Original Science Article/Unblinded Study

Cort

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Thanks to Sue for Transcribing the article. Hopefully Science will let us (generally poor) patients check it out on the forums. I added my own comments (such as they are)]. It really is a very impressive paper. Annette Whittemore said they had to jump through a lot of hoops in order to get CFS in there- they certainly looked at this thing from a lot of different angles.

Science Express, 8 October 2009
www.sciencexpress.org/

Detection of an Infectious Retrovirus, XMRV, in Blood Cells of Patients with Chronic Fatigue Syndrome

Vincent C. Lombardi,1* Francis W. Ruscetti,2* Jaydip Das Gupta,3 Max A. Pfost,1 Kathryn S. Hagen,1 Daniel L. Peterson1 Sandra K. Ruscetti,4 Rachel K. Bagni,5 Cari Petrow-Sadowski,6 Bert Gold, 2 Michael Dean, 2 Robert H. Silverman, 3 Judy A. Mikovits1†

1Whittemore Peterson Institute, Reno, NV 89557, USA. 2Laboratory of Experimental Immunology, National Cancer Institute-Federick, Frederick, MD 21701, USA. 3Department of Cancer Biology, The Lerner Institute, The Cleveland Clinic, OH 44106, USA. 4Laboratory of Cancer Prevention, National Cancer Institute-Frederick, Frederick, MD 21701, USA. 5Advanced Technology Program, National Cancer Institute-Frederick, Frederick, MD 21701, USA. 6Basic Research Program, Scientific Applications International Corporation, National Cancer Institute-Frederick, Frederick, MD 21701, USA

*These authors contributed equally to this work.

†To whom correspondence should be addressed. E-mail: judym@wpinstitute.org


Chronic fatigue syndrome (CFS) is a debilitating disease of unknown etiology that is estimated to affect 17 million people worldwide. Studying peripheral blood mononuclear cells (PBMCs) from CFS patients, we identified DNA from a human gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), in 68 of 101 patients (67%) compared to 8 of 218 (3.7%) healthy controls. Cell culture experiments revealed that patient-derived XMRV is infectious and that both cell-associated and cell-free transmission of the virus are possible. Secondary viral infections were established in uninfected primary lymphocytes and indicator cell lines following exposure to actived PBMCs, B cells, T cells, or plasma derived from CFS patients. These findings raise the possibility that XMRV may be a contributing factor in the pathogenesis of CFS.

Chronic fatigue syndrome (CFS) is a disorder of unknown etiology that affects multiple organ systems in the body. Patients with CFS display abnormalities in immune system function, often including chronic actiation of the innate immune system and a deficiency in natural killer (NK) cell activity (1,2). A number of viruses, including ubiquitous herpesviruses and enteroviruses have been implicated as possible environmental triggers of CFS (1). Patients with CFS often have active  herpesvirus infections, suggesting an underlying immune deficiency.

The recent discovery of a gammaretrovirus, XMRV, in the tumor tissue of a subset of prostate cancer patients prompted us to test whether XMRV might be associated with CFS. Both of these disorders, XMRV-positive prostate cancer and CFS, have been linked to alterations in the antiviral enzyme RNase L (3-5). Using the Whittemore Peterson Institute’s (WPI) national tissue repository, which contains samples from well-characterized cohorts of CFS, we isolated nucleic acids from PBMCs and assayed the samples for XMRV gag sequences by nested PCR (5, 6).

Cort - This transcription does not have the supplement which listed the patient characteristics more fully. Here they are: The patients meet both the Fukuda and Canadian Consensus criteria for chronic fatigue syndrome. They were severely disabled, had various immunological abnormalities (RNase L dysregulation, natural killer cell cytotoxicity, increased IL-6 and levels and extremely low VO2 max levels during exercise testing.

(The WPI later stated that not all patients had all these immunological abnormalities. The Klimas/Fletcher research group just published a study finding increased Il-6 but decreased Il-8 cytokine levels in chronic fatigue syndrome patients). My understanding was that the WPI used blood samples in their national tissue repository to test for the virus but I think there's some controversy over this.)
Of the 101 CFS samples analyzed, 68 (67%) contained XMRV gag sequence. Detection of XMRV was confirmed in 7 of 11 WPI CFS samples at the Cleveland Clinic by PCR-amplifying and sequencing segments of XMRV env (352 nt) and gag (736 nt) in CFS PBMC DNA (Fig. 1A) (6).

