• Welcome to Phoenix Rising!

    Created in 2008, Phoenix Rising is the largest and oldest forum dedicated to furthering the understanding of and finding treatments for complex chronic illnesses such as chronic fatigue syndrome (ME/CFS), fibromyalgia (FM), long COVID, postural orthostatic tachycardia syndrome (POTS), mast cell activation syndrome (MCAS), and allied diseases.

    To become a member, simply click the Register button at the top right.

Lo and Alter respond to contamination and other methodology questions in PNAS"

CBS

Senior Member
Messages
1,522
There are a series of letters published in today's (9/30/10) early edition of PNAS:

I have not yet had a chance to read them - they appear to require a subscription - I have full copies.

Erlwein, McClure and Co.
Martin
Lo and Alter
I'm sure that as we read through and analyze these there will be more posts relatively soon.
 

CBS

Senior Member
Messages
1,522
I'm going to try and break this down with brief quotes and a paraphrase or two:

Erlwein -

It is known that, to allow the “hot start,” platinum Taq polymerase from Invitrogen contains mouse monoclonal antibodies and may contain traces of mouse DNA. Several laboratories have reported contamination on amplifying XMRV sequences with platinum Taq. This enzyme was used by Lo et al. (1) in the PCR and, although negative samples were negative, assurance that control samples were assayed simultaneously with the positives in a blinded, randomized way is missing. The fact that the analysis in figure 1 (on cases) (1) and in figure 2 (on controls) (1) has not been carried out on the same primer sites may be trivial, but it is not standard practice. Multiple bands on the PCR gels are difficult to explain if, as suggested by the sequencing data, only one virus was present in these samples. It is fortuitous that readable sequences were generated from the PCR products shown in figure 1 (1) without cloning, which was not mentioned in the Methods section (1).
Paraphrase - It's contamination and we don't think you handled your patient and control samples in the same manner. And for good measure, we'll throw in a jab at something they claim wasn't included in their methods section.

Using the same gag primers as Lombardi et al. (6) for XMRV detection, it is surprising that Lo et al. (1) failed to detect XMRV sequences in any of their CFS cases and, conversely, Lombardi et al. (6) failed to detect any of the MLV sequences described by Lo et al. (1). One might have expected some crossdetection. This is more perplexing as our own study and that of the Centers for Disease Control failed to link CFS to retroviral infection using generic primers that would have detected MLV sequences (2, 5).
Taken together, the data linking CFS and MLV-like virus gene sequences, rather than strengthening the case for XMRV involvement in CFS, raises more questions than answers.
Martin:
The use of the nested PCR at near the limits of its sensitivity, however, does not exclude a lower than detectable level of mouse endogenous retroviruses in many healthy individuals. The numerous “nonspecific” bands generated in the nested PCR assays on control and patient samples, as reported by Lo et al. (1), mean that any retroviral sequences would need to compete for primers with the nonspecific reactions occurring with normal DNA. The primer cross-reactivity with DNA sequences other than those of murine retroviruses limits the possible detection of authentic murine retroviral sequences potentially present in DNA of healthy controls.
Paraphrase - Lo et al. used such a nested PCR near the limits of its sensativity and there fore we cannot rule out the possibility that everyone in society has some level of MLV infection

Lo Atler response:
We were aware that the SuperScript III One-Step RT-PCR System (cat. no. 12574–030; Invitrogen) had come under question for contamination with mouse DNA. For that reason, werigorously examined the Platinum Taq polymerase we used (cat. no. 10966–034, lot 727463; Invitrogen) for any possible mouse DNA contamination. First, as stated in the article (3), no murine leukemia virus (MLV)-related gene amplicon was produced in more than 300 negative controls tested in parallel. Second, we tested the polymerase and our PCR assay systemrepeatedly using the highly sensitive mouse DNA-specific seminested PCR targeting the mitochondria DNA described; we never detected any mouse DNA contamination.

