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Multiple Sources of Contamination in Samples from Patients Reported to Have XMRV..

Discussion in 'XMRV Research and Replication Studies' started by magnetronnie, Nov 22, 2011.

  1. magnetronnie

    magnetronnie

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    This was posted on ERV:
    http://scienceblogs.com/erv/2011/11/xmrv_and_chronic_fatigue_syndr_30.php#comment-5915108

    http://antiviralresistance.org/abstract26_2011.pdf

    --

    So if I get it right, Coffin and his group discovered that Lombardi et al. suffered from two types of contamination (XMRV from cell lines and mouse genomic DNA), as predicted by various other studies in Retrovirology?
  2. Firestormm

    Firestormm Senior Member

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    Multiple Sources of Contamination in Samples from Patients Reported to Have XMRV

    Multiple Sources of Contamination in Samples from Patients Reported to Have XMRV Infection: http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030889

    Mary F. Kearney1*, Jonathan Spindler1, Ann Wiegand1, Wei Shao2, Elizabeth M. Anderson1, Frank Maldarelli1, Francis W. Ruscetti3, John W. Mellors4, Steve H. Hughes1, Stuart F. J. Le Grice1, John M. Coffin5

    Abstract

    Xenotropic murine leukemia virus (MLV)-related retrovirus (XMRV) was reported to be associated with prostate cancer by Urisman, et al. in 2006 and chronic fatigue syndrome (CFS) by Lombardi, et al. in 2009.

    To investigate this association, we independently evaluated plasma samples from 4 patients with CFS reported by Lombardi, et al. to have XMRV infection and from 5 healthy controls reported to be XMRV uninfected.

    We also analyzed viral sequences obtained from supernatants of cell cultures found to contain XMRV after coculture with 9 clinical samples from 8 patients.

    A qPCR assay capable of distinguishing XMRV from endogenous MLVs showed that the viral sequences detected in the CFS patient plasma behaved like endogenous MLVs and not XMRV.

    Single-genome sequences (N = 89) from CFS patient plasma were indistinguishable from endogenous MLVs found in the mouse genome that are distinct from XMRV.

    By contrast, XMRV sequences were detected by qPCR in 2 of the 5 plasma samples from healthy controls (sequencing of the qPCR product confirmed XMRV not MLV).

    Single-genome sequences (N = 234) from the 9 culture supernatants reportedly positive for XMRV were indistinguishable from XMRV sequences obtained from 22Rv1 and XMRV-contaminated 293T cell-lines.

    These results indicate that MLV DNA detected in the plasma samples from CFS patients evaluated in this study was from contaminating mouse genomic DNA and that XMRV detected in plasma samples from healthy controls and in cultures of patient samples was due to cross-contamination with XMRV (virus or nucleic acid).

    Received: October 11, 2011; Accepted: December 22, 2011; Published: February 20, 2012

    Full paper: http://www.plosone.org/article/fetc....1371/journal.pone.0030889&representation=PDF
  3. Firestormm

    Firestormm Senior Member

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    Discussion:

    'Our analyses of plasma samples independently collected from CFS patients previously reported to be XMRV-infected [2,29] and from healthy controls reported to be XMRV-uninfected [2], and of virus isolation co-culture supernatants identified three different types of sample contamination, leading to false positive detection of XMRV.

    First, we detected high levels of mouse genomic (both IAP and MLVs) and mouse mitochondrial DNA, but no XMRV sequences, in plasma samples from CFS patients, leading us to conclude that contaminating mouse genomic DNA in this set of plasma samples led to false positive PCR results for XMRV.

    Second, although the 5 plasma samples from healthy controls were free of mouse DNA, two of them contained contaminating XMRV nucleic acid most likely plasmid or a PCR amplified DNA
    product as shown by the PCR amplification profiles and confirmed by sequencing of the amplified product.

    Third, our analyses of sequences from viruses reportedly isolated from 8 patients with putative XMRV infection [29] revealed that the sequences did not differ among the patients and were indistinguishable from sequences of XMRV in XMRV-infected cell lines indicating that the cultures were cross-contaminated from infected cell lines used in the same laboratory.

