Viral Testing Complexities

Introduction
The main roadmap section on viral testing is here. This page instead focuses on certain complexities and pitfalls involved in viral testing, and is more advanced reading.

The main viruses linked to ME/CFS are: certain enteroviruses (namely coxsackievirus B and echovirus), certain herpesviruses (specifically Epstein-Barr virus, HHV-6, cytomegalovirus, varicella zoster virus), and parvovirus B19. And since the COVID pandemic, it appears that the SARS-CoV-2 coronavirus can trigger ME/CFS as well. The bacterium Chlamydia pneumoniae is also associated with ME/CFS.

Most viruses associated with ME/CFS are commonly found in the general population: Epstein-Barr virus, for example, is found in 95% of adults (this virus is typically acquired when you are in your teens or twenties). But in the general population, these viral infections are usually fully suppressed and controlled by the immune system, so they become largely inactive and dormant, and are classified as past infections or latent infections.

However, in ME/CFS patients, evidence suggests these common infections are active rather than dormant, and may thus be playing a causal role in this disease. 



PCR Viral Testing
PCR blood tests for enterovirus and herpesvirus are generally not useful in ME/CFS. PCR directly detects the presence of a pathogen in the sample tested (eg, in a blood sample or tissue sample). However, PCR blood tests for viruses typically come out as negative in ME/CFS.

This negative result might be explained by the fact that ME/CFS is linked to non-cytolytic enterovirus infections, and to partial reactivation of HHV-6, and might involve abortive herpesvirus infections. These are all intracellular infections which remain inside human cells within the tissues, and do not produce many new viral particles. So there may be very few viral particles in the blood, and this could be why PCR is often negative in ME/CFS.

For ME/CFS involving chronic enterovirus infection, Dr John Chia found even with the most sensitive PCR blood tests, enterovirus was only detected around 30% of the time (in severe bedbound patients this virus was detected 70% of the time, but in milder patients only 12% of the time).1 So Dr Chia's research showed that blood PCR is not a reliable method of detecting enterovirus in ME/CFS patients.

In the rare case of ME/CFS linked to parvovirus B19, here the virus can actually be found in the blood, so blood PCR is useful for parvovirus ME/CFS.

Generally ME/CFS specialist doctors will use antibody blood tests, rather than blood PCR tests. Antibody tests measure the immune response to a pathogen, in contrast to PCR which detects the pathogen itself. The advantage of antibody tests is that even if the pathogen is hiding away in the tissues as an intracellular infection, if the infection is extensive enough, tests may detect an elevated antibody response.



Viral Antibody Testing
Viral antibody blood tests are used by ME/CFS doctors, but ME/CFS doctors interpret the results of these tests differently to the way regular doctors and infectious disease specialists interpret them, as will now be explained.
 
IgM and IgG antibodies are some of the main players for fighting infection. When you first catch an infectious pathogen, within days of the acute infection starting, IgM antibodies which specifically target that pathogen are created by the immune system.

As the acute infection is slowly brought under control by the immune response, and the pathogen is either completely eliminated from the body or has been reduced to an inactive dormant state (where the pathogen is hiding in a latent state inside cells), levels of IgM go down to zero, and at the same time, IgG antibodies start being produced which target that pathogen.

These IgG antibodies remain mildly raised for life, providing lifelong protection against reinfection with that particular pathogen. And for viruses which cannot be fully eliminated from the the body and hide in cells in a latent state, this mildly elevated IgG helps stop the virus from reactivating from latency.

Thus in a blood test, finding high IgM and low IgG antibody levels indicates a new acute infection. Finding low IgM and mildly raised IgG indicates a past infection that has been brought under control. And if both IgM and IgG are raised, this indicates a past infection that has reactivated from latency.

So these are the normal general rules used by infectious disease (ID) doctors for interpreting antibody levels, and they are summarized in the follow table.

Normal Rules for Interpreting Antibody Levels
IgM IgG Interpretation
High Low A new acute infection that has just been caught.
Low Mildly Raised A probable past infection, either inactive (in a latent state) or completely eliminated from the body and cured.
High High A previous infection that has reactivated from a latent state.

High IgM indicates a normal active infection as we normally understand them: an infection that is producing lots of new viral particles, either as an initial acute infection, or as a flare up and reactivation of an old infection. Such infections with high IgM typically show up positive if you test the blood for the presence of the actual virus by PCR.

In the case of ME/CFS and chronic Lyme disease, however, IgM is typically low, but IgG is usually substantially raised (not just mildly raised) on a long-term basis.

To an ID specialist, finding low IgM and chronically high IgG is curious, but they do not see it as an active infection (especially because blood PCR tests are usually negative, as you do not get much virus in the blood in ME/CFS).

