Tuesday, January 14, 2014

Unfinished Blog-Post: A Post-Mortem for Lombardi et al. 2009 - The Addendum

A long time ago I started some blog-posts, but never found the time nor energy to finish them. Now I have long lost interest in the matter and will publish them all "as is" – maybe someone will find these raw thoughts helpful.

Reposted is here the study "Mikovits et al. 2010", the so called "Addendum" to "Lombardi et al. 2009". This is work in progress.

Please note:
  • All emphasis, color and comments mine
  • Comments are in square brackets
  • Colors denote possible origin of research results
    (WPI/Mikovits, NCI/Ruscetti and CC/Silverman)
  • IFC possibly done at NCI/Ruscetti (not ruled out: UniN, Reno)

Some remarks:
  • The study and the "Addendum" were most likely written by different persons. "PBMCs" in the study versus "PMCs" in part of the Addendum
  • Silverman is not mentioned as co-author of the Addendum
  • Mikovits is author of the Addendum (PI for the study) 
  • Lombardi is co-author (was author for the study)
  • Ruscetti is PI (was co-author for the study)
  • The other groups from NCI are not mentioned as co-authors of the Addendum
  • The quote:
    In our October 2009 publication, we established XMRV infection in the blood products of our patient population by five different methods. Of these methods, single-round PCR on DNA from peripheral blood mononuclear cells (PBMCs), the least sensitive method, required us to use samples from a subset of chronically ill patients we had observed to have persistent viremia.
  • asdf
    "performing the multiple methods on the same 57 blood samples"
    yet only 35 samples were tested by all methods.

Virulence 1:5, 386-390; September/October 2010; © 2010 Landes Bioscience
Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome
Judy A. Mikovits,1,* Vincent C. Lombardi,1 Max A. Pfost,1 Kathryn S. Hagen 1 and Francis W. Ruscetti 2

1 Whittemore Peterson Institute; Reno, NV USA;
2 Laboratory of Experimental Immunology; Cancer and Inflammation Program; National Cancer Institute-Frederick; Frederick, MD USA

Addendum to: Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, et al. Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syn- drome. Science 2009; 326:585–9; PMID: 19815723; DOI: 10.1126/science.1179052.


In October 2009, we reported the first direct isolation of infectious xenotropic murine leukemia virus-related virus (XMRV). In that study, we used a combination of biological amplification and molecular enhancement techniques to detect XMRV in more than 75% of 101 patients with chronic fatigue syndrome (CFS).

[This is a misrepresentation of Lombardi et al. 2009, after all it reported a 67% detection rate.]

Since our report, controversy arose after the publication of several studies that failed to detect XMRV infection in their CFS patient populations. In this addenda, we further detail the multiple detection methods we used in order to observe XMRV infection in our CFS cohort. 

Our results indicate that PCR from DNA of unstimulated peripheral blood mononuclear cells is the least sensitive method for detection of XMRV in subjects’ blood. We advocate the use of more than one type of assay in order to determine the frequency of XMRV infection in patient cohorts in future studies of the relevance of XMRV to human disease.

Patient selection poses a challenge to any study of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). In our October 2009 paper, samples banked from 2006 to 2008 were selected for our study from severely disabled patients who fulfilled the 1994 CDC Fukuda Criteria for chronic fatigue syndrome1 as well as the 2003 Canadian Consensus Criteria (CCC) for ME/CFS.2 The CCC requires
post-exertional malaise, which many clinicians feel is the sine qua non of ME/ CFS. Furthermore the CCC further requires that patients exhibit post exertional fatigue, unrefreshing sleep, pain and neurological/cognitive manifestations, rather than these being optional symptoms.3 Many clinicians interested in CFS are switching to the Canadian criteria because they feel it is more descriptive of the clinical entity being defined. The Fukuda criteria have the advantage of a longer period of usage and existence of many publications that have added modifications. Suffice it to say that the clinician author of the Science paper elected to use both criteria, thus bypassing the argument of which criteria were better. Moreover, the emphasis in the Science paper was directed toward the virology, not the clinical description of ME/CFS.

In our October 2009 publication, we established XMRV infection in the blood products of our patient population by five different methods. Of these methods, single-round PCR on DNA from peripheral blood mononuclear cells (PBMCs), the least sensitive method, required us to use samples from a subset of chronically ill patients we had observed to have persistent viremia

[How was this "observation" of "persistent viremia" take place? qRT-PCR? Gut feeling? CFS illness score? Tea leaf reading? Or did they test their "least sensitive" method to produce consistent results? And what were these?]

In Figure 1A of our Science paper, we showed that DNA of 7 of 11 patients exhibited the expected gag and env PCR amplification products from single-round PCR with XMRV primers. We included this figure to demonstrate that samples exhibited gag products upon nested PCR, though PCR with nested env primers did not result in detectable products from these samples (Table 1).

Table 1. XMRV detection using cDNA from 22 unstimulated PBMCs gag gene

Samples that are negative for XMRV by one of our PCR assays are sometimes positive by other assays. For example, in Figure 1A of the Science paper, patient 1118 was negative by single round PCR on DNA from unstimulated PBMCs, but positive in other assays (Science Figs. 2A and D, 4A and S5). Of the 34 patients whose PBMCs were negative for XMRV by DNA or cDNA PCR, 17 were positive for infectious virus when co-cultured with the LNCaP indicator cell line, as XMRV gag and env PCR products were detected in the cell line following their infection with XMRV from the patient PBMCs (Table 2). Both gag and env products obtained from either single-round or nested PCR were sequenced and shown to be 99% identical to XMRV VP62.