CORT- Initially they just looked at one sequence (gag) which is not as comprehensive as one might like but when they sent the samples to the Cleveland clinic the Cleveland clinic looked at two sequences (gag and env). Both sequences matched up with XM RV and in similar proportions of chronic fatigue syndrome patients (about 2/3rds). That added reliability to the results in two ways; a different group did their own PCR and they did a more comprehensive search.

They did not, however, do an unblinded study which is one of the things its been suggested to me that may have gotten De Freitas in trouble. I don't know know how important it is to do 'double blinded studies' with regards PCR; is there much room for subjective error? I don't know. I have been told that the study's upcoming likely be double blinded; ie no one knows which samples came from patients or healthy controls.
In contrast, XMRV gag sequences were detected in 8 of 218 (3.7%) PBMC DNA specimens from healthy individuals. Of the 11 healthy control DNA samples analyzed by PCR for both env and gag, only one sample was positive for gag and none for env (Fig. 1B). In all positive cases, the XMRV gag and env sequences were more than 99% similar to those previously reported for prostate tumor-associated strains of XMRV (VP62, VPf35, and VP42) (fig. S1) (5).

Cort -About 4% of the 218 specimens from healthy individuals tested positive for XMRV gag. They didn't look at those 8 samples again; instead they took 11 more samples and analyze them for both env and gag. None of them had both.
Sequences of full-length XMRV genomes from two CFS patients and a partial genome from a third patient were generated (table S1). CFS XMRV strains 1106 and 1178 each differed by six nucleotides (nt) from the reference prostate cancer strain XMRV VP2 (EF185282), and with the exception of one nt, the variant nucleotides mapped to different locations within the XMRV genome, suggesting independent infections.

Cort - They then generated sequences of the XMRV found in two patients and compared them to the standard reference XMRV strain. Because the differences found in the XMRV strains were found in different locations in the genome they weren't copies of each other; ie the XMRV in each patient had a different history and was probably from a different infection. This suggested to me that one bug likely did not pervade all the positive samples - which was probably a question given that there was high genetic similarity between the different samples - but apparently not too high. This
By comparison, prostate cancer-derived XMRV strains VP35 and VP42 differed from VP62 by 13 and 10 nt, respectively. Thus, the complete XMRV genomes in CFS patients are > 99% identical in sequence to those detected in patients with prostate cancer.

To exclude the possibility that we were detecting a murine leukemia virus (MLV) laboratory contaminant, we determined the phylogenetic relationship between endogenous (nonecotropic) MLV sequences, XMRV sequences, and sequences from CFS patients 1104, 1106 and 1178 (fig. S2). XMRV sequences from the CFS patients clustered with the XMRV sequences from prostate cancer cases and formed a distinct branch from nonecotropic MLVs common in cases of inbred mouse strains. Thus, the virus detected in the CFS patients’ blood samples is unlikely to be a contaminant.

Cort They determined that the XMRV was very much like prostrate cancer XMRV and quite a bit different from the murine leukemia virus found in ; ie the XMRV they found is not a contaminant that derived from laboratory mice. This was a big question because XMRV is believed, if I have my facts right, to come from MLV.
To determine whether XMRV proteins were expressed in PBMCs from CFS patients, we developed intracellular flow cytometry (IFC) and Western blot (WB) assays, using antibodies (Abs) with novel viral specificities. These antibodies included among others: (i) rat monoclonal antibody (mAb) to the spleen focus-forming virus (SFFV) envelope (Env), which reacts with all polytropic and xenotropic MLVs (7), (ii) goat antisera to whole mouse NZB xenotropic MLV; and (iii) a rat mAb to MLV p30 Gag (8). All of these Abs detected the human VP62 XMRV strain grown in human Raji, LNCaP and Sup-T1 cells (fig. S3) (5). IFC of activated lymphocytes (6, 9) revealed that 19 of 3 PBMC samples from CFS patients reacted with the anti-MLV p30 Gag mAb (Fig. 2A). The majority of the 19 positive samples also reacted with antisera to other purified MLV proteins (fig. S4A). In contrast, 16 healthy control PBMC cultures tested negative (Fig. 2A, fig. S4A).

