1. All of the controls were not screened by all of the labs.
Response: Controls were screened by at least five labs: WPI, National Cancer Insitute/NCI-Ruscetti, Food and Drug Administration/FDA-Lo, Centers for Disease Control & Prevention (CDC) and NCI/Drug Resistance Program (DRP).
"Blood specimens were collected multiple times from three consenting laboratory controls under local IRB approval and WB, plasma and PBMCs were tested by all of the participating laboratories for XMRV/MLVs, using nucleic acid amplification testing (NAT), serology and virus culture techniques." - SOM
There were 3 lab techs that were used as controls. Only those 3 were screened by all labs, using all methods. A lab tech working with the viruses is likely to become infected. So they
could have been negative before blinding and positive after. The other controls were not screened with all methods by all labs and the blood group are not saying if any clinically validated method was used on any of the controls.
"Similarly, fifteen control specimens from blood donors (n=12) or laboratory controls (n=3) that had been established as negative for XMRV and MLVs by PCR, serology and culture by multiple laboratories, were collected, processed and aliquoted in parallel (17)." - Main text
The 'or' indicates they could choose either the 3 lab controls or the 12 blood donors to send to those labs. Note, they screened for XMRV and MLVs. Not MRVs, more on this at the end of this post.
2. Control peripheral blood mononuclear cells (PBMCs) were not screened prior to blinding, so could not have been ruled as negative.
Response: Three out of the 15 did have their PBMCs extensively screened prior to blinding, yet two of these were still called “positive” in various assays by the WPI and NCI/Ruscetti in the study.
The 3 were the lab techs, not the people who were used as controls. Lab techs are exposed to the virus. They should not have been used as controls. The other 12 did not have their PBMCs screened prior to blinding.
3. No cryopreservative was used for the storage of the PBMCs, which would prevent the WPI’s assay from working. No Trizol was used.
Response: Due to the short-term nature of the study it was not felt that preservatives were required for PBMC cryopreservation. The Lo/Alter study detected sequences in PBMCs stored for 15 years in the absence of preservatives. Trizol is for the extraction of nucleic acid and laboratories were given the option of choosing their own extraction methods
The study should not have been short term. They should be protecting the blood supply, and it doesn't matter how long it takes, only that it is done correctly. No trizol and no preservative was used, which stopped the WPIs assay from working.
Because the PBMCs were not preserved in trizol and also exposed to a slow thaw, the samples would have been useless as far as the sophisticated techniques of Dr Ruscetti were concerned. Dr Lombardi being inexperienced simply did not notice. Matrix effects induced by poor sample prep affect the avidity of antibodies and hence the antigen antibody bonding will not survive the various washing processes.
4. The length of time allotted for the serology and culture assays was massively reduced, so that the WPI or NCI/Ruscetti assays were not performed as desired.
Response: All the laboratories were allowed as much time as required to perform their desired assays. The culture and serological assays were performed by WPI and NCI/Ruscetti to their own specifications.
This changed the assays from being clinically validated to not being clinically validated. They rushed the study. The study has to be done again, correctly this time with only clinically validated assays.
5. The WPI was not given the opportunity to complete virus culture assays.
Response: The WPI encountered mycoplasma contamination of their target cell population, and used the plasma samples without results. This was very unfortunate. There were no further stocks left to perform repeat cultures with. It was deemed by both the WPI and the working group that performing the studies on freeze/thawed material would be invalid.
Again they did not design the study well enough and should have had these available, if they wanted to get an accurate result. This is the blood supply, not a science fair. The study should be redone correctly!
6. Samples and collection tubes were handled in the same laboratory as 22Rv1 cells used to spike the analytical controls.
Response: As stated in the paper, 22Rv1 cells were handled in a separate facility to where all other activities were performed. The fact that only one laboratory detected PCR and virus culture in clinical samples supports the fact that 22Rv1 contamination did not occur at the central laboratory.
22Rv1 was in the CDC lab with a portion of collection tubes, so yes one labs samples could be contaminated.
"Spiked controls
22Rv1 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics. In order to create positive controls for NAT assays for PBMC and WB, ten 22Rv1 cells per aliquot of PBMC and WB from one of the three laboratory controls were added.
