Negative XMRV CFS study with Ila Singh's name on it (University of Utah)

[liquid sky]

I felt like Cort's XMRV buzz page had hinted that the Singh study was looking negative... maybe that was just my imagination, but this doesn't now come as a big surprise."

Your the third person that I have heard say that Cort was hinting at the Singh study looking negative. How would he know that? Are some privy to study results ahead of time?

I am looking forward to the BWG and Lipkin studies also, but that will be quite some time. I think the Lipkin study will take 2-3 years. I wish they would publish some of the positive studies and info that is backlogged. That would at least make it a fair playing field.

I keep getting a message that I need 10 characters to post, huh? So, I am typing in more to see if that's the real problem.

Well, it's still not working. I see a new post and I agree. Singh has shown she could not find any positives with her methods.

 

[Wonko]

Dr Singh's study shows that her new assays are unable to detect HGRVs in known positives


http://networkedblogs.com/hsc3y

True, as far as it goes, depends on how positive a "known positive" is I suppose, she was unable to detect XMRV in a blinded sample that the WPI says is positive. That may not be the same thing as a "known positive" IF the WPI is wrong.

 

[Ernie]

They didn't look for MLV's that's why they didn't find them. They used new assay's, frozen samples, They only tested two patients from the original science study. 13 negative controls and 2 positive controls were also 350 included. Unfortunately for her one of those patients was the source of a full length clone and EM showing viral budding. There are many things wrong with this paper.

 

[Esther12]

Your the third person that I have heard say that Cort was hinting at the Singh study looking negative. How would he know that? Are some privy to study results ahead of time?

I don't know... a while back Buzz had been hinting that Singh was going to be positive, then it seemed to move to hinting Singh was going to be negative. I've got no inside information here, but maybe Cort had been chatting to her?

 

[liquid sky]

If she is trying to find positive samples in a study, then she MUST calibrate her methods to a known positive sample. That is what they do in all other diseases. A clone will not do. Why was this not done?

 

[Jemal]

I don't know... a while back Buzz had been hinting that Singh was going to be positive, then it seemed to move to hinting Singh was going to be negative. I've got no inside information here, but maybe Cort had been chatting to her?

It could be he has spoken with her or others that may have made certain hints...
However, the Singh study took so long I presumed it was negative as well. I had hope it would be positive though

 

[liquid sky]

I didn't realize he was on speaking terms with Singh. Isn't she also the one who was going to do autopsies to check for possible places in the body where the virus lives?

 

[Jemal]

I didn't realize he was on speaking terms with Singh. Isn't she also the one who was going to do autopsies to check for possible places in the body where the virus lives?

I don't know if he is on speaking terms with her.

And yes, she is (was?) doing the autopsy study. I am also wondering if this means the end of her research into XMRV and prostate cancer.

 

[liquid sky]

Surely, if she meant what she said about a possible viral cause for ME/CHF, she will still do the autopsies. If she stops now, that would look like she is not really trying to get to the answer.

 

[Jemal]

Surely, if she meant what she said about a possible viral cause for ME/CHF, she will still do the autopsies. If she stops now, that would look like she is not really trying to get to the answer.

I think she was doing the autopsies in relation to XMRV. If she now thinks XMRV is contamination, I don't think she will continue that research. But I am not her spokesperson, so this is just what I think and deduce

 

[dannybex]

They used new assay's, frozen samples, They only tested two patients from the original science study.

Quoting from the study:

"Using a third party phlebotomy service that collected blood samples in home visits, we obtained blinded whole blood and serum samples from 14 individuals. These individuals had repeatedly tested positive in the last two years when tested by the labs at the WPI, though this information was not available to us till the completion of our study.

The Clinical Research department at ARUP Laboratories received these specimens and processed the blood using the same protocols as for our healthy volunteers and CFS patient samples. Thus the samples were never opened in a research lab where XMRV might be present until they reached us. We tested these samples using all of the assays we developed four qPCR assays, ELISA and Western blots. None of the samples contained any evidence of XMRV."

It sounds to me like she might be implying that the WPI's lab was contaminated at some point? I don't know. All this is way above my head.

 

[dannybex]

If she now thinks XMRV is contamination, I don't think she will continue that research. But I am not her spokesperson, so this is just what I think and deduce

And if XMRV is contamination, how does that affect her work -- and her patents -- re XMRV and cancer?

 

[Ernie]

Quoting from the study:

"Using a third party phlebotomy service that collected blood samples in home visits, we obtained blinded whole blood and serum samples from 14 individuals. These individuals had repeatedly tested positive in the last two years when tested by the labs at the WPI, though this information was not available to us till the completion of our study.

The Clinical Research department at ARUP Laboratories received these specimens and processed the blood using the same protocols as for our healthy volunteers and CFS patient samples. Thus the samples were never opened in a research lab where XMRV might be present until they reached us. We tested these samples using all of the assays we developed four qPCR assays, ELISA and Western blots. None of the samples contained any evidence of XMRV."

It sounds to me like she might be implying that the WPI's lab was contaminated at some point? I don't know. All this is way above my head.