These results were confirmed by Western blots (Fig. 2B and C) (6) using Abs to SFFV Env, mouse xenotropic MLV and MLV p30 Gag. Samples from five healthy donors exhibited no expression of XMRV proteins (Fig. 2C). The frequencies of CFS cases vs. healthy controls that were positive and negative for XMRV sequences were used to calculate a Pearson 2 value of 154 (two-tailed P value of 81 x 10-35). These data yield an odds ratio of 541 (95% confidence interval of 23.8-122). Suggesting a non-random association with XMRV and CFS patients.

To determine which types of lymphocytes in blood express XMRV, we isolated B and T cells from one patient’s PBMCs (6). Using mAb to MLV p30 Gag and IFC, we found that both activated T and B cells were infected with XMRV (Fig. 2D, fig. S4A). Furthermore, using mAb to SFFV Env, we found that >95% f the cells in a B-cell line developed from another patient were positive for XMRV Eng (Fig. S4B). XMRV protein expression in CFS patient-derived activated T and B cells grown for 42 days in culture was confirmed by Western blots (fig. S4C) using Abs to SFFV Env and xenotropic MLV.

Cort - Now that they had evidence XMRV DNA was present in the immune cells of chronic fatigue syndrome patients they searched for evidence that those reacting to it. If the cell is infected with XMRV it should produce antibodies to it . RFirst they looked for antibodies (to other mouse viruses) which they knew also reacted with XMRV. They found that depending on which antibody they looked for from 100% to 2/3rds to 50% of the 30 samples tested positive for them. None of the 20 samples from healthy controls did.

Next they tried to determine which of the immune cells in the samples they had contained the virus. They found that both T. and B. cells reacted positively to the antibody tests noted above.

We next investigated whether the viral proteins detected in PBMCs from CFS patients represent infectious XMRV. Activated lymphocytes (6) were co-cultured with LNCaP, a prostate cancer cell line with defects in both the JAK-STAT and the RNase L pathways (10, 11) that was previously shown to be permissive for XMRV infection (12). After co-culture with activated PBMCs from CFS patients, LNCaP cells expressed XMRV Env and multiple XMRV Gag proteins by Western Blot (Fig. 3A) and IFC (fig. S5A).

Transmission electron microscopy (EM) of the infected LNCaP cells (Fig. 3B) as well as virus preparations from these cells (Fig. 3C) revealed 90-100 nm diameter budding particles consistent with a gamma (type C) retrovirus (13).

We also found that XMRV could be transmitted from CFS patient plasma to LNCaP cells when we applied a to enhance infectivity. (6, 14, 15). Both XMRV gp70 Env and p30 Gag were abundantly expressed in LNCaP cells incubated with plasma samples from 10 of 12 CFS patients, whereas no viral protein expression was detected in LNCaP cells incubated with plasma samples from 12 healthy donors (Fig. 3A). Likewise, LNCaP cells incubated with patient plasma tested positive for XMRV p30 Gag in IFC assays (fig S5B).

We also observed cell-free transmission of XMRV from the PBMCs of CFS patients to the T-cell line SupT1 (Fig. 4B) and both primary and secondary transmission of cell-free virus from the activated T cells of CFS patients to normal T cell cultures (Fig. 4C). Together, these results suggest that both cell-associated and cell-free transmission of CFS-associated XMRV are possible.

Cort - Infectious or Latent? But was XMRV infectious in the body? The WPI had not yet identified the virus outside of the cells and until they did so they couldn't say that the patient had an ‘active’ infection - ie that it was moving from cell to cell. They put XMRV infected white blood cells into a culture containing XMRV-free cells and then were able to detect XMRV sequences in the previously non-infected cells; XMRV had left those infected lymphocytes and infected the new cells.

Then in probably the most important step they were able to show that cell free blood from an ME/CFS patient was able to infect several different types of white blood cells. Without showing actual evidence of XMRV in the plasma the WPI had shown that cell to cell transmission of XMRV to a variety of cell types (not just prostate cancer cells) was possible and even likely.

Finally when they looked at these cells under an electron microscope they found what looked like mouse type viruses budding from them.