As for the “nonspecific” side bands seen in the gel, we have found that virus gag gene-specific primer sets could sometimes amplify human DNA nonspecifically and produce amplicons of different sizes. Thus, Martin was correct that any murine retroviral sequences would need to compete for the primers with the nonspecific reactions also occurring with normal human DNA. This would likely lower our assay sensitivity of detecting authentic murine retroviral sequences in blood of patients and healthy controls. We designed and studied multiple primer sets targeting MLV-related virus gag genes and carefully compared their performance. We chose the primer sets with the greatest sensitivity for this study. The analysis in figures 1 (cases) and 2 (controls) were always
carried out using the same external primer set and both of the internal primer sets, as described in the text (3).
We fully understand that our nested PCR targeting the MLVrelated virus genes, like any other assay, had a limitation of sensitivity in detecting very low copies of the virus target genes in the clinical samples. Thus, it is true that our study, using the nested PCR at or near the limits of its sensitivity, does not necessarily exclude the possibility of a lower than detectable level of MLVs being present in many healthy individuals. The finding of markedly different frequencies of positivity in the CFS cases and the healthy controls could be a quantitative, not a qualitative, distinction.

The finding of mouse retrovirus genes or the viruses in people is still at an early stage of exploration. Further studies are needed to better elucidate this distinction as well as the pathogenic role, if any, of MLV-related viruses in human disease.
These are the key points. Sounds like Lo and Alter are quite comfortable with their study as it was and with the acknowledge limitations.
 

CBS

Senior Member
Messages
1,522
On Sept 13th, Myra McClure was interviewed by Vincent Racaniello forr his TWiV postcast. McClure was quite convinced that the WPI needed to prove there was no contamination.

Was she aware of Lo and Alter's soon to be published response? Did she make her case for contamination after the episode of TWiV?

Will this latest response from Lo/Alter make any difference to her on the issue of contamination? As Lo and Alter state, we are "still at a very early stage of exploration." However, continued claims that there is nothing to see ("just a bunch of CFS patients being CFS patients - complaining while we can't figure out why") are becoming harder and harder to defend.
 

eric_s

Senior Member
Messages
1,925
Location
Switzerland/Spain (Valencia)
Thanks CBS. That's very interesting. I'm still fully behind Lo/Alter, when their study came out i said that as far as i'm concerned there is an association between CFS and HMRV.

Here the critics really provide technical arguments, which i'm not able to understand, but i like that way. Done in such a way, i don't have any problem with people criticizing positive results.
 

CBS

Senior Member
Messages
1,522
There are too many big names that have been looking at this for years and are gearing up for greater involvement (eg. Lipkin & Singh). You don't do that if you're not finding anything (if you can't find anything apparently you take your negative results and go on a speakers tour - maybe if McClure was spending more time in the lab and less time defending her absence of evidence she'd be a bit more open to changing her tune).
 

voner

Senior Member
Messages
592
cbs: thanks for the info and explanation...

Since Dr. Ila singh used to work at Columbia, and knows Dr. Vincent Racaniello, lets hope He interviews her on TWIV when her studies are published. Hopefully, this will clarify the muddy water. I was struck by Dr. Lombardi's confidence in his results, etc when he was interviewed on the radio show .... Nevada Newsline --interviewed by Mike Hillerby... Dr. Lombardi was proceeding aggressively and didn't act as though there was ANY doubt in his mind....

it really sounds like the research world is heading in 2 directions, one small group are agressively pursuing the XMRV connection and the rest are quibbling. Some researchers love to quibble (using the word "quibble may be a bit harsh here)...and it certainly is valid at this point.

the big question will be::: "WHO GETS THE FEDERAL RESEARCH $$ ?????? & WHY?
 

CBS

Senior Member
Messages
1,522
quantitative versus qualitative distinction?

Lo and Alter state:

The finding of markedly different frequencies of positivity in the CFS cases and the healthy controls could be a quantitative, not a qualitative, distinction.
This is an interesting comment. It has significant potential to change the meaning of detecting MLV's in controls and other disease states. It also means that dismissing MLV's as being 'passenger viruses' or co-infections may be overlooking the importance of quantitative distinctions.



On an administrative note, I have asked that the title of this thread be changed. My first thought was:


"Lo and Alter MLV smackdown with McClure and Friends in PNAS"


Instead I'm asking for a change to : "Lo and Alter respond to contamination and other methodology questions in PNAS"
 

anciendaze

Senior Member
Messages
1,841
Once again it sounds like "if you were doing things the way we are you wouldn't get these disturbing results."

The possibility of many similar endogenous sequences is scarcely news, this is a fundamental problem for the entire field. It also says rather clearly that humans have been infected with very similar retrovirus over long periods in the past.

On contamination: you need to test your reagents and equipment before you use them, not try to untangle the mess afterward. (Did the "esteemed" Dr. Weiss do this in 1997?) As for working at or near the limits of sensitivity, I must ask "are you running a research laboratory or an ordinary clinical laboratory?" (Funding can be adjusted as appropriate.)