Actually I meant this and it's from the paper. They could not even detect the 2 positives with their methods:

348 inoculated LNCaP cells with 100 ?l of plasma from 31 patients and 34 healthy volunteers, and
349 passaged the cells weekly for 6 weeks. 13 negative controls and 2 positive controls were also
350 included. Only one culture was handled at a time to prevent any cross-contamination. After
351 weeks 2, 4 and 6, cultures were lysed and analyzed by Western blots (Fig. 4) and by qPCR for
352 XMRV. No XMRV protein or DNA was detected in any of the cultures.[/B]

 

[dipic]

Yet another garbage zero-zero paper where even the author admitted that they used novel assays not able to detect XMRV. Seriously, WTF? Can we please get some true replication studies? This is becoming truly sad and pathetic; I'm beginning to lose all faith in the scientific community.

As long as scientists continue to use their own, different methods, we'll keep getting junk negative papers like this. F--king depressing.

 

[Jemal]

And if XMRV is contamination, how does that affect her work -- and her patents -- re XMRV and cancer?

Very good questions, especially about the patents. Maybe she thinks they are junk now? I am very interested to know if she continues XMRV research or not.

 

[Esther12]

I don't think we can just go on insisting that we have known XMRV positive patients whose blood needs to be used to validate all testing.

It's looking increasingly likely that those patients thought to be positive are actually negative. This hasn't been settled yet, but it's certainly possible that the 0/0 studies are getting it right, and the other studies have had trouble with contamination.

We've yet to hear back from Silverman and Klein about the claims that some of their PC work is best explained by contamination... it seems to me that there's not much we can say about XMRV with any certainty.

 

[dannybex]

Actually I meant this and it's from the paper. They could not even detect the 2 positives with their methods:

348 inoculated LNCaP cells with 100 ?l of plasma from 31 patients and 34 healthy volunteers, and
349 passaged the cells weekly for 6 weeks. 13 negative controls and 2 positive controls were also
350 included. Only one culture was handled at a time to prevent any cross-contamination. After
351 weeks 2, 4 and 6, cultures were lysed and analyzed by Western blots (Fig. 4) and by qPCR for
352 XMRV. No XMRV protein or DNA was detected in any of the cultures.[/B]

Thanks Ernie. I guess I was reading a different section of the paper.

So my question is, didn't she find an XMRV - cancer connection? And if so, were different techniques used in that study or studies?

 

[Cort]

I felt like Cort's XMRV buzz page had hinted that the Singh study was looking negative... maybe that was just my imagination, but this doesn't now come as a big surprise."

Your the third person that I have heard say that Cort was hinting at the Singh study looking negative. How would he know that? Are some privy to study results ahead of time?

I am looking forward to the BWG and Lipkin studies also, but that will be quite some time. I think the Lipkin study will take 2-3 years. I wish they would publish some of the positive studies and info that is backlogged. That would at least make it a fair playing field.

I keep getting a message that I need 10 characters to post, huh? So, I am typing in more to see if that's the real problem.

Well, it's still not working. I see a new post and I agree. Singh has shown she could not find any positives with her methods.

No hinting at all actually. All I knew was that Dr. Light told me - that the study was done and being published soon. I had absolutely NOO IDEA what the results would be.

Dr. Singh started this study over a year ago I believe. The fact that it was taking so much longer than expected did suggest problems might have occurred. However we did know that she felt that she found XMRV early on - which was encouraging. The Alberta study is in the same situation - it was supposed to have been completed much earlier.

Dr. Singh has been studying murine retroviruses throughout her career and was one of the top XMRV prostate researchers. She is a very careful researcher and this study was thought to be one of the best, if not the best underway. It's very unfortunate..This was one of the biggies...
'
Here's an article on the study titled "The Best XMRV Study Under Way?" http://forums.phoenixrising.me/content.php?202-Dr-Singh-Talks-XMRV-and-ME-CFS-with-Dr-Racaniello that was published in August of last year.

The BWG, Lipkin, Glaxo Smith Kline, Joliceur XMRV positive, Univ of Alberta, NCI and probably some other studies remain.

 

[Esther12]

No hinting at all actually. All I knew was that Dr. Light told me - that the study was done and being published soon. I have absolutely NOO IDEA what the results would be.

I was just reading tea leaves then. Sometimes these arbitrary patterns can fall in to place!

 

[Cort]

A riff on the Racaniello article

http://www.virology.ws/2011/05/04/ila-singh-finds-no-xmrv-in-patients-with-chronic-fatigue-syndrome/

From Dr Racaniello - after noting all the problems with the earlier studies, Dr. Racaniello called the Singh study the 'most comprehensive study to date' and, importantly, that it took into account a number of potentially confounding factors that could have contributed to past negative results (differences in patient characterization, geographic locations, clinical samples used, and methods). That was its purpose all along. The killer here is the inclusion of 'methods' since this implies that Dr. Racaniello believes this study did all the methods right.