    Specifically, the viruses reportedly isolated from patient samples exhibited very little diversity and were closely related to the 22Rv1 virus, consistent with a virus highly similar or identical to that found in the 22Rv1 cell line after a few cycles of virus replication in culture.

    These findings indicate that the putative isolations of replicating XMRV from patient samples were likely false positives as a result of cross-contamination of the cultures with XMRV from infected cell lines.

    PCR and tissue culture are sensitive methods, and are, as a consequence, susceptible to contamination. Care must be taken both to prevent such contamination and to ensure that the analysis includes proper controls to exclude false positive results.

    The experimental samples and controls must be collected at the same time, using the identical materials, and processed together under identical conditions.

    Furthermore, it is important to develop strict criteria for declaring a sample positive, including a requirement that multiple methods should yield positive and negative results for the same samples, and the results should be reproducible.

    Our independent analyses of samples from patients reported to be XMRV-infected (12), refutes prior evidence of XMRV infection in these patients and argues against XMRV infection of human populations.

    Our data show that there are at least three different ways contamination can be misinterpreted as XMRV or MLV infection of humans.

    The first is mouse DNA, which is a ubiquitous environmental contaminant that can find its way into experimental samples in many different ways.

    Examples include monoclonal antibodies or other bioproducts prepared in mice or mouse cells, chemicals, disposables, or other materials stored where mice can have access [3,13,14,30,31,32], or handling of mouse specimens or cell lines in the same laboratory where human samples are being processed [12].

    Inbred strains of mice contain around 60 MLV proviruses per haploid genome that can be detected by PCR with an env-specific probe [33], and some wild subspecies contain even more.

    Given that approximately 50% of the proviruses may be deleted in the env region (Fig. 3), one cell may contain over 200 proviruses that can be detected by PCR with gag, pro, or pol primers, increasing the potential that trace amounts of mouse DNA can give rise to a positive PCR signal.

    The second source of contamination is cloned or amplified XMRV DNA, including DNA being used as a positive control in diagnostic tests. A microgram of XMRV DNA is approximately 1013 copies. Any laboratory that works with either cloned or amplified XMRV DNA is a potential source of contamination.

    The third source of contamination is inadvertent spread of XMRV originating from 22Rv1 cells to indicator cells co-cultivated with clinical samples. Although this virus is quite sensitive to human restriction factors such as tetherin and APOBECs 3F and 3G, many established cell lines, like 293T, do not express these factors, and cross-contamination can occur even in laboratories with considerable virology experience, leading to subsequent spread to other cell lines, as was observed for the 293T-XMRV cells reported here.

    Inadvertent contamination of other human cell lines provides a plausible explanation for XMRV contamination even in laboratories that have never cultured the 22Rv1 cell line.'

    Author Contributions

    Conceived and designed the experiments: MK JMC. Performed the experiments: MK AW JS FR. Analyzed the data: MK JS AW JMC WS EA. Contributed reagents/materials/analysis tools: FM SL FR. Wrote the paper: MK JMC JWM SH.
  4. Jemal

    Jemal Senior Member

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    This is not new, it was already published in november. There's another thread on this forum about it:
    http://forums.phoenixrising.me/show...n-Samples-from-Patients-Reported-to-Have-XMRV

    The other thread got no discussion though, so maybe it's for the better that we have another thread about this report.

    What's interesting is that Ruscetti is one of the authors. Does it mean he now believes XMRV is contamination or is he still working on it? Questions, questions...
  5. Firestormm

    Firestormm Senior Member

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    Thanks Jemal and sorry for not spotting it (have asked for the threads to be merged). I guess as this has the full paper attached now, it's the reason for it (?).

    Was reading through the paper earlier, and I couldn't help but ask 'Why did this take so long to publish?' Perhaps I'm missing something, but this began in 2010, right?

    'Plasma samples from 4 patients with chronic fatigue syndrome (CFS) reported to be XMRV-infected by PCR and virus isolation, performed at the Whittemore-Peterson Institute (WPI) and the Leukocyte Biology Section (LBS), NCI-Frederick, respectively, and from 5 MRV-uninfected, healthy controls were obtained from F. Ruscetti, CI-Frederick with permission from J. Mikovits, WPI (Table 1).

