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

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].

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>

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
• Fig 2 from the PLOS-article [2]

New Treatment for CRPS?

Complex Regional Pain Syndrome (CRPS) is a multi-symptom, multi-system syndrome that remain poorly understood. As I have mentioned previously , it was called reflex sympathetic dystrophy (RSD) when I first learned about it. I still catch myself calling it RSD.

For a complete review of CRPS, please refer to my previous post on the topic. This post is to look at an article published in the February issue of the journal Annuals of Internal Medicine (full reference below).

A research team at the Pain Research Institute at the University of Liverpool note that there is some evidence for “for immune activation in the affected limb, peripheral blood, and cerebrospinal fluid.”

This lead them to conduct a randomized, placebo-controlled, double-blind, single-center, 2-period crossover trial study. The study included13 eligible participants were randomly assigned between November 2005 and May 2008; 12 completed the trial. Participants were selected from confirmed CRPS patients who had pain