Table 2. Co-Culture with LNCaP of PBMCs from 12 patients PCR negative for env

Subsequent to our October 2009 publication, two papers from the United Kingdom 4,5 and a paper from the Netherlands 6 have appeared in which the authors report the lack of detection of XMRV PCR products from DNA of unstimulated PBMCs, using patient populations selected by only the Fukuda criteria or the Oxford criteria rather than both Fukuda and CCC criteria. We regret that these authors did not request positive control samples of our patients who exhibit XMRV PCR products even when assayed by the least sensitive detection method, namely PCR of DNA from unstimulated PBMCs. Given that only 7% of our 101 patients’ PBMCs exhibit products upon DNA PCR (Table 3 and 4), and that a number of patients were included in the UK studies who do not fulfill the CCC criteria, very few, if any, of the samples would be expected to be positive by DNA PCR. We also note that both studies followed different methods than ours for blood collection, DNA quantities and isolation and PCR, possible sources of the disparate results. The XMRV detection results of the 101 patients are listed in Table 4.

The negative reports of PCR tests for XMRV has raised questions whether our findings could be due to contamination of our PCR experiments by mouse genomic XMRV-gag specific PCR products and no env specific PCR products following single round DNA PCR of DNA of unstimulated PBMCs

In contrast, when cDNA was prepared from PBMCs, 67% of the nested PCR, which inevitably raises questions of contamination, is not essential to detect XMRV in highly viremic ME/ CFS patients. 
[This sentence makes no fucking sense. None whatsoever.]
[And again, how were these "highly viremic ME/ CFS patients" determined? qRT-PCR? Gut feeling? CFS illness score? Tea leaves? Or did they test their "least sensitive" method to produce consistent results? And what were these?]

The remaining 90 samples described in the paper exhibited very few DNA, which contain gag and env sequences highly similar to XMRV. Positive PCR results for XMRV were obtained independently in multiple laboratories led by co-authors of the Science paper. In the summer of 2006, prior to work on XMRV at the Reno Whittemore-Peterson Institute (WPI), 30 mL of heparinized peripheral blood were obtained from patients resid- ing in the US, Canada and Europe coming to be treated at the well-known Sierra Internal Medicine practice, located in Incline Village, NV. Once collected, 48 of these blood samples were shipped directly to NCI where cDNA was prepared for planned microarray experiments. After the WPI observed an XMRV PCR product from a patient sample in 2009, the NCI began testing these stored samples by PCR. cDNA from 42 of the 48 samples sent to the NCI lab in February 2007 tested posi- tive for XMRV gag by nested PCR. Neither the WPI nor NCI labs where PCR was per- formed had ever worked with mouse tissues or had been exposed to XMRV from other sources. The env sequences amplified from LNCaP cells infected by patient PBMCs exhibit less similarity to mouse genomic DNA than to XMRV VP62, further indi- cating the presence of XMRV infection rather than mouse genomic DNA contami- nation. After we developed a sensitive cell culture assay for detection of XMRV, we assayed our cell lines and patient material with a highly sensitive assay (developed and kindly provided by Bill Switzer, CDC) to detect the presence of mouse tissue con- tamination by the identification of murine mitochrondial cytochrome oxidase by real time PCR. All of the cell lines and 101 patient materials tested negative for mouse contamination.

Table 3. Summary of multiple viral assays from a group of 57 patients

Table 4. XMRV detection results of 101 patients 

In our experience from performing the multiple methods on the same 57 blood samples [Only 35 samples were tested by all methods!], the most sensitive blood-based assays for detection of XMRV in decreasing order (Table 3) are:
(1) performing nested PCR for gag sequences from LNCaP cells that have been co-cultured with subject’s plasma or activated PBMCs,
(2) the presence of antibodies to XMRV Env in subject’s plasma,
(3) presence of gag products by nested PCR on stimulated PBMCs or detection of viral proteins expressed by activated PBMCs with appropriate antisera,
(4) nested RT-PCR of plasma nucleic acid or PCR from cDNA from unactivated PBMCs and
(5) PCR of DNA from unactivated PBMC prepared from subject’s blood.

Despite association with both prostate cancer and CFS, many questions remain regarding the prevalence of XMRV in the human population, the incidence of XMRV in disease, and the extent of genetic variation between XMRV isolates. The genetic variation between XMRV isolates currently identified is only 0.03%, despite the fact that the viral sequences were obtained from isolates from two vastly different diseases in patients from geographically distinct areas. This variation is smaller than the variation observed between HTLV-1 isolates.7 As in the case with HTLV, the lack of diversity implies that XMRV recently descended from a common ancestor. 8

[That'll be VP62, which recently descended from 22Rv1.]

The high degree of similarity to xenotropic murine leukemia virus suggests that a cross-species transmission event was likely involved in the evolution of XMRV.9

[Not cross-species transmission, but rather cross-tissue transmission while xeno-grafting 22Rv1.]

Further examination of XMRV from human subjects may reveal more extensive sequence variation, which also may confound its detection unless PCR primers are designed with this possibility in mind.

We have not claimed in our October 2009 publication or in other venues that XMRV is the cause of CFS, only that its detection in the majority of our ME/CFS patient cohort allows us to form a testable hypothesis as to an infectious basis for this devastating disease. Future work should establish what role XMRV may play in development of prostate cancer, ME/CFS and other diseases.

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