We next investigated whether XMRV stimulates an immune response in CFS patients. For this purpose, we developed a flow cytometry assay that allowed us to detect antibodies to XMRV Env by exploiting its close homology to SFFV Env (16). Plasma from 9 out of 18 CFS patients infected with XMRV reacted with a mouse B cell line expressing recombinant SFFV Env (BaF3ER-SFFV-Env) but not to SFFV Env negative control cells (BaF3ER), analogous to the binding of the SFFV Env mAb to these cells (Fig. 4D and S6A).

In contrast, plasma from seven healthy donors did not react (Fig. 4D and fig. S6A). Furthermore, all nine positive plasma samples from CFS patients but none of the plasma samples from healthy donors blocked the binding of the SFFV Env mAb to SFFV Env on the cell surface (fig. S6B). These results are consistent with the hypothesis that CFS patients mount a specific immune response to XMRV.

Neurological maladies and immune dysfunction with inflammatory cytokine and chemokine upregulation are some of the most commonly reported features associated with CFS. Several retroviruses including the MLVs and the primate retroviruses, HIV and HTLV-1, are associated with neurological diseases as well as cancer (17).

Studies of retrovirus-induced neurodegeneration in rodent modes have indicated that vascular and inflammatory changes mediated by cytokines and chemokines precedes the neurological pathology (18, 19). The presence of infectious XMRV in lymphocytes may account for some of these observations of altered immune responsiveness and neurological function in CFS patients.

Cort - They indicate that in mice retroviruses first alter vascular (blood vessel) functioning and cause inflammation by triggering the release of cytokines and chemokines. (Again we have the scenario of the immune system causing the damage not the virus per se!). Neurodegeneration comes later This is a nice tie in between inflammation and vascular problems and central nervous system problems. This would seem to make sense - people with more severe chronic fatigue syndrome seem to have more and more neurological problems

The evidence for cytokine problems in CFS is pretty scattered; its hard to get consistent cytokine abnormalities. At some point the WPI is going to want to tie XMRV infection to the immune response and then tie this to the problems in chronic fatigue syndrome. Perhaps the varied cytokine results are caused by a heterogeneous population. But then again we've been told that 95% of patients will have XMRV - which doesn't sound very heterogeneous. Just who has this and who doesn't is of course one of the big questions
In summary, we have discovered a highly significant association between the XMRV retrovirus and CFS. This observation raises several important questions.

Is XMRV infection a causal factor in the pathogenesis of CFS or a passenger virus in the immunosuppressed CFS patient population? What is the relationship between XMRV infection status and the presence or absence of other viruses that are often associated with CFS (e.g., herpesviruses)? Conceivably these viruses could be cofactors in pathogenesis, as is the case for HIV-mediated disease, where co-infecting pathogens play an important role (20). Patients with CFS have an elevated incidence of cancer (21). Does XMRV infection alter the risk of cancer development in CFS?

As noted above, XMRV has been detected in prostate tumors from patients expressing a specific genetic variant of the RNASEL gene (5). In contrast, in our study of this CFS cohort, we found that XMRV infection status does not correlate with the RNASEL genotype (6) (table S2).

Cort XMRV infection does not correlate with the RNase L. genotype - they don't say anything about the RNase L abnormality found in chronic fatigue syndrome patients. At least according to this paper we don't know if having high rates of RNase L. fragmentation could contribute to the likelihood of XMRV infection
Finally, it is worth noting that 3.7% of the healthy donors in our study tested positive for XMRV sequences. This suggests that several million Americans may be infected with a retrovirus of as yet unknown pathogenic potential.


References and Notes.