    All donors signed informed consent forms and the study was approved by the Institutional Review Board at the WPI (Approval ID# IRB00000215). Blood samples were drawn into EDTA-containing tubes by Phlebotomy Services International at the donors homes on January 21, 2010 and shipped overnight to LBS...'

    I also have to ask what this study says about the other 'independent' one claimed to have taken place by was it Mikovits? I can't remember right at this moment, but there was a claim that contamination had been ruled out I think.

    Would be great to have been able to see that paper if only to now compare...
  6. Bob

    Bob

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    I haven't looked at this study in detail yet, but what i've been thinking about recently is, what happens if (hypothetically) you've got MLV and XMRV (VP-62) contamination, but there is also non-contamination XMRV or PMRV etc (HGRVs) present? Does what you find, depend on what you are looking for?

    The other question is, were their primers refined to detect XMRV VP-62 rather than a wider range of XMRVs, PMRVs and any other potential HGRVs? I'll have a look at the details more closely soon.
  7. Firestormm

    Firestormm Senior Member

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    Addenda to the Science Paper: Posted June 5th 2010: http://phoenixrising.me/forums/showthread.php?5428-Addenda-to-the-Science-paper

    So.... Switzer's assay wasn't enough then? Or what?
  8. RRM

    RRM

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    I have read (elsewhere) that some people are wondering about Frank Ruscetti's part in this study. What is important to note for clarity is that LBS (Leukocyte Biology Section) is the abbreviation used for Ruscetti's lab in this paper. Everytime "LBS" is mentioned they mean Rusetti('s lab).

    Basically, Ruscetti tested the plasma 4 patients as HGRV positive and 5 controls as XMRV negative himself. Then he blinded these samples and sent them to Mary Kearney's lab (DRP).

    Ruscetti also tested 9 co-cultures (from 8) patients as being positive for HGRV's. This not only included ME/CFS patients but cancer patients as well, and did not only include blood but tissue (2 samples) and B cells (1 sample) too. He sent these co-cultures (with no controls and thus unblinded) to the DRP lab.

    DRP "confirmed" Ruscetti's patients to be positive for MLV-like sequences in plasma in the blinded setting, but they also found mouse DNA in all 4 positive samples. Contamination was determined through three different, independent methods: testing for mouse mitochondrial DNA, testing for IAP sequences through phylogenetic analysis.

    The 9 co-cultures were also examined by DRP. In this case, they didn't find MLV-like sequences but only XMRV (and in the plasma samples they did not find XMRV but only MLV-like sequences). However, these XMRV sequences were identical or essentially identical to "22Rv1 XMRV". The problem with this part of the study is that a)it wasn't blinded and b) you cannot physically test for 22Rv1 contamination at this moment like you can for mouse DNA contamination, so the authors mostly relied on phylogenetic analysis in this part of the study. I say "mostly" because they targeted a crossover region of 22Rv1, which does make their conclusions stronger.


    Even without all of the negative studies or the BWG study or any of the other findings, this is convincing evidence of Ruscetti contaminating his samples on its own. Still, because of the earlier findings, it doesn't really add or matter much.


    The problem with this argument is that it really "works" anytime. Any proof of contamination in any study could be "refuted" by saying that you have detected something "real" anyway and that the contamination assay was reacting to a (almost) identical contaminant.


    I could contaminate a lab with Coca Cola and later say that the fact that Coca Cola contaminated my samples does not mean that we have not genuinely found something extremely similar to Coca Cola in these samples after all.

    They did not detect VP62 but MLV-like sequences (comparable to Lo's) as well as "22Rv1 XMRV".
  9. Firestormm

    Firestormm Senior Member

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    Hi RRM,

    I was saying elsewhere that this seems to have taken an awfully long time to produce. I mean samples in Jan 2010, more experiments from LBS in Feb 2011; and published Nov 2012 as an extract....?

    Any comment on the time it has taken? I just wonder if this had been available before now (had been afforded maximum warp) it might have saved a lot of hassle.