But to an ME/CFS specialist, low IgM with chronically high IgG signifies there may be ongoing active infection in the tissues — not a normal active infection which produces lots of viral particles, but a chronic active infection in the form of a non-cytolytic enterovirus infection, an abortive herpesvirus infection, or a partial reactivation of a virus.

So in this way, ME/CFS specialists interpret antibody blood tests differently to ID specialists. Thus if you are an ME/CFS patient and your blood tests indicate low IgM, but with high IgG, an ID specialist will tell you that you do not have an active infection. But an ME/CFS specialist will view your high IgG as evidence of an ongoing active infection somewhere in the body.

So in summary, ME/CFS specialists use a fourth rule (in addition to the three rules in the table above), which is the following:

Extra Rule for Interpreting Antibody Levels in ME/CFS
IgM IgG Interpretation
Low High Chronic low-level active infection in the tissues.

This fourth rule is a controversial though, as some researchers believe the high IgG antibody levels in ME/CFS are of no significance, and do not believe ME/CFS is caused by ongoing infection. However in the case of enterovirus ME/CFS, there is considerable evidence for chronic active low-level infection in the tissues, which are the likely cause of these chronically high IgG.



Threshold Antibody Level for Diagnosing Active Infection
We have established that ME/CFS specialists view persistently high IgG as evidence for chronic active infection. Thus in order to determine which chronic active viral infections may be underpinning your ME/CFS, you will generally check for high IgG antibody levels in viral antibody tests.

But what exactly do we mean by high IgG antibody levels? How do we know if the IgG levels in our viral test lab reports are high, or merely mildly raised? What threshold value indicates high IgG, and in turn suggests active infection?

The typical laboratory report usually does not tell you if your antibody levels are high. The reference range on the lab report only relates to whether antibodies are absent or present. The lab report will specify a certain reference value for a negative result — for example the lab report may say: negative < 3.0 U/ml — and if your antibody levels are below that value, then you do not have those antibodies present in the blood. So this reference value only tell you if antibodies are absent or present; it does not indicate if your antibody levels are high.

Usually the experience and expertise of an ME/CFS specialist doctor will enable them to diagnose high IgG antibody levels. Some ME/CFS doctors like Dr Chia have performed in-house studies to properly calibrate the threshold for high IgG antibody levels, and have publicized the threshold value. But other ME/CFS doctors have not publicized the threshold values they use.

The proper method for calibrating the threshold for high IgG involves measuring the antibody levels in a group of ME/CFS patients, and also in a group of healthy people, and comparing the average IgG antibody levels for each of these two groups. The ME/CFS patients' average antibody levels will be high, and the healthy peoples' average antibody levels will be substantially lower. You then set the threshold value for high IgG antibody levels somewhere between these two averages. Dr John Chia used precisely this method to determine the threshold for high antibody levels in the coxsackievirus B and echovirus ARUP Lab tests (see this video at 10:34).

Alternatively some doctors like Dr Daniel Dantini set the threshold value for high IgG as being 4 times the average IgG antibody level of the group of healthy people.1 But unless you have information on the average antibody levels in healthy people for a given test (and your lab report will not tell you this), this method is of little help.

So, apart from the coxsackievirus B and echovirus ARUP Lab tests, for all other lab tests we do not have this threshold value rigorously calibrated and made available to ME/CFS patients. Thus for ME/CFS patients who are unable to see an ME/CFS specialist doctor, it is hard to get an accurate diagnosis for high IgG. As patients we can only guess at what constitutes high IgG antibody levels.

However, we can make an educated guess based on Dr Chia's calibration of the ARUP Lab coxsackievirus B and echovirus tests: Dr Chia determined that antibody levels in the region of 1:160 to 1:320 and higher are the threshold for chronic active infection. Now ARUP Lab state that the reference range for negative in these tests is 1:10. So if you divide 160 by 10, you get 16. So here the threshold antibody level Dr Chia uses for chronic active infection is 16 times higher than the reference antibody level for negative.

So if we assume we can extrapolate to other tests, we can state an approximate rule: IgG antibody levels are deemed high if they are at least 16 times the reference threshold for negative. However, note that this factor of 16 has not been validated by any ME/CFS specialist doctors, and does not have much of a scientific basis, but at least provides something to work with in the absence of guidance from an ME/CFS doctor.



Special Note on Enterovirus Testing in ME/CFS
When testing for the chronic enteroviruses infections (coxsackievirus B and echovirus) found in the tissues of ME/CFS patients, there are some special considerations: Dr John Chia discovered that only the neutralization method of antibody testing, which is the gold standard in terms of sensitivity, is able to detect chronic enterovirus infections in ME/CFS patients. Dr Chia uses the ARUP Lab antibody tests for coxsackievirus B and echovirus, which employ the sensitive micro-neutralization method (details here).

Other methods of antibody testing such as EIA, ELISA, IFA may not be sensitive enough to detect chronic enterovirus. And CFT is definitely not sensitive enough to detect chronic enterovirus infections. Thus for reliable chronic coxsackievirus B and echovirus testing in ME/CFS patients, a neutralization antibody test is required.

