Finally a Viral Cause of Chronic Fatigue Syndrome? Or Not? – How Results Can Vary and Depend on Multiple Factors

15 02 2010

Last week @F1000 (on Twitter) alerted me to an interesting discussion at F1000 on  a paper in Science, that linked Chronic fatigue syndrome (CFS) to a newly discovered human virus XRMV [1]ResearchBlogging.org.

This finding was recently disputed by another study in PLOS [2], that couldn’t reproduce the results.  This was highlighted in an excellent post by neuroskeptic “Chronic Fatigue Syndrome in “not caused by single virus” shock!

Here is my take on the discrepancy.

Chronic fatigue syndrome (CFS) is a debilitating disorder with unknown etiology. CFS causes extreme fatigue of the kind that does not go  away after a rest. Symptoms of CFS include fatigue for 6 months or more and experiencing other problems such as muscle pain, memory problems, headaches, pain in multiple joints and  sleep problems. Since other illnesses can cause similar symptoms, CFS is hard to diagnose. (source: Medline Plus).

No one knows what causes CFS, but a viral cause has often been suspected, at least in part of the CFS patients. Because the course of the disease often resembles a post-viral fatigue, CFS has also been referred to as post-viral fatigue syndrome (PVFS).

The article of Lombardi [1], published in October 2009 in Science, was a real breakthrough. The study showed that two thirds of patients with CFS were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV). XMVR was previously linked to prostate cancer.

Lombardi et al  isolated DNA from white blood cells (Peripheral Blood Mononuclear Cells or PBMCs) and assayed the samples for XMRV gag sequences by nested polymerase chain reaction (PCR).

The PCR is a technique that allows the detection of a single or few copies of target DNA by amplifying it across several orders of magnitude, generating thousands to millions of copies of a particular DNA. Nested PCR amplifies the resultant amplicon several orders of magnitude further. In the first round external primers are used (short DNA-sequences that fit the outer end of the piece of DNA to be amplified) and an internal set of primers is used for the second round. Nested PCR is often used if the target DNA is not abundantly present and to avoid the comtamination with products that are amplified as a spin-off due to the amplification of artifacts (sites to which the primers bind as well)

[I used a similar approach 15-20 years ago to identify a lymphoma-characteristic translocation in tonsils and purified B cells of (otherwise) healthy individuals. By direct sequencing I could prove that each sequence was unique in its breakpoint sequence, thereby excluding that the PCR-products arose by contamination of an amplified positive control. All tumor cells had the translocation against one in 100,000 or 1,000,000 normal cells. To be able to detect the oncogene in B cells, B cells had to be purified by FACS. Otherwise the detection limit could not be reached]

Lombardi could detect XMRV gag DNA in 68 of 101 patients (67%) as compared to 8 of 218 (3.7%) healthy controls. Detection of gag as well as env XMRV was confirmed in 7 of 11 CFS samples at the Cleveland Clinic (remarkably these are only shown in Fig 1A of the paper, thus not the original PCR-results).
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, only one sample was positive for gag and none for env. The XMRV gag and env sequences were more than 99% similar to those previously reported for prostate tumor–associated strains of XMRV. The authors see this as proof against contamination of samples with prostate cancer associated XMRV-DNA.

Not only PCR experiments were done. Using intracellular flow cytometry and Western blot assays XMRV proteins were found to be expressed in PBMCs from CFS patients. CFS patiens had anti-XMRV antibodies and cell culture experiments revealed that patient-derived XMRV was infectious. These findings are consistent with but do not prove that XMRV may be a contributing factor in the pathogenesis of CFS. XMRV might just be an innocent bystander. However, unlike XMRV-positive prostate cancer cells, XMRV infection status did not not correlate with the RNASEL genotype.

The Erlwein study was published within 3 months after the first article. It is much simpler in design. DNA was extracted from whole blood (not purified white blood cells) and subjected to a nested PCR using another set of primers. The positive control was an end-point dilution of the plasmid. Water served as a negative control. None of the 186 CSF samples was positive.

