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New Paper - Gammaretroviruses - Maureen Hanson, David Bell

VillageLife

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Sensitivity of PCR Assays for Murine Gammaretroviruses and Mouse Contamination in Human Blood Samples


http://www.plosone.org/article/info...seases+(PLoS+ONE+Alerts:+Infectious+Diseases)

Li Ling Lee1, Lin Lin1, David S. Bell2, Susan Levine3, Maureen R. Hanson1*

Abstract
Gammaretroviruses related to murine leukemia virus (MLV) have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs) or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.
 

Bob

Senior Member
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Ah, the Maureen Hanson paper, at last!
Thanks for posting it VillageLife.
It's a nice long & complex paper for us all to read through!
I'll try and look through it tomorrow.
Will be interesting to find out exactly why she concluded that there wasn't any XMRV present in the blood.
I'm also very interested in reading her conclusions re the antibodies.
 

SOC

Senior Member
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7,849
I haven't read the paper, yet, but I want to thank these researchers for making clear, accurate, scientific statements about their conclusions
While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.

We've seen far too many conclusions lately that extrapolate far beyond the data -- the ones of the "We didn't find it, so it doesn't exist" ilk -- so it's refreshing to see sound research conclusions.
 
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Just read through this. It highlights what a complex and cutting-edge question this is; they used lots of different assays and did a whole lot of work, and they did initially find suggestive results with more positives in patients than controls, but then weren't able to reproduce those results using other assays, and in the end they weren't able to conclude they weren't contamination but also weren't able to identify the contamination, although they did find some evidence of some contamination.

A couple of quotes...


Like a number of other investigators, we have identified MLV-like gag sequences following PCR of nucleic acid samples from whole blood or PBMCs from human subjects. Nested PCR analysis of our initial batch of 30 samples resulted in a significant difference in frequency of gag PCR products between patients and controls; however, continued analysis failed to maintain this association.

So they too found higher levels in patients than in controls. It looks like 50% of the 10 severely affected, 30% of 10 'recovered', and 15% of the 20 controls, with 1 severe and one recovered testing 'double positive'.

But subsequent analysis generally failed to detect anything using many different methods, and they couldn't maintain this association they found initially.

This is highly relevant to recent discussions:


If sporadic contamination of reagents and/or environmental contamination was the source of the gagsequences, then a possible explanation for the initial association could be due to non-random receipt of patient and control samples. Although we were blinded to subject health status, we did not receive equal numbers of patient and control samples on the same day. If, for example, mouse DNA or MLVs were present on a day that 5 patient and 1 control samples were received, but absent on a day when 5 controls and 1 patient sample were received, then a higher frequency of patient versus control samples would appear positive for MLV-like DNA fragments. However, we did not observe any clear correlation between day of receipt of samples and whether they were positive by PCR for MLV-like sequences. While it is impossible to know whether or not contamination has occurred in another laboratory, one possible explanation for the much higher frequencies of positive patients versus controls in both the Lombardi et al. [1] and Lo et al. [9] studies could be the existence of reagent or environmental contamination at the time of assay of patient samples, but not when controls were assayed. In both of those studies, large batches of patient and control samples were assayed at separate times.

So they have a specific hypothesis here for systematic higher levels in patients vs controls, both in their own study and in Lombardi et al and Lo et al. But they weren't able to confirm their analysis in their own case.

Interestingly, they propose a highly plausible theory for the different levels of detection in both Lombardi et al and Lo et al: in both, large batches of patient and control samples were assayed at different times. So different levels of reagent or environmental contamination at the time of the assays are a possible explanation.

I think this is interesting because it is a different hypothesis to "patient samples were handled more frequently", which I never bought. This hypothesis is more subtle than that: it is the potential for different levels of environmental contamination at a stage in the process that nobody previously suspected might be subject to contamination. This is significant because it emphasises that whatever 'mistakes' may have been made, there were not obvious risks to anybody before these experiments - that removes the burden of blame placed on Dr Mikovits by some.

 
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While it now appears likely that the virus named XMRV originated as a result of passage of cell lines through mice [23], the inquiry into the possible connection of this virus to prostate cancer and CFS/ME has generated some important information. Before the attempts to replicate the Lombardi et al. [1], it was not known that a number of cell lines were infected with XMRV or related MLVs, possibly causing misinterpretation of data when such lines were used in various experimental protocols. How readily this type of virus can spread from one cell line to other cell lines was also not appreciated [24]. Furthermore,the frequent contamination of reagents [12], [25][27] and lab environments with mouse DNA [10], [11], [28], despite standard precautions, has been unexpectedly high. The necessity for screening for the presence of mouse DNA has led to the development of a useful assay [10]. Should there be a future zoonotic transmission of MLV or other mouse viruses into the human population, the scientific community will be better prepared to verify its presence.