    Of course Paprotka wasn't out until March 2011. Published in May 2011 or thereabouts from memory. So that part couldn't have formed a key component of this - but even so....!!
  10. Bob

    Bob

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    So you think that if there was contamination and a wild infective virus, then they wouldn't be able to distinguish the two?

    I don't agree with what you've said RRM... If the primers are designed to detect a specific variant of virus (eg VP-62/22RV1), but they are detecting similar variants, then this can be determined. Thus it is possible to determine if they are detecting 22RV1 XMRV or other variants. I haven't read this study yet, so I don't know if they have done this.

    This is something that clearly needs further research to understand exactly what's going on.


    The "22RV1 XMRV" variant is VP-62, isn't it?
  11. Sam Carter

    Sam Carter Guest

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    Hi Bob,

    My understanding is that VP62 is one example of a 22RV1-derived XMRV, as are VP42, VP35, WPI-1178 and WPI-1106.

    There is also Paprotka's "Xenotropic MuLV-related virus 22Rv1/CWR-R1" which may be ancestral (I haven't checked) to the others, but they all differ by only a few nucleotides.
  12. Bob

    Bob

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    Hi Sam,

    Thanks for that.

    I thought that only VP-62 was discovered in 22RV1, and that the others (VP-35 etc) were part of Silverman's research but not found in 22RV1.

    So I didn't think that all of Silverman's variants had been found in 22RV1.

    If I'm wrong then i've got some more reading to do!

    Can anyone clarify?
  13. Sam Carter

    Sam Carter Guest

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    Hopefully this will help, Bob.

    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018392/?tool=pubmed

    Disease-associated XMRV sequences are consistent with laboratory contamination
    Retrovirology. 2010; 7: 111.
    Stphane Hu,...,Greg J Towers


    """"""""""""""""""""""""""""""""
    Phylogenetic analysis

    22Rv1 cell line derived gag (1605 nt; n = 11), pol (1635 nt; n = 15) and env (1935; n = 8) unique sequences were manually aligned with 6 full-length XMRV sequences apparently amplified from PC samples (GenBank DQ241301, DQ241302, DQ399707, EF185282, FB579966, NC_007815), 2 full-length XMRV sequences from CFS patient samples (GenBank GQ497343, GQ497344)

    ....

    We provide several independent lines of evidence that XMRV detected by sensitive PCR methods in patient samples is the likely result of PCR contamination with mouse DNA and that the described clones of XMRV arose from the tumour cell line 22Rv1, which was probably infected with XMRV during xenografting in mice
    """"""""""""""""""""""""""""""""
    (emphasis added)

    DQ241301=VP35 DQ241302=VP42 DQ399707=VP62 GQ497343=WPI-1178 GQ497344=WPI-1106
  14. RRM

    RRM

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    They should under normal circumstances. However, in the case of the HGRV findings, the argument by proponents hinges on the idea that the "real" pathogen is (virtually) indistinguishable from known laboratory contaminants.


    I don't really understand what you are trying to say here. Can you clarify this a little, perhaps?

    In any case, Mary Kearney's lab found both "22Rv1 XMRV" (in all of Ruscetti's cultures as well as in two of the healthy controls' plasma) and the broader MLV-related variants (in all of Ruscetti's patients' plasma samples).

    It would be illogical to find 22Rv1 XMRV (or VP62 or any other "true XMRV" variant) and then report that this is the result of mouse contamination, by the way. After all, 22Rv1 (or VP62, etc) is negative for either mouse mitochondrial DNA or IAP sequences).


    No.

    VP62 is a variant that was found in the sample of a single patient and this is not the same patient as 22Rv1 was derived from. However, the sequence of VP62 is almost (99.9%) identical to 22Rv1 XMRV, and phylogenetic analysis indicates that 22Rv1 XMRV is ancestral to VP62.

    The thing that seperates VP62 from other variants (like VP42 or WPI-1106, or whatever) is that Silverman managed to turn VP62 into a plasmid after he isolated it from his patient sample. Because of this, VP62 was relatively easy to distribute to other labs to act as a positive control. Of course, 22Rv1 XMRV is equally easy to distribute (or even easier), but it was only later that 22Rv1 was found to contain XMRV as well.
    Firestormm likes this.
  15. Bob

    Bob

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    OK, I'll try... I haven't yet read the paper that started this discussion, so what i'm saying might be totally irrelevant... But the point i was trying to make was...