The question then is: which study is true? (although it should be stressed that the Science paper just shows a link between the virus and CFS, not a causal relationship)

Regional Differences

Both findings could be “real” if there was a regional difference in occurrence of the virus. Indeed XMRV has previously been detected in prostate cancer cells from American patients, but not from German and Irish patients.

Conflict of Interest

Lombardi’s clinic [1] offers $650 diagnostic test to detect XMRV, so it is of real advantage to the authors of the first paper that the CSF-samples are positive for the virus. On the other hand Prof. Simon Wessely of the second paper has built his career on the hypothesis that CFS is a form of psychoneurosis, that should be treated with cognitive behavior therapy. The presence of a viral (biological) cause would not fit in.

Shortcomings of the Lombardi-article [1]

Both studies have used nested PCR to detect XMRV. Because of the enormous amplification potential, PCR can easily lead to contamination (with the positive control) and thus false positive results. Indeed it is very easy to get contamination from an undiluted positive into a weakly positive or negative sample.

Charles Chiu who belongs to the group detecting XMRV in a specific kind of hereditary prostate cancer, puts it like this [5]:

In their Dissenting Opinion of this article, Moore and Shuda raise valid concerns regarding the potential for PCR contamination in this study. Some concerns include 1) the criteria for defining CFS/ME in the patients and in controls were not explicitly defined, 2) nested PCR was used and neither in a blinded nor randomized fashion, 3) the remarkable lack of diversity in the six fully sequenced XMRV genomes (<6 nucleotide average difference across genome) — with Fig. S1 even showing that for one fully sequenced isolate two of the single nucleotide differences were “N’s” — clearly the result of a sequencing error, 4) failure to use Southern blotting to confirm PCR results, and 5) primary nested PCR screening done in one lab as opposed to independent screening from start to finish in two different laboratories. Concerns have also been brought up with respect to the antigen testing

Shortcomings of the Erlwein-article [2]

Many people have objected that the population of CSF patients is not the same in both studies. Sure it is difficult enough to diagnose CSF (which is only done by exclusion), but according to many commenters of the PLOS study there was a clear bias towards more depressed patients. Therefore, a biological agent is less likely the cause of the disease in these patients. In contrast the US patients had all kinds of physical constraints and immunological problems.

The review process was also far less stringent: 3 days versus several months.

The PLOS study might have suffered from the opposite of contamination: failure to amplify the rare CSF-DNA. This is not improbable. The Erlwein group did not purify the blood cells, used other primers, amplified another sequences and did not test DNA of normal individuals. The positive control was diluted in water not in human DNA. The negative control was water.

Omitting cell purification can lead to a lower relative amount of the XMRV-DNA or to inhibition (often seen this with unpurified samples). Furthermore the gel results seem of poor quality (see Fig 2). The second round of the positive PCR sample results in an overloaded lane with too many aspecific bands (lane 9), whereas the first round leads to a very vague low molecular band (lane 10). True that the CSF-samples also run two rounds, but why aren’t the aspecific bands seen here? It would have been better to use a tenfold titration of the positive control in human DNA (this might be a more real imitation of the CSF samples: (possibly) a rare piece of XMRV DNA mixed with genomic DNA) and to use normal DNA as control, not water.Another point is that the normal XMRV-incidence of 1-3,7% in healthy controls is not reached in the PLOS study, although this could be a matter of chance (1 out of 100).

Further Studies

Anyway, we can philosophize, but the answer must await further studies. There are several ongoing efforts.

References

  1. Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, & Mikovits JA (2009). Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science (New York, N.Y.), 326 (5952), 585-9 PMID: 19815723
  2. Erlwein, O., Kaye, S., McClure, M., Weber, J., Wills, G., Collier, D., Wessely, S., & Cleare, A. (2010). Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008519
  3. http://f1000biology.com/article/yxfr5q9qnc967kn/id/1166366/evaluation/sections
  4. http://neuroskeptic.blogspot.com/2010/01/chronic-fatigue-syndrome-in-not-caused.html
  5. Charles Chiu: Faculty of 1000 Biology, 19 Jan 2010 http://f1000biology.com/article/id/1166366/evaluation

Photo Credits

Nested PCR ivpresearch.org
Advertisements