1. L.D. Devanur, J.R. Kerr, J. Clin. Virol. 37, 139 (2006).
2. T.L. Whiteside, D. Friberg, Am. J. Med 105, 27S (1998).
3. R.J. Suhadolnik et al., J. Interferon Cytokine Res. 17, 377 (1997).
4. G. Casey et al., Nat. Genet. 32, 580 (2002).
5. A. Urisman et al., PLoS Pathog. 2, 211 (2006).
6. Materials and methods are available as supporting material on Science Online.
7. Wolff, R. Koller, S. Ruscetti, J. Virol. 43, 472 (1982).
8. B. Chesebro et al., Virology 127, 134 (1983).
9. K.A. Smith and F.W. Ruscetti, Adv. Immunol. 31, 137 (1981).
10. G. Dunn, K. Sheehan, L. Old, R. Schreiber, Cancer Res. 65, 3447 (2005)
11. Y. Xiang et al., Cancer Res. 63, 6795 (2003)
12. B. Dong et al., Proc. Nat. Acad. Sci. U.S.A. 104, 1655 (2007)
13. B.J. Poiesz et al., Proc. Natl. Acad. Sci. U.S.A. 77, 7415 (1980).
14. G.R. Pietroboni, G.B. Harnett, M.R. Bucens, J.Virol. Methods 24, 85 (1989)
15. S.M. Yoo et al, J. Virol. Methods 154, 160 (2008).
16. L. Wolff, E. Scolnick, S. Ruscetti, Proc. Natl. Acad. Sci. U.S.A. 80, 4718 (1983)
17. C. Power, Trends Neurosci. 24, 162 (2001)
18. X. Li, C. Hanson, J. Cmarik, S. Ruscetti J. Virol. 83, 4912 (2009)
19. K.E. Peterson, B. Chesebro Curr. Top. Microbiol. Immunol. 303, 67 (2006)
20. A. Lisco, C. Vanpouille, L. Margolis Curr. HIV/AIDS Rep. 6, 5 (2009)
21. P.H. Levine et al., Cancer Res. 52, 5516s (1992)
22. We thank D. Bertolette, Y. Huang, C. Hanson and J. Troxler for expert technical assistance; K. Nagashima for electron microscopy; and C. Ware and K. Hunter for thoughtful discussions. Funded by the Whittemore Peterson Institute and the Whittemore Family Foundation; the National Cancer Institute; the National Institutes of Health (under contract HHSN26120080001E); and grants to R.H.S. from NCI/NIH (CA104943) the U.S. Department of Defense Prostate Cancer Research Program W81XWH-071338, V Foundation for Cancer Research, Charlotte Geyer Foundation, and Mal and Lea Bank. The content of this publication does not reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. R.H.S. may receive royalty payments in the future from Abbott Laboratories, Inc. GenBank accession numbers are as follows: WPI-1130, GQ483508; WPI-1138, GQ483509; WPI-1169, GQ483510; WPI-1178, GQ497343; WPI-1106, GQ497344; WPI-1104 GQ497345.


Supporting Online Material

www.sciencemag.orgcgi/content/full/1179052/EC1
Materials and Methods
Figs. S1 to S6
Tables S1 and S2
References

14 Jul 2009; accepted 31 August 2009
Published online 8 October 2009; 10.1126/science.1179052
Include this information when citing this paper.

Fig. 1. XMRV sequences in PBMC DNA from CFS patients. Single round PCR for gag, env and gapdh sequences in PBMCs of (A) CFS patients and (B) healthy controls. The positions of the amplicons are indicated and DNA markers (ladder) are shown. Representative results from one group of 20 patients are shown.

Fig. 2. Expression of XMRV Proteins in PBMCs from CFS patients. (A) PBMCs were activated with PHA and IL-2, reacted with a mAb to MLV p30 Gag and analyzed by IFC. (B) Lysates of activated PBMCs from CFS patients (lanes 1-5) were analyzed by Western blots with rat anti-SFFV Env mAb (top panel), goat anti-xenotropic MLV (middle panel) or goat anti-MLV p30 Gag (bottom panel). Lane 7: lysate from SFFV-infected HCD-57 cells. At left: molecular weight markers in kD. (C) Lysates of activated PBMCs from healthy donors (lanes 1, 2, 4, 5, and 7) or from CFS patients (lanes 3 and 6) were analyzed by Western blots using rat anti-SFFVEnv mAb (tp panel) or goat anti-MLV p30 Gag (bottom panel). Lanes 8: SFFV-infected HCD-57 cells. At left: molecular weight markers in kD (D) CD4+ T cells (left) or CD19+ B cells (right) were purified, activated and examined by flow cytometry for XMRV Gag using an anti-MLV p30 Gag mAb.

Fig. 3. Infectious XMRV in PBMCs from CFS patients. (A) Lysates of LNCaP cells co-cultured with PBMCs from CFS patients (lanes 1, 3, nd 5) or healthy donors (lanes 2 and 4) were analyzed by Western blots with rat anti-SFFV Env mAb (tp panel) or goat anti-xenotropic MLV (bottom panel). Lane 6: uninfected LNCaP; ane 7: SFFV-infected HCD-57 cells. At left: molecular weight markers inn kD. (B) Transmission electron micrograph of LNCaP cells infected by incubation with an activated T cell culture from a CFS patient. (C) Transmission electron micrograph of virus particles released by infected LNCaP cells.