For the herpesviruses linked to ME/CFS (EBV, HHV-6 and CMV), the EIA, ELISA or IFA methods of antibody testing are fine, and there is no need to use the neutralization method.

Here are the various methods of antibody testing, in order of highest sensitivity:
Note in the neutralization method, results for IgM and IgG are usually pooled together to give a single figure for antibody level. Whereas other methods may provide two figures, one for IgM and another for IgG.

As an alternative to antibody blood tests, chronic enterovirus infections in the tissues of ME/CFS patients can be detected using a sample of the infected tissue itself. Dr Chia's lab offers testing of stomach biopsy tissue samples by means of immunoperoxidase staining (a gastroenterologist is required to take a sample of stomach tissue using an endoscope, which is then sent to Dr Chia's lab).



Antiviral Treatment of Herpesvirus ME/CFS
Antiviral treatment of ME/CFS involving EBV, HHV-6 or CMV takes a long time. In Dr Martin Lerner's clinical trials using Valtrex or Famvir to treat ME/CFS associated with EBV, and Valcyte to treat ME/CFS where HHV-6 and/or CMV infection are involved, it would take 3½ months before the first signs of improvement in symptoms would appear, and around 1 or 2 years for the full benefits of antiviral therapy to manifest.

Why does it take so long to treat ME/CFS associated with these herpesviruses, when under normal circumstances, antivirals are able to bring infections under control in a matter of weeks? The answer may lie in the unusual type of herpesviruses infections Dr Lerner theorized cause ME/CFS, namely abortive herpesvirus infections. A normal productive viral infection produces new viral particles, whereas an abortive infection does not create viral particles, but an abortive infection can nevertheless chronically persist within cells.

Antivirals such as Valtrex and Valcyte do not have any direct antiviral effect against abortive herpesvirus infections, but are efficacious against productive herpesvirus infections. Dr Lerner hypothesized that in ME/CFS there is a low-level productive infection alongside the abortive infection, and that viral particles from the productive infection constantly re-seed the abortive infection, so the abortive infection cannot be cleared.1

When you take herpesvirus antivirals, they target the productive infection, and so greatly reduce the number of viral particles produced. This stops the abortive infection from being re-seeded, so that eventually the immune system very slowly clears the abortive infection (or at least reduces the abortive infection). This according to Dr Lerner is why antivirals take such a long time to work in ME/CFS linked to EBV, HHV-6 and CMV. This is only a theory, but Lerner's ideas that ME/CFS may be caused by abortive rather than productive herpesvirus infections are currently being further researched by a team at Ohio State University.

The exception to this lengthy treatment time is ME/CFS caused by reactivation of varicella zoster virus (VZV) in the dorsal root ganglia next to the spine. This form of ME/CFS rapidly responds to antivirals such as Valtrex within weeks. But the difference is that VZV reactivation is a normal productive infection which produces viral particles, and Valtrex is effective against productive VZV. It is only abortive infections which Valtrex and Valcyte have no direct effect against.

In this video at 6:58, Dr Chia tells the story of an executive who became bedridden with severe ME/CFS. After 11 months in this bedridden state, Dr Chia noticed two little shingles blisters appearing on this patient, and thus suspected VZV reactivation. Reactivation of VZV is typically diagnosed just by the appeared of shingles blisters, rather than through viral blood tests. Dr Chia gave her some acyclovir to take daily, and within 3 weeks she was back to full-time work. That's how quickly VZV ME/CFS responds to antivirals.



Antiviral Treatment of Enterovirus ME/CFS
Antiviral or immunomodulatory treatment of enterovirus takes 2 or 3 months. Treatments such as oxymatrine, Epivir, tenofovir and interferon beta which target the enteroviruses found in ME/CFS (namely coxsackievirus B or echovirus) take around 2 or 3 months to work. It is the intracellular non-cytolytic form of enterovirus which is found in ME/CFS, an unusual form of the virus which lives inside cells, and which these treatments target.



The RNA Viruses Linked to ME/CFS
Interestingly, there are several positive-sense single-stranded RNA viruses (+ssRNA) which are linked to chronic fatiguing illnesses:

Enterovirus — known to cause non-cytolytic infections, and linked to ME/CFS
Dengue virus — known to cause non-cytolytic infections, and known to cause a chronic ME/CFS-like illness
Hepatitis C virus — known to cause non-cytolytic infections, and known to cause a chronic fatiguing illness

SARS-CoV-1 coronavirus — known to cause a chronic ME/CFS-like illness
SARS-CoV-2 coronavirus — known to cause a chronic ME/CFS-like illness (long COVID)

Ross River virus — linked to ME/CFS, and such alphaviruses are known to cause non-cytolytic infections
West Nile Virus — linked to ME/CFS