    If they detect a virus, but don't sequence it, then they don't know exactly which variant they have found.
    So my point was that, with further investigation, it is possible to determine exactly which variants they are detecting.
    And if VP-62 or 22RV1 XMRV is being detected, but other variants are also being detected, with wider genetic variation, then this raises lots of questions.
    Such as: Are all of the viruses/variants coming from the same source?
    This seems like quite a crucial issue to me.
    If there is a wide genetic variation in the variants being detected, then it suggests that they are not all originating from the 22RV1 cell line.


    I thought they had decided that the variant found in 22RV1 was effectively the same as VP-62...

    I also didn't know that 22RV1 is thought to be ancestral to VP62, so that's an interesting bit of information, and raises even more questions!
    Although, with a brain like mine, I can never remember what I used to know and have forgotten, and what I never knew! Anyway, it's always useful to brush up on it all!
    Firestormm likes this.
  16. Bob

    Bob

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    Thanks for that Sam. I think I'm gonna have to read that again.
    From skimming it, I can't work out how much variation there is supposed to be between all of the variants studied.

    Skimming through it briefly, it's interesting to see that two of the variants (VP-29 & VP-184) are recombinations of MLVs and Moloney MLVs. That's exactly what Switzer also found in his study, in which he concluded that they were wild human viruses.
  17. RRM

    RRM

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    It's a good question. It seems like a sensible experiment to do and they initiated it quite early, but it is unclear to me why this has taken such a long time to publish.
  18. Firestormm

    Firestormm Senior Member

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    A 'comment' has been posted at Plos One about this paper from a 'R. Roberts' (wonder if that's anyone we know?):

    http://www.plosone.org/annotation/l...notation/645c4122-165f-4956-9134-e605adf47359

    1 March 2012. It's a bit long to repost here in full but here's his summary:

    'SUMMARY

    The evidence in this paper indicates that although contamination of these samples probably did occur from multiple sources present in the DRP lab during testing, this was not the source of the MRV sequences found to be infecting the four CFS patients.

    The authors should have at least considered this a more parsimonious explanation for the inconsistent results and should have therefore retested fresh samples using the same assays without contaminated cells or mouse products present in the lab.

    The simplest way of confirming the presence or absence of contamination in the Lombardi samples is to test the Lombardi samples. It is difficult to understand why this simple orthodox scientific protocol was not used.

    MRV sequences found in patients is not found in any infected cell line or mouse ERV.

    Lombardi samples independently tested negative for mouse contamination.

    This study used unvalidated mouse contamination assays.

    The experiment needs to be repeated by an independent team of researches testing the actual samples used in Lombardi et al. and ensuring that multiple sources of contamination are not present in the study, unlike the situation in the study critiqued here.

    The HFF cells used here need to be screened for the presence of IAP particles.

    The cycling reagents, cycling conditions and primer combinations used in Lombardi et al. would allow repetition of the methods used and eliminate a major confounding variable.

    The performance of any novel real-time assay can then be objectively compared to the performance of the assay, which appears to be the most successful in Lombardi et al., and their specificities and sensitivities compared.

    Testing the samples for mouse contamination should be the first step to avoid the very real possibility in this experiment that samples were handled differently because the testing history was known.

    Two real-time assays should be used targeting a sequence unique to the VP62 strain of XMRV and using a set of primers to allow for sequence variability. This would allow the performance of the novel X-SCA assay used here to be compared to a real-time assay, which has demonstrated the ability to detect VP62 like sequences in people shown to be infected using IHC.

    The flow cytometry assay used to detect monoclonal antibodies to SFFV env needs to be repeated.'
  19. Firestormm

    Firestormm Senior Member

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  20. RustyJ

    RustyJ Contaminated Cell Line 'RustyJ'

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    I haven't the concentration to really understand the points of the response. Is it a valid comment? Who is ME Advocacy Association? Who is R. Roberts?

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