Since the first association of the retrovirus XMRV with chronic fatigue syndrome in 2009 in the US, subsequent studies have failed to detect evidence of infection in patients from the US, Europe, and China. These studies were potentially compromised by a number of factors, such as differences in patient characterization, geographic locations, clinical samples used, and methods used to detect the virus. These and other potentially confounding conditions have been addressed in the most comprehensive study to date on the association of XMRV with CFS.

Study parameters

To address these issues, the authors collected blood from 105 CFS patients and 200 healthy volunteers in the Salt Lake City area. One hundred of the patients fulfilled both the CDC-Fukuda and the Canadian consensus criteria for diagnosis of ME/CFS. The patients were selected from a clinic that specializes in the diagnosis and management of CFS and fibromyalgia.

The clinic, of course, was Dr. Bateman's clinic - a clinician who knows how to characterize CFS patients.

They looked for XMRV using four different PCR assays and ELISA antibody and they tried to grow the virus in cell cultures; the same approaches, Dr. Racaniello reported, used by Lombardi et. al in the original paper.

New blood samples were also collected (by a third party) from 14 patients from the original study by Lombardi et al. The samples were blinded for subsequent study. Detection of viral nucleic acids was done using four different PCR assays. Anti-XMRV antibodies in patient sera were detected by ELISA. Finally, virus growth from clinical specimens was attempted in cell culture. The authors used the multiple experimental approaches reported by Lombardi and colleagues

.

As was noted many moons ago - the pol region is the most specific for XMRV since that region rarely changes in viruses. They also looked for the gag and env regions (as the WPI did). As with many of the other studies, everybody was negative via PCR and antibodies - patients and controls.

PCR for viral nucleic acids. Four different quantitative PCR assays were developed that detect different regions of the viral genome. The assay for pol sequences has been used by several groups and is the most specific PCR assay for XMRV. Three other PCR assays were also used that target the LTR, gag and env regions of XMRV DNA. These assays could detect at least 5 viral copies of XMRV DNA. The precision and reproducibility of the PCR assays, as well as their specificity for XMRV, were also demonstrated. DNA prepared from white blood cells of 100 CFS patients and 200 controls were negative for XMRV. For every 96 PCR reactions, 12 water controls were included; these were always negative for XMRV DNA.

The inability to find XMRV via PCR and antibodies has, of course, happened numerous times before. This time Singh also tried to use culture - for up to six weeks - the time Dr. Mikovits stated was necessary. Unfortunately no viral protein or DNA from XMRV was found either in people in with CFS, healthy controls or people who previously tested positive for XMRV by the WPI.

Infectious XMRV in human plasma. It has been suggested that the most sensitive method for detecting XMRV in patients is to inoculate cultured cells with clinical material and look for evidence of XMRV replication. The XMRV-susceptible cell line LNCaP was therefore infected with 0.1 ml of plasma from 31 patients and 34 healthy volunteers; negative and positive controls were also included. Viral replication was measured by western blot analysis and quantitative PCR. No viral protein or DNA was detected in any culture after incubation for up to 6 weeks.

Then they used the nested PCR techniques used by Lo (which the WPI endorsed). They did find some positives but concluded they came from trace amounts of mouse DNA in their reagents

Presence of mouse DNA. After not finding XMRV using qPCR, serological, and viral culture assays, the authors used the nested PCR assay described by Lo et al. Although positives were observed, they were not consistent between different assays. This led the authors to look for contamination in their PCR reagents. After examination of each component, they found that two different versions of Taq polymerase, the enzyme used in PCR assays, contained trace amounts of mouse DNA.

Dr. Racaniello basically concluded that this study was good enough to conclude that XMRV was not found in these patients and suggested suggested that with regards to researchers the XMRV case was pretty closed but that for patients sake (non-scientists)
the WPI should look to their reagents etc. in order to clear up where the XMRV in their study came from.

Given the care with which these numerous assays were developed and conducted, it is possible to conclude with great certainty that the patient samples examined in this study do not contain XMRV DNA or antibodies to the virus. Its not clear why the 14 patients resampled from the original Lombardi et al. study were negative for XMRV in this new study. The authors suggest one possibility: presence of trace amounts of mouse DNA in the Taq polymerase enzymes used in these previous studies. I believe that it is important to determine the source of XMRV in samples that have been previously tested positive for viral nucleic acid or antibodies. Without this information, questions about the involvement of XMRV in CFS will continue to linger in the minds of many non-scientists.

Dr. Singh, Dr. Bateman and the Lights took a strong stand against the use of antiretrovirals stating that they were 'forced to conclude that prescribing antiretroviral agents to CFS patients is insufficiently justified and potentially dangerous.' and that much data suggests pathogens play a role in CFS and that work should continue.

Given the lack of evidence for XMRV or XMRV-like viruses in our cohort of CFS patients, as well as the lack of these viruses in a set of patients previously tested positive, we feel that that XMRV is not associated with CFS. We are forced to conclude that prescribing antiretroviral agents to CFS patients is insufficiently justified and potentially dangerous.

They also note that there is still a wealth of prior data to encourage further research into the involvement of other infectious agents in CFS, and these efforts must continue.