As I've said before, you can guarantee you will not get positive results due to contamination if you exclude all positive results, and this proposition does not require expensive research to prove. This now appears to have happened at the CDC, which has an extremely sensitive test for mouse DNA. The contamination problem in the laboratory where they were working with samples from DeFreitas went unreported in their publications. At different times the contaminant has been identified as either gibbon ape leukemia virus or murine leukemia virus. After the contaminant was completely excluded, their results turned so thoroughly negative they started fiddling with her lab. protocol until it produced garbage. (Failure of this modified protocol was believed to discredit her.) Like the contamination later plaguing Dr. Weiss, the specific route for contamination was never identified.
 

Cort

Phoenix Rising Founder
There are too many big names that have been looking at this for years and are gearing up for greater involvement (eg. Lipkin & Singh). You don't do that if you're not finding anything (if you can't find anything apparently you take your negative results and go on a speakers tour - maybe if McClure was spending more time in the lab and less time defending her absence of evidence she'd be a bit more open to changing her tune).

Well said!
 

anciendaze

Senior Member
Messages
1,841
There are too many big names that have been looking at this for years and are gearing up for greater involvement (eg. Lipkin & Singh). You don't do that if you're not finding anything (if you can't find anything apparently you take your negative results and go on a speakers tour - maybe if McClure was spending more time in the lab and less time defending her absence of evidence she'd be a bit more open to changing her tune).
This is a case where CBS and I agree in broad outline, but differ on specific wording. We wouldn't be having this discussion if major authorities had not made major blunders many years ago. Most people simply never noticed that virtually all information came from a very small number of sources which substituted opinions for facts. Once the bandwagon was rolling, it was easy to get others to sign on to the idea. Colossal mistakes spanning years do happen.

What has dismayed me about the whole series of responses since last October is the paucity of new ideas, (compared to those getting positive results,) and the unwillingness to grant opponents the bare possibility of having real results, even temporarily and hypothetically. I have tried to make contamination arguments work. The only place where I have had some success is with Huber's study, and there I had to assume she had an underlying set of data with modest positive results mixed in with contamination to get probabilities I could swallow. The probabilities elsewhere would make being dealt a pat hand at bridge look like a sure thing. One group might make systematic errors to cause badly skewed results, but, as more diverse groups become involved, it is less and less likely they will make the same errors without noticing.

I have assumed the negative results came from people who were honestly doing what they said they were doing to the best of their abilities. We have now reached the point where these criticisms verge on charges of gross incompetence, fraud and conspiracy. If I am forced to make a choice between competing conspiracy theories, I know which I would choose. I will not reject data simply because someone else says "these can't be right, we got different results." (This reminds me of the bit in the book and movie MASH about "We're the pros from Dover". Assertion and bluff work better than we like to admit.) It looks more and more to me like the contamination called into question is contamination by the idea of organic causation.
 

Otis

Señor Mumbler
Messages
1,117
Location
USA
It looks more and more to me like the contamination called into question is contamination by the idea of organic causation.

What a great finish to that post anciendaze! And the this contamination is very widespread indeed. Anyone who would argue that science is objective doesn't have this disease.
 

alex3619

Senior Member
Messages
13,810
Location
Logan, Queensland, Australia
As I've said before, you can guarantee you will not get positive results due to contamination if you exclude all positive results, and this proposition does not require expensive research to prove. This now appears to have happened at the CDC, which has an extremely sensitive test for mouse DNA. The contamination problem in the laboratory where they were working with samples from DeFreitas went unreported in their publications. At different times the contaminant has been identified as either gibbon ape leukemia virus or murine leukemia virus. After the contaminant was completely excluded, their results turned so thoroughly negative they started fiddling with her lab. protocol until it produced garbage. (Failure of this modified protocol was believed to discredit her.) Like the contamination later plaguing Dr. Weiss, the specific route for contamination was never identified.

Hi anciendaze

I find the bolded (my bolding) bit highly disturbing. Do you have a reference for this? Taken in a particular way, it could imply that the CDC found XMRV way back then, and dismissed it as contamination, and then shut down all research in this area. So I just want to ask: can you elaborate on this? I might actually have read something on this in the past, my memory is atrocious, but it would have been before I knew about XMRV or all sorts of red lights would have been flashing in my mind.