This is important because it is only fair to emphasise again that nobody knew about these risks before Lombardi et al, in that the levels of contamination of lab products with XMRV and related MLVs are now extremely widespread. That's significant because again it shows that Dr Mikovits could not have been expected to expect this to be the case - it is a shock to everyone. It's an even more significant point, IMO, because the completely unexpected and unknown spread, which has been going on for 15 years or more, ofthis particular lab-created retrovirus that infects human cells, is a big surprise finding, and it is surely an important one - and should be bigger news. How much more of this has been going on? And has any such agent, ever in history, infected anything which has been distributed for human use (such as specific cell lines used to grow vaccines)? The scale of this problem and the length of time it has been occurring, by the law of averages suggests: Almost certainly, yes. No pathogenic example has yet been identified, but that is not to say that one does not exist. Sporadic cases of H1N1-induced novel forms of narcolepsy, anyone?


Whether there are unknown retroviruses that are inciting factors in CFS/ME remains unknown. The PCR primers that we and others have employed for screening for XMRV and MLV-like sequences will allow detection of only a subset of viruses related to MLV. These PCR assays would not have amplified sequences from common feline leukemia viruses or gibbon ape leukemia viruses, even though they also are in the gammaretrovirus family. In fact, Elfaitouri et al [29] have pointed out that most of the primer sets that have been used to study CFS/ME samples would not even detect all groups of MLVs. Had it not been for the 2009 report [1] associating XMRV with CFS/ME, we would not have chosen PCR amplification for identification of viruses in this population. Less specific methods such as virus microarrays or high-throughput DNA sequencing are more suitable for detection of unknown agents that may be associated with disease states. Their application should be fruitful in identification of pathogens that may more frequently infect CFS/ME patients, either as a cause or consequence of the illness, and will be instrumental in verifying whether or not gammaretrovirus infections exist in humans and/or whether or not an unknown viral infection is associated with CFS/ME.

Two points I've recently highlighted elsewhere on these forums are re-iterated again here.

It is still unknown whether unkown retroviruses are inciting factors in ME/CFS. Obviously, but seems to need stating.

Most of the studies of ME/CFS samples have not used primers that would detect all groups of MLVs. So MLVs, other than XMRV specifically, have not been much searched for.

And as a final comment: after all this time, after all the analysis in this study, all the experiments performed, it still was not possible to nail the question of whether the sequences they sporadically detected really did come from the patients' blood, or not. That just illustrates how cutting-edge and difficult the science of this is, and shows that there is still a great deal of work to be done before all the mysteries around these viruses will be fully explained.
 
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I haven't read the paper, yet, but I want to thank these researchers for making clear, accurate, scientific statements about their conclusions


We've seen far too many conclusions lately that extrapolate far beyond the data -- the ones of the "We didn't find it, so it doesn't exist" ilk -- so it's refreshing to see sound research conclusions.
Absolutely - I couldn't agree more: it is just so refreshing to see clear, accurate, scientific statements that don't overreach.

When that is not the case, it is clearly visible to even non-experts such as ourselves, and this engendered an enormous amount of quite legitimate distrust of many researchers in the early days. When you can clearly see a scientist's bias on display, from the illogicality of their conclusions which are not just not remotely justified by their own findings, on points of simple logic alone, that is a very frightening thing to see.

Reading a paper like this one, where the language is sober, logical, reasonable, and fair, and the conclusions are justified, is such a massive breath of fresh air that it really takes my breath away. I do hope that this sort of thing is the norm in science, and that what we see regularly from the likes of White and Wessely et al, and what we saw from McClure and many other early researchers and commentators on XMRV, is very much the exception.
 
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One other point I meant to add, which seems to me quite crucial.

Although there have now been many published studies (I can count at least 7, I think there are more), in both ME/CFS and prostate cancer, which have reported statistically significant higher levels of XMRV/MLV detection in patients vs controls, I have yet to hear of a single example of a study or even part of a study which found more XMRV/MLVs in controls rather than patients.

Now: while the explanations of differential handling of samples do make sense, there are now quite enough results to make it seem most peculiar that this is a one-way street.

This could potentially be explained by many studies having been done which did find much more XMRV or MLVs in controls than in patients, but which were not published due to embarrassment. But it seems most odd that anyone should not publish in this circumstance, because such a study would massively affect the debate and would be very significant. I doubt this is the case, but if anyone has evidence of a counter-example, let them come forward...