Fig. 4. Infectious XMRV and antibodies to XMRV in CS patient plasma. (A) Plasma from CS patients (lanes 1-6) were incubated with NCaP cells an lysates prepared after six passages. Viral protein expression was detected by Western blots with rat anti-SFFV Env mAb (top panel) or goat anti-MLV p30Gag (bottom panel). Lane 7: uninfected LNCaP; lane 8: SFFV-infected HCD-57 cells. At left: molecular weight markers in kD. (B) Cell-free transmission of XMRV to the SupT1 cell line was demonstrated using transwell co-culture with patient PBMCs followed by nested gag PCR. Lane 1: MW marker. Lane 2: SupT1 co-cultured with Raji. Lanes 3-7 SupT1 c-cultured with CS patient PBMCs. Lane 8: No template control (NTC). C) Normal T cells were exposed to cell-free supernatants obtained from T cells (anes 1,5,6) or B cells (lane 4) from CFS patients. Lanes 7 and 8 are secondary infections of normal activated T cells. Initially, uninfected primary T cells were exposed to supernatants from patients WPI-1220 (lane 7) and WPI-1221 (lane 8) PBMCs. Lanes 2 and 3: uninfected T cells; Lane 9: SFFV-infected HCD-57 cells. Viral protein expression was detected by Western blot using a rat anti-SFV Env mAb. At left: molecular weight markers in kD. (D) Plasma samples from a CFS patient or from a healthy control as well as SFFV Env mAb or control were reacted with BaF3ER cells (top) or BaF3ER cells expressing recombinant SFFV Eng (bottom) and analyzed by flow cytometry.

www.sciencexpress.org/8 October 2009
 

Cort

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Thanks - the paper really is very impressive in its breadth; they tested this thing back and forth. Whatever they found showed up again and again.

We're hearing all sorts of rumors but at the worst it looks to me - a laymen - like they must have found SOMETHING. I don't know how you can build up all the stuff they did on NOTHING. Maybe you can, I'm no scientist, but the number of things they did successfully was quite impressive.
 

Cort

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Blinded vs double-blinded

The PCR study was unblinded - which I've been told was a problem with DeFreitas retrovirus study; once they blinded it (no one knew where any one sample came from) her results tanked. That's not necessarily because the original test was unblinded - but it could apparently have played a role.

My question is how much subjectivity is there in interpreting these tests? I assumed they were all computer driven. Does anyone know?
 

Esther12

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The PCR study was unblinded - which I've been told was a problem with DeFreitas retrovirus study; once they blinded it (no one knew where any one sample came from) her results tanked. That's not necessarily because the original test was unblinded - but it could apparently have played a role.

My question is how much subjectivity is there in interpreting these tests? I assumed they were all computer driven. Does anyone know?
I'm really unsure on this too. Especially considering the varying/conflicting results from prostate cancer.
 
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I'm not certain, but I think its pretty common not to be blinded or at least not to report it.

DeRisi wants a fully-blinded replication, and its certainly a good idea.

Its quite possible to be biased in a subtle way. You know, youre aware yhat youre handling the patient samples right now, and whoops, you are a little more careful to handle them carefully and not get a false negative result.
 

Parismountain

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XMRV Science Article

I got awarded the best science student award in high school by the science teachers. I then took a college clep test which with my knowledge of science, I was able to skip science completely in college. So I had promise but my other interests kept me away from something I had aptitude in.

I mention that because my ignornace of scientific method is large at this point. Can somebody explain to me in the sampling population that was chosen for the XMRV paper, were the DNA lab folks blind as to which group the blood they were testing came from? Is that just standard in studies to blind the samples so a lab tech can't know if he's looking at a sample with a high chance of positive result or a low chance?

I've just got to assume the lab techs do not know when working on a sample the population it came from. If I make that assumption I don't see how in the world three different labs can be wrong about XMRVs link the CFS. Stated positively, I can't imagine a way for this research to be wrong.

You test the blood, it's positive or negative for XMRV. Then you match the identifier on the blood sample back to the patient and the group they reside in.

