Nov 8, 2007 2:58:15 GMT 10
[For those who wish to read the original text: www.vicd.info/testing.html ]
TESTING
TESTING FOR HHV-6 & EBV
Viruses are difficult to detect when they are chronic. It is very difficult to detect chronic infections of HHV-6 and EBV. One reason is that 90% of us have been exposed to these viruses by early childhood and the viruses remain latent in our cells. Therefore, in order to differentiate active from latent infection, tests must be done in the serum (the clear liquid portion of the blood) on the theory that in actively replicating virus, viral particles will leave the cell and spill into the plasma. The problem is that, unlike most viruses, HHV-6 and EBV are very “cell associated” which means that the virus rarely leaves the cell. In fact, with HHV-6, transmission is largely cell-to-cell or directly through the cell walls. The net result is that it becomes very difficult to detect active infection because there are very few viral particles in the serum.
The other reason chronic infections are difficult to detect is that these viruses (especially HHV-6A) migrate to the central nervous system and other organs and away from the blood. For example, HHV-6A has been found to persist in the spinal fluid long after it has disappeared from the plasma.
This means that indirect signs of the virus (antibodies to the viral proteins) become more important and antibody assays are more sensitive than molecular assays (PCR) for differentiating latent from active chronic infections.
PCR tests for HHV-6 & EBV.
These tests are currently not useful for detecting most cases of Virus Induced CNS Dysfunction. While PCR tests can easily detect primary HHV-6B roseola infections and acute mononucleosis, they are not sensitive enough to detect chronic, persistent central nervous system infections. A negative PCR test done in serum or plasma is meaningless because the test itself is hopelessly insensitive. At the same time, a positive PCR test on whole blood is also meaningless until a threshold is established that can distinguish between healthy controls and patients. Since 90% of us have latent virus in the cells, a whole blood test will detect latent virus in the cells of healthy controls as well as patients. Many healthy individuals, especially young adults, have detectable levels of latent virus in their whole blood.
Only a positive test in the plasma or serum is a significant result, although extremely rare. Not one of Montoya’s patients who benefited from antiviral treatment (and were subsequently determined to have active infections) was positive by PCR. A high positive result for HHV-6 may mean that the patient has chromosomally integrated HHV-6. This is a rare form of HHV-6 that is inherited and was found in 0.2% of the population in one large study in Japan.
HHV-6 Antibody Tests
IgG Antibodies.
These antibody levels indicate that a person has had an infection at some point in the past; at high levels they can suggest (but not prove) that the virus is active. Much more research is needed on this question but in one study of HHV-6 in CFS patients, 89% of the patients with HHV-6 IgG antibody titer of 1:320 and above were found to have active infections by culture (Wagner 1996). Results vary by laboratory, but at most commercial labs, healthy adults have titers of 1:40 to 1:160. Sometimes a different ELISA assay is used and the results are reported as an index. These index scores can also vary by laboratory depending on the manufacturer of the kit. Young adults may have unusually high antibodies due to reactivation during mononucleosis.
IgM Antibodies.
This is not a very useful test. Although it is worth testing once, this result is rarely positive in patients with chronic viral infections. Typically IgM titers persist only for a few weeks after the primary infection. Just because the IgM is normal does not mean that you don’t have an active infection; many clinicians are not aware of this fact.
EBV Antibody Tests
Antibody testing for EBV is more complicated. There are three different types of EBV assays offered at commercial laboratories. These antigens are the viral capsid antigen (VCA), the early antigen (EA), and the EBV nuclear antigen (EBNA). In addition, differentiation of immunoglobulin G and M subclasses to the VCA can often be helpful for confirmation. The optimal combination of EBV serologic testing consists of the antibody titers to all four markers: IgM and IgG to the VCA, antibody to the EA
Viral Capsid Antibodies (VCA).
IgG antibodies to the viral capsid antigen develop 2 to 4 weeks after onset of the initial and then persist for years, at gradually declining levels. Drs. Montoya and Kogelnik at Stanford have found that patients with elevated antibodies to VCA IgG and HHV-6 antibodies respond to antivirals, and the VCA titers dropped significantly with treatment, suggesting that elevated VCA titers represent active infection. This test is not definitive since many healthy adults have relatively high antibodies as well. A high titer in a 45 year old is far more significant than the same titer in a 20 year old. IgM antibody tests are not very useful since they are typically found only in primary or initial infections, not chronic or reactivated infection.