If we approach this question on the balance of probabilities: The odds of flipping a coin and getting heads 8 times in a row are one in 128.

So we might reasonably conclude that it is more than 99% probable that the unexplained discrepancies are something specifically to do with the patient samples themselves, and not due to random contamination of batches at the time when the assay was performed.
 

alex3619

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Although there have now been many published studies (I can count at least 7, I think there are more), in both ME/CFS and prostate cancer, which have reported statistically significant higher levels of XMRV/MLV detection in patients vs controls, I have yet to hear of a single example of a study or even part of a study which found more XMRV/MLVs in controls rather than patients.

Hi Mark, this is the crux of why I do not buy the counter-claims as proved. They might be right, but until questions like this are answered they are not "proved" right. What has happened though is the standard of evidence to justify the XMRV hypothesis has been raised substantially, so high perhaps that it will be hard to justify the hypothesis even if it is correct.

Pop quiz: how long did it take the hypothesis that H. pylori caused peptic ulcers to be accepted? Read the answer in my new blog due out soon - others have said it before, but not many seem aware of the actual facts. In case anyone is looking at the Wikipedia, its very very wrong.

Bye, Alex
 

currer

Senior Member
Messages
1,409
Tissue sampling anybody?
Time for a different approach. The problems with blood samples seem insurmountable

On another note, it is scarey how all the laboratory materials are contaminated with mouse DNA.
If this contamination is impossible to eradicate, the human population has certainly been exposed to it." Sterile" takes on a new meaning.
.
 

currer

Senior Member
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1,409
I've just thought - have Hanson and Bell taken on board the recent discovery by Maureen O'Keefe that commonly used PCR reagents inhibit the reproduction of MRV's? Remember that one?
Could this explain the diminishing positives with each round of testing?
http://okeefe-lab.blogspot.co.uk/p/current-lab-projects.html
Can anyone recover the link to that report on her blog? I'm too busy to look atm.
 
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I've just thought - have Hanson and Bell taken on board the recent discovery by Maureen O'Keefe that commonly used PCR reagents inhibit the reproduction of MRV's? Remember that one?
Could this explain the diminishing positives with each round of testing?
http://okeefe-lab.blogspot.co.uk/p/current-lab-projects.html
Can anyone recover the link to that report on her blog? I'm too busy to look atm.
Wow, I like that currer, I missed that one. It also seemed odd to me that out of all the different tests they ran, the only one that found results was the very first one. Bit of an odd coincidence.

Now wouldn't that be a thing: if reagents you were using progressively suppressed replication, the more pre-test calibration you did before running the tests proper, the less chance you would find anything - a potential explanation for negative results, and it'd almost be like the more carefully and rigorously you prepare and the harder you try, the less likely you are to find anything at all. That appeals to my sense of irony...:)
 

Sam Carter

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I've just thought - have Hanson and Bell taken on board the recent discovery by Maureen O'Keefe that commonly used PCR reagents inhibit the reproduction of MRV's? Remember that one?
Could this explain the diminishing positives with each round of testing?
http://okeefe-lab.blogspot.co.uk/p/current-lab-projects.html
Can anyone recover the link to that report on her blog? I'm too busy to look atm.

I've just skim read the paper so I may have misunderstood things, but they do say:

"Nevertheless, we are confident that negative results are not due to PCR inhibition because we regularly observed non-specific amplified fragments of variable sizes."

ETA: They also write: "While one group used the dUTP-UDG method to prevent carryover of PCR products [20], our preliminary experiments suggested that this technique reduces the sensitivity of our PCR assays, which is consistent with a subsequent report [21]. We chose instead to reduce the possible environmental contamination of PCR assays by developing a single-round assay that can detect spiked plasmid DNA with the same sensitivity as the previously described gagO/gagI nested PCR."

Reference 21 is Bacich DJ, Sobek KM, Cummings JL, Atwood AA, O'Keefe DS (2011) False negative results from using common PCR reagents. BMC Res Notes 4: 457.
 

natasa778

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This bit is also worth quoting. Great thinking currer!


