My Cooperative Diagnostics Test

January 4, 2010

Posted by Cort Johnson

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I received my test result back from Cooperative Diagnostics. I was, like everyone else who has taken the poll thus far, tested negative. I was able to get the test done because I was part of an XMRV study CD is doing.

Given the results of the Imperial College tests the results are illuminating because both groups appear to be using a similar strategy; ignoring the sequences the WPI tested for and instead focusing on sequences they know (or believe they know) are there. This is certainly a legitimate way to test for ‘XMRV’ as we know it. Whether its a good test for what the WPI found is another question.

This is what they said.

Quote:
The two strains of XMRV found by Lombardi et al in chronic fatigue syndrome that were sequenced had roughly 6 bases that were different from prostate cancer XMRV. We used these strains in addition to prostate cancer strains and several strains of murine leukemia viruses (MLV) in the design of our test. In contrast to published tests for XMRV that have been designed in highly variable DNA regions in the MLV family … we chose to design our initial test to use the pol gene so it could detect all MLV species, including XMRV. This means we tested a DNA sequence that has not changed in hundreds or even thousands of years on an evolutionary scale in the MLV family. This test would be more apt to detect XMRV than any other test used. Therefore, we would likely have more positives than previously reported in PCR tets for CFS when XMRV is actually present.

We also used a superior test sensitivity, enabling the detection of 10 to 25 times few viruses in a blood sample than previously reported tests for XMRV. This was proven when we were able to detect XMRV in 1 infected 22Rv1 cell from a commercially available prostate cancer cell line in a background of 2.5 ug of extracted DNA from blood. We also designed a second test with the same performance characteristics of the first. This allowed us to also evaluate the env genes, creating an extra confirmation step in our testing.

A Laymen’s Assessment of the XMRV Situation (Be very wary of Laymen’s Assessments :) )

Replication vs Validation Studies – the Imperial College and CD tests are validation ‘studies’ not replication studies and there’s a big difference between them. Replication studies follow the first studies techniques to the letter.They’re using the same processes to find the same genetic sequences that the original study found.

Validation studies, on the other hand, simply try to validate the first studies assertion – in this case the WPI’s assertion that they found XMRV. They’re not trying to replicate the original techniques; in fact, it’s better if they don’t. Virus hunters can use any number of techniques to find a virus; if a virus is there any of these techniques should be able to find it. Thus, they’re often using different kinds of PCR tests and are often looking for different genetic sequences than in the original study. When Imperial College or CD attempts to detect the virus using a different means than the WPI does, its trying to validate their assertion that they’ve found XMRV – not find the same genetic sequences WPI did.

Variable Genetic Sequences – It’s intriguing that CD states that the tests thus far developed for XMRV focused on ‘highly variable’ regions of the DNA (we heard this from another researcher). The WPI tested ‘gag’ sequences which, as I remember, are from the coat of the virus. Since this part of the virus is constantly interacting with the environment it’s not surprising that it might be variable.

Cooperative Diagnostics states, though, that ALL XMRV tests, not just the WPI test, have focused on variable regions of the genome. The upside to that approach appears to be that these tests can differentiate between different types of murine retroviruses; ie they can pick out XMRV from other murine retroviruses. The downside is that because they are variable they could conceivably differ from strain to strain and patient to patient – they might not show up in some tests.

Cooperative Diagnostics Search for XMRV – Cooperative Diagnostics is taking a very conservative approach by focusing on highly stable regions which are shared with other retroviruses. These were gene sequences in the pol gene that are believed to have been stable for ‘hundreds or even thousands’ of years. Since several viruses share these regions they expected higher levels of false positives ; ie if anyone carried those viruses they would show as testing positive for ‘XMRV’.

  • Highly Sensitive ]- They also developed the most highly sensitive test yet. They demonstrated the test was able to identify even very low levels of the XMRV virus found in standard preparations of prostate cancer cells.
  • Another Gene Sequence - They also looked at at the env gene sequence found in the XMRV. This is the sequence that the Cleveland Clinic – in small number of samples – demonstrated was in the WPI samples – and helped convince them the WPI had found XMRV. Because Cooperative Diagnostics is looking two stable sequences they appear to be providing the most comprehensive test for XMRV commercially available.
  • At least in my sample and the 12 other people who voted in the poll none of us tested positive for these sequences; ie we did not have XMRV.

Not XMRV? Leaving aside different patient cohorts, geographical locations, and other confounding factors the Imperial College and CD findings thus far refute the WPI’s claims that they found XMRV. Instead they suggest either that the WPI found a different type of XMRV or some gene sequences that are similar to those found in XMRV but not the actual bug itself.

The problem is that the WPI fully sequenced two strains of the bug that they found – and they both came out to be XMRV! This suggests that they did find XMRV but it doesn’t give us any clue as to why XMRV did not show up for Imperial College or thus far for CD.

The Weak Link?*- it could be that XMRV is more variable than researchers know; that they’re looking for gene sequences that are slightly different than the standard XMRV sequence. That standard XMRV sample, after all, must have come from prostate cancer studies from a small probably localized set of patients and may not reflect what XMRV looks like in the rest of the population. The problem with this argument is that Cooperative Diagnostics tried to account for this by looking for a genetic sequence that they don’t believe changes between several viruses alone within a virus – and didn’t find it.*Still, this is why a standardized test that takes genetic variation in the virus into account is so important.

The BATTLE

  • Evidence For the WPI’s Assertion: they identified a ‘gag’ sequence that appeared to come from XMRV. The Cleveland clinic confirmed that seven of 11 patients also had an ‘env’ sequence. Importantly, the WPI sequenced 2 1/2 strains of the bug from their patients and it turned out to be XMRV. The weak point here was that the ‘gag’ sequence is variable in XMRV but they have that full sequencing to back them up – strong evidence in their favor. Plus while its not evidence for XMRV the WPI did find something that was able, to jump from an infected cell to an uninfected cell in a culture test; this doesn’t appear to be a fragment of something – it appears to be ‘something’.
  • Evidence Against the WPI’s Assertion: One study and the poll results from another labs test that looked for very stable genetic sequences in XMRV have not found them; ie no XMRV.

Contamination - Its also possible that the WPI is picking up an endogenous retrovirus that has some similar genetic sequences to XMRV. This is the ‘contamination’ theory which no one really seems to take seriously. For one thing the WPI fully sequenced two samples of XMRV and parts of third and they had very close genetic similarity to XMRV.

A Conundrum – There doesn’t appear to be any good answer – we’re at a conundrum. Short of someone having made a technical error its hard for this layman to understand these conflicting results. Perhaps the best way to leave it is that XMRV is a newly discovered retrovirus and much remains to be learned about it.

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