Early Antigen (EA) Antibodies. IgG.
The early antigen (EA) antibody appears during active replication phase and generally falls to low levels after 3 to 6 months. Most healthy adults have very low levels of early antigen; higher levels however can be a sensitive marker of active infection in chronic disease. There is consensus that a titer of 1:640 is indicative of active disease, and that a titer of <1: 80 suggests there is a far smaller chance of an active infection. Results vary by laboratory and much more testing needs to be done to establish cutoff values, but the early antigen IgG antibody test remains our best clue for active infection. Again, IgM elevations are typically found only in a primary infection, not a reactivated infection, so the IgM test is usually not very meaningful for chronic infections.
Epstein-Barr Nuclear Antigen Antibodies (EBNA).
Antibody to EBNA is not seen in the acute phase, but slowly appears 2 to 4 months after onset, and persists for life. Most physicians use of this test is to determine if an EBV infection is the initial infection or a secondary/ reactivated infection. A low EBNA in the presence of a high VCA titer suggests an immune deficiency. For reactivated infections, only the absolute level of the IgG titer usually significant.
Interpreting Results from Commercial Laboratories
There are several commercial laboratories that will test for HHV-6 and EBV. However, not all labs use the same metric. For example, Focus Diagnostics and Specialty Laboratories use an IFA method with results reported as titers (1:80, 1:160 etc). Other labs such as Mayo Clinic, Quest and Labcorp use the ELISA method and report with an index. There is no standard data publicly available that would allow patients to compare IFA and ELISA scores. The Stanford group currently conducting a trial of Valcyte for these viruses uses Focus Diagnostics as their reference laboratory because they are familiar with their scale and can interpret the results.
The best way to interpret the results from your laboratory is to ask your doctor to find out median and range values at the laboratory for controls or blood donors. If your result is in the top quartile you are more likely to have an infection, but there is not way to know for certain. Patients who are immunosuppressed and have a low IgG may show up with low antibody levels in spite of active disease. If the IgG is low normal or below normal, then the HHV-6 and EBV antibody test results may also be suppressed. Similarly, some patients with very high IgG may have high EBV and HHV-6 antibody levels that do not indicate active disease. These considerations need to be evaluated by a knowledgeable physician.
TESTING
TESTING FOR HHV-6 & EBV
Viruses are difficult to detect when they are chronic. It is very difficult to detect chronic infections of HHV-6 and EBV. One reason is that 90% of us have been exposed to these viruses by early childhood and the viruses remain latent in our cells. Therefore, in order to differentiate active from latent infection, tests must be done in the serum (the clear liquid portion of the blood) on the theory that in actively replicating virus, viral particles will leave the cell and spill into the plasma. The problem is that, unlike most viruses, HHV-6 and EBV are very “cell associated” which means that the virus rarely leaves the cell. In fact, with HHV-6, transmission is largely cell-to-cell or directly through the cell walls. The net result is that it becomes very difficult to detect active infection because there are very few viral particles in the serum.
The other reason chronic infections are difficult to detect is that these viruses (especially HHV-6A) migrate to the central nervous system and other organs and away from the blood. For example, HHV-6A has been found to persist in the spinal fluid long after it has disappeared from the plasma.
This means that indirect signs of the virus (antibodies to the viral proteins) become more important and antibody assays are more sensitive than molecular assays (PCR) for differentiating latent from active chronic infections.
PCR tests for HHV-6 & EBV.
These tests are currently not useful for detecting most cases of Virus Induced CNS Dysfunction. While PCR tests can easily detect primary HHV-6B roseola infections and acute mononucleosis, they are not sensitive enough to detect chronic, persistent central nervous system infections. A negative PCR test done in serum or plasma is meaningless because the test itself is hopelessly insensitive. At the same time, a positive PCR test on whole blood is also meaningless until a threshold is established that can distinguish between healthy controls and patients. Since 90% of us have latent virus in the cells, a whole blood test will detect latent virus in the cells of healthy controls as well as patients. Many healthy individuals, especially young adults, have detectable levels of latent virus in their whole blood.
Only a positive test in the plasma or serum is a significant result, although extremely rare. Not one of Montoya’s patients who benefited from antiviral treatment (and were subsequently determined to have active infections) was positive by PCR. A high positive result for HHV-6 may mean that the patient has chromosomally integrated HHV-6. This is a rare form of HHV-6 that is inherited and was found in 0.2% of the population in one large study in Japan.