The further refinement that went to the high 90s in the CFS group, has there been a statement as to how the 3.6%ish healthy number rose when the refined test was run? I haven't run across Peterson mentioning that but maybe he has.
 

fresh_eyes

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Thanks for this question, Parismountain. I need some clarification on this too. My understanding is that the study was not blinded. I don't know if that means that the actual lab techs knew which samples were which, or just the people running the study. Anybody?
 
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George

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Unblinded

Yes they knew which ones were which. This study was not a medical study. They will get to the blind and double blind test later. This paper was specifically designed to be peer reviewed by Science. (this was well thought out politics)

The rigor was in showing the following;

  1. That XMRV was a valid retrovirus and not a undefined particle
  2. That is was infectious in PMBC, specifically in T, B and NK cells. Therefore it acted in a way associated with retrovirus'.
  3. That it was highly infectious in an activated state.

They chose to use CFS patients versus healthy controls because they were more or less sneaking this (Us) information in. They already knew more or less what the results were going to be. Because they had already started testing and got specific results then went back and did a completely redesigned study incorporating the NCI and Cleavland Clinic for an extra layer of protection.

The reason they did not try to do a medical blind study was because of the problem of criteria. The criteria has become so distorted over the last 20 years world wide that the first hurdle they have to over come is redefining the criteria.

So they designed a scientific paper that would catch the attention of the appropriate people, they raised questions rather than produced answers. The scientific community can not resist questions and the political community can't resist doing something for infectious diseases.

This was the first step that had to be taken in order to get others to take the next steps. And it worked. The next step will be validating, then redefining the criteria for the illness, through blind and double blind. Then establishing a definition (probably a rename) next up testing, then treatments. It doesn't always roll that way but they are all the boxes that have to be checked off.

That's why we all need to settle in and wait.:(:(:(
 

Marylib

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George!

Thanks again.

So this means that WPI and those researchers already working on duplicating the study will indeed be redefining CFS? At least for the purposes of science? -- I won't even "go there" with the CDC... Am I wrong or is this just peachy -- terribly lovely wonderful news? or is is old news and I am just now getting it. :eek:
 
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George

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You knew you just weren't thinking about it.

Thanks again.

So this means that WPI and those researchers already working on duplicating the study will indeed be redefining CFS? At least for the purposes of science? -- I won't even "go there" with the CDC... Am I wrong or is this just peachy -- terribly lovely wonderful news? or is is old news and I am just now getting it. :eek:
I think sometimes we are just too close to what's going on. I know the more time that's passed the more I've been able to step back and see the bigger picture.

I realized, watching all the interviews and reading all the papers, there was no way they could do a medical CFS study with XMRV because they would have set themselves up to be attacked by whoever wanted to jump on them regarding how they were defining CFS. So they had to take the approach of dealing with the retrovirus as a scientific evidence and introducing the PWC's more as a coincidence than the main event.

Even being so careful they are still attacked from time to time regarding the cohort. And of course that was the first thing that Reeves mentioned when he said that the paper wasn't replicable.

And he's right, not using his definition. The big question the WPI paper asked was who are these people who are sick and have this virus? So now the HHS, the NCI, and researchers around the world are going to be grappling with that question. CFS will be poked, prodded, bent, mushed, mashed, broken and reassembled in order to define who are the people who are ill who have this virus.

At the same time they are going to have to wade through every aspect of the definitions that are out their now and that means that even someone without XMRV/XAND is going to be scrutinized over how they ended up with a CFS lable.

It's going to take a very long while. (grins)
 

Parismountain

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Thanks

Thanks Cort for linking my question to the thread that was already going.

I've read today's explanation about slipping in CFS, political angles, how to define the group to be studied, that all makes sense what WP did. I'm dreading though that it wasn't blinded. Yeah they knew what they were going to end up because they had already discovered the link and so they did what they did for the publication but I'm scratching my head over why they didn't blind the lab as to control versus probable CFS blood. I could list probably 5 doctors who I'd trust to pony up 20 patients each of their strongest CFS diagnosis candidates and pick 100 healthy controls but boy at least don't let the technicians know which is which. Or do it twice with different techs. One set knows, one set doesn't. Then compare.

Maybe we'll know shortly how other researchers and labs are coming along with attempts to validate. I know I sound skeptical but I completely missed the blinded part.