MLVs previously found to be present in patient samples have been observed at very low levels. Therefore we wanted to examine the inhibitory effect that minute levels of primer-dimer contamination (10-7 dilution) can have on Gag PCR product formation from MLVs that are present at low copy number. Figure 3b demonstrates that contamination of MLV present at 200, 000 copies or less with 10-7 dilution of Gag PCR primer-dimers from a previous PCR reaction almost completely inhibits PCR product formation. This data suggests that contamination with extremely small quantities of primer-dimers generated from previous PCR reactions can inhibit PCR product formation, thus leading to false negative results.
Taken together, these data suggest that inhibition of legitimate target occurs from UNG-digested PCR products or from contamination with primer-dimers from a previous PCR reaction, regardless of the presence of UNG. Thus contamination from any previous PCR samples can result in either false positives (which may or may not be detected in the negative control depending upon the samples that are contaminated and whether UNG is used) or false negatives which will not be detected using standard PCR controls.
 

natasa778

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I've just skim read the paper so I may have misunderstood things, but they do say:

"Nevertheless, we are confident that negative results are not due to PCR inhibition because we regularly observed non-specific amplified fragments of variable sizes."


This is what O'Keefe paper is suggesting, not sure if Hanson study took these steps

To circumvent the potential for PCR inhibition, a positive control included in each PCR reaction is necessary to confirm that the PCR is not inhibited. To accomplish this, a synthetic target template with identical primer-binding sites but different size could be constructed, and spiked into each test sample. This would result in potential amplification of two bands: one band to detect the target DNA and the other to detect the synthetic target. In addition, the copy number of the synthetic target spiked into the test samples would need to be at the level of detection of the assay, so as any inhibition would result in lack of amplification. None of the 38 MLV-related publications we examined used internal PCR controls. Given the data presented in this manuscript, we would propose that in cases such as the debate regarding the existence of novel infectious viruses, failure to find a virus is not the equivalent of evidence against its existence. The potential for false negative results needs to be considered and carefully controlled in PCR experiments, just as the potential for false positive results already is.

Also we are not talking about total PCR inhibition here, but rather as Mark put it "the harder you try, the less likely you are to find anything at all". At what stage/s were those fragments amplified, and does the 'strength' of PCR (the degree of inhibition) have effect on WHAT is amplified?
 

natasa778

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ok Hanson paper address - at least seems to - the PCR inhibition problem identified by O'Keefe:

While one group used the dUTP-UDG method to prevent carryover of PCR products [20], our preliminary experiments suggested that this technique reduces the sensitivity of our PCR assays, which is consistent with a subsequent report [21 - O'Keefe paper]. We chose instead to reduce the possible environmental contamination of PCR assays by developing a single-round assay that can detect spiked plasmid DNA with the same sensitivity as the previously described gagO/gagI nested PCR. When this assay, which requires less manipulation than nested PCR, was used on a new set of DNA samples, we did not detect MLV-like gag sequences.


Anyone?

Does this come anywhere close to possibly explaning the difference in rates of detection btw patients and controls?
 

Sam Carter

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This is what O'Keefe paper is suggesting, not sure if Hanson study took these steps



Also we are not talking about total PCR inhibition here, but rather as Mark put it "the harder you try, the less likely you are to find anything at all". At what stage/s were those fragments amplified, and does the 'strength' of PCR (the degree of inhibition) have effect on WHAT is amplified?

Perhaps you could write this up as a comment / question, Nastasa, and get a definitive answer from the authors? I think it would be useful to hear in more detail what steps they took to avoid the problems highlighted by Dr. O'Keefe.
 

Bob

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ok Hanson paper address - at least seems to - the PCR inhibition problem identified by O'Keefe:

While one group used the dUTP-UDG method to prevent carryover of PCR products [20], our preliminary experiments suggested that this technique reduces the sensitivity of our PCR assays, which is consistent with a subsequent report [21 - O'Keefe paper]. We chose instead to reduce the possible environmental contamination of PCR assays by developing a single-round assay that can detect spiked plasmid DNA with the same sensitivity as the previously described gagO/gagI nested PCR. When this assay, which requires less manipulation than nested PCR, was used on a new set of DNA samples, we did not detect MLV-like gag sequences.

Anyone?

Does this come anywhere close to possibly explaning the difference in rates of detection btw patients and controls?

So that does directly address the O'Keefe paper doesn't it.

From my memory (which isn't reliable), of reading the O'Keefe paper, single round PCR can be affected if the PCR slide has been used before, either in the manufacturers' or the researchers' calibration, or if a negative slide is reused. If there is even a tiny amount of contamination, then the results can be false-negative. (I might have some of this wrong though - my memory is notoriously useless!)

Talking about XMRV research in the 'discussion and conclusions', the O'Keefe paper states:
"Therefore, amplification of the high copy number positive control could occur while samples positive for a low copy number virus could be inhibited by carry-over PCR contamination."