For spiking of plasma, a large batch of supernatant from 22Rv1 cells was prepared from a T-75 flask plated at 60% confluency and grown for four days. Supernatant was collected and 0.45 μm filtered. An aliquot was tested by the CDC laboratory using six different real-time RT-PCR assays (19, 24-26). " - SOM
"A single lot of EDTA BD Vacutainer Blood collection tubes (BD Biosciences) was purchased and distributed to two laboratories (CDC and NCI/DRP) for testing to ensure contamination with XMRV or mouse DNA was not present." - SOM
7. Patients were on additional therapies that would produce false negatives.
Response: Lo/Alter patients were not on any additional treatments. It is unclear what additional treatments patients were on at the time of Lombardi et al. There is no published evidence that additional treatments would have positive or negative effects.
The WPI patients were on additional therapies. We don't have the information about the others. There are loads of studies that show these treatments would affect results! Many drugs have an anti-retroviral effect, from Doxycycline on. Hands up who's not got a pill-sorter nearby?
8. FDA/Lo used the wrong assay from Lo et al. and instead used the one that could not detect positives.
Response: Lo et al. used their own criteria to decide on which assay(s) to use, but it is clear that both primer sets in their paper are equally capable of amplifying diverse polytropic murine leukemia viruses (MLVs), so it is not obvious that one would be better that the other at detecting “positives.”
It doesn't matter that they chose it. They still chose to use an assay that had never found a patient positive... Primers are only one variable that can be altered to change the sensitivity of an assay. There is buffer, salts, magnesium, annealing temperature...
9. The NCI-Ruscetti did no PCR and could not use their clinically validated serology and culture assays.
Response: NCI-Ruscetti felt that they were not sufficiently experienced at PCR to participate in the study. They did perform their serology and culture assays – just as performed in Lombardi et al.
So did the NCI tell Ruscetti he could not run PCR? That reply suggests they did.
The culture and serology found positives. The issue is the messing up of controls previously. There were no true controls in the study. Those people are positive for the viruses!
10. All the SRWG labs optimized their assays to VP62. VP62 does not exist in nature and Lombardi et al. is now known to have discovered HGRVs. Does your study include HGRVs? Or how do HGRVs relate to XMRV?
Response: As demonstrated in an earlier slide, although this study was initiated after Lombardi et al. as a study of XMRV, as soon as Lo et al. was published the mission of the study was broadened to include all MLV-like viruses. Thus, almost all of the assays were designed to perform against MLVs in general and were optimized and tested as such. As our study has demonstrated there is no such thing as an independently validated clinically positive sample against which to test. Currently there is no such thing as human gammaretroviruses (HGRV). No published virus has been isolated, cloned or sequenced from a human.
An assay designed to perform against MLVs is an assay designed to detect mouse viruses, not MRVs. The retroviruses found in ME patients are MRV's. The Genbank bank WPI env show this. They could not have broaded the assays. All PCR assays were optimised to a synthetic virus (VP62), not a wild type virus, or any other virus. See Hanson's study. She used a clinical positive to validate.
Lombardi, who performed testing for this study, changed the PCR assay and it was not the assay from Lombardi et al. The assay he used here is not clinically validated.
Lombardi et al. isolated the viruses. Others have isolated too, cloned and sequenced. VP42 and VP35 are clones of the prostate cancer HGRVs. They are not VP62. VP35 was cloned before VP62.
I have a whole selection of quotes where countries admit to HGRVs. Let me know if you'd like to see them.
Bell's patients in the Hanson study were the MRV positives. There was no positive in Levine's patients. Bell uses the CCC, Levine the Fukuda. What diagnostic protocol did the BWG III use? Apart from the Lo patients, it was Fukuda, which
diagnoses +/-40% wrongly. Alter and Lo seem to have used Fukuda too, though it is not explicitly spelled out in the paper as far as I can see. Certainly the only diagnostic reference is to Fukuda.
And then we come onto the question of coding - who, how, where, when? And that's another nest of worms, for BWG III and for Lipkin.
We know very little about the Lipkin study. If it is a replication of Lombardi, even then it cannot be called definitive. We already know that the doctors providing the patients used Fukuda, which means it's not a replication, as Lombardi et al used the Canadian. I do hope the full paper is open access...
You cannot have a definitive study. That is politics, not science. Science is a process of seeking to get as close to the truth as possible, but one cannot ever say "It is this way, and there is no other way" as our understanding of the natural world is continually evolving. Unless, of course, you want to shut the door on progress...