Bye
Alex
 

anciendaze

Senior Member
Messages
1,841
...I find the bolded (my bolding) bit highly disturbing. Do you have a reference for this? Taken in a particular way, it could imply that the CDC found XMRV way back then, and dismissed it as contamination, and then shut down all research in this area. So I just want to ask: can you elaborate on this? I might actually have read something on this in the past, my memory is atrocious, but it would have been before I knew about XMRV or all sorts of red lights would have been flashing in my mind...
There is a quote in "Osler's Web" which appears to be have taken from a recorded interview with Tom Folks referring to possible Gibbon Ape Leukemia Virus. He also says that the decision to end the lab. work came from upper floors in the administration building. Leonard Jason has made statements about the CDC contamination being MLV in published interviews, but I don't have a good reference.

I want to make it clear I am not describing this as a clear conspiracy. This is the kind of unchallenged assumption I've found in organizational mistakes big enough to kill people in completely unrelated situations. Another clue is Folks' statement that the test started jumping on endogenous material after they changed the protocol. With 94% homology to MuLV and 95% homology to an endogenous sequence, it would be easy for them to set up criteria for test assays which would also exclude XMRV. After enough people have accepted such criteria it is virtually impossible to get an organization to change them. The circular reasoning has been expanded to the point the circle passes over the horizon for individuals with limited authority and responsibility. Those higher up the hierarchy don't have the time and interest to challenge detailed assumptions originating at a lower level. About 80-90% of their time goes to internal politics and funding battles.

When the fertilizer hits the turbofan, you can expect to hear that "mistakes were made" without any personal responsibility for those mistakes.
 

alex3619

Senior Member
Messages
13,810
Location
Logan, Queensland, Australia
Hi anciendaze, If this is true, then I agree its not a conspiracy. Its a case of total stuff up and incompetence. It doesn't mean they aren't trying to cover it up now though if they've realized what they have done , we simply don't know one way or the other. I've read "Osler's Web" but that was many years ago. Maybe its time I got it down off the shelf again. Bye, Alex
 

natasa778

Senior Member
Messages
1,774
When the fertilizer hits the turbofan, you can expect to hear that "mistakes were made" without any personal responsibility for those mistakes.

when the fertilizer hit the turbofan on the BSE/CJD story, these were the conclusions of subsequent enquiry:

[replace BSE with XMRV and insert spooky music for full reading experience]

http://collections.europarchive.org...iry.gov.uk/report/volume1/execsum2.htm#669592

# BSE has caused a harrowing fatal disease for humans. As we sign this Report the number of people dead and thought to be dying stands at over 80, most of them young. They and their families have suffered terribly. Families all over the UK have been left wondering whether the same fate awaits them.
...
# BSE developed into an epidemic as a consequence of an intensive farming practice - the recycling of animal protein in ruminant feed. This practice, unchallenged over decades, proved a recipe for disaster.

# In the years up to March 1996 most of those responsible for responding to the challenge posed by BSE emerge with credit. However, there were a number of shortcomings in the way things were done.

# At the heart of the BSE story lie questions of how to handle hazard - a known hazard to cattle and an unknown hazard to humans. The Government took measures to address both hazards. They were sensible measures, but they were not always timely nor adequately implemented and enforced.

# The rigour with which policy measures were implemented for the protection of human health was affected by the belief of many prior to early 1996 that BSE was not a potential threat to human life.

# The Government was anxious to act in the best interests of human and animal health. To this end it sought and followed the advice of independent scientific experts - sometimes when decisions could have been reached more swiftly and satisfactorily within government.
...
# At times officials showed a lack of rigour in considering how policy should be turned into practice, to the detriment of the efficacy of the measures taken.

# At times bureaucratic processes resulted in unacceptable delay in giving effect to policy
.
# The Government introduced measures to guard against the risk that BSE might be a matter of life and death not merely for cattle but also for humans, but the possibility of a risk to humans was not communicated to the public or to those whose job it was to implement and enforce the precautionary measures.

# The Government did not lie to the public about BSE. It believed that the risks posed by BSE to humans were remote. The Government was preoccupied with preventing an alarmist over-reaction to BSE because it believed that the risk was remote. It is now clear that this campaign of reassurance was a mistake. When on 20 March 1996 the Government announced that BSE had probably been transmitted to humans, the public felt that they had been betrayed. Confidence in government pronouncements about risk was a further casualty of BSE.