HHV-6 Antibody Tests
IgG Antibodies.
These antibody levels indicate that a person has had an infection at some point in the past; at high levels they can suggest (but not prove) that the virus is active. Much more research is needed on this question but in one study of HHV-6 in CFS patients, 89% of the patients with HHV-6 IgG antibody titer of 1:320 and above were found to have active infections by culture (Wagner 1996). Results vary by laboratory, but at most commercial labs, healthy adults have titers of 1:40 to 1:160. Sometimes a different ELISA assay is used and the results are reported as an index. These index scores can also vary by laboratory depending on the manufacturer of the kit. Young adults may have unusually high antibodies due to reactivation during mononucleosis.
IgM Antibodies.
This is not a very useful test. Although it is worth testing once, this result is rarely positive in patients with chronic viral infections. Typically IgM titers persist only for a few weeks after the primary infection. Just because the IgM is normal does not mean that you don’t have an active infection; many clinicians are not aware of this fact.
EBV Antibody Tests
Antibody testing for EBV is more complicated. There are three different types of EBV assays offered at commercial laboratories. These antigens are the viral capsid antigen (VCA), the early antigen (EA), and the EBV nuclear antigen (EBNA). In addition, differentiation of immunoglobulin G and M subclasses to the VCA can often be helpful for confirmation. The optimal combination of EBV serologic testing consists of the antibody titers to all four markers: IgM and IgG to the VCA, antibody to the EA
Viral Capsid Antibodies (VCA).
IgG antibodies to the viral capsid antigen develop 2 to 4 weeks after onset of the initial and then persist for years, at gradually declining levels. Drs. Montoya and Kogelnik at Stanford have found that patients with elevated antibodies to VCA IgG and HHV-6 antibodies respond to antivirals, and the VCA titers dropped significantly with treatment, suggesting that elevated VCA titers represent active infection. This test is not definitive since many healthy adults have relatively high antibodies as well. A high titer in a 45 year old is far more significant than the same titer in a 20 year old. IgM antibody tests are not very useful since they are typically found only in primary or initial infections, not chronic or reactivated infection.
Early Antigen (EA) Antibodies. IgG.
The early antigen (EA) antibody appears during active replication phase and generally falls to low levels after 3 to 6 months. Most healthy adults have very low levels of early antigen; higher levels however can be a sensitive marker of active infection in chronic disease. There is consensus that a titer of 1:640 is indicative of active disease, and that a titer of <1: 80 suggests there is a far smaller chance of an active infection. Results vary by laboratory and much more testing needs to be done to establish cutoff values, but the early antigen IgG antibody test remains our best clue for active infection. Again, IgM elevations are typically found only in a primary infection, not a reactivated infection, so the IgM test is usually not very meaningful for chronic infections.
Epstein-Barr Nuclear Antigen Antibodies (EBNA).
Antibody to EBNA is not seen in the acute phase, but slowly appears 2 to 4 months after onset, and persists for life. Most physicians use of this test is to determine if an EBV infection is the initial infection or a secondary/ reactivated infection. A low EBNA in the presence of a high VCA titer suggests an immune deficiency. For reactivated infections, only the absolute level of the IgG titer usually significant.
Interpreting Results from Commercial Laboratories
There are several commercial laboratories that will test for HHV-6 and EBV. However, not all labs use the same metric. For example, Focus Diagnostics and Specialty Laboratories use an IFA method with results reported as titers (1:80, 1:160 etc). Other labs such as Mayo Clinic, Quest and Labcorp use the ELISA method and report with an index. There is no standard data publicly available that would allow patients to compare IFA and ELISA scores. The Stanford group currently conducting a trial of Valcyte for these viruses uses Focus Diagnostics as their reference laboratory because they are familiar with their scale and can interpret the results.
The best way to interpret the results from your laboratory is to ask your doctor to find out median and range values at the laboratory for controls or blood donors. If your result is in the top quartile you are more likely to have an infection, but there is not way to know for certain. Patients who are immunosuppressed and have a low IgG may show up with low antibody levels in spite of active disease. If the IgG is low normal or below normal, then the HHV-6 and EBV antibody test results may also be suppressed. Similarly, some patients with very high IgG may have high EBV and HHV-6 antibody levels that do not indicate active disease. These considerations need to be evaluated by a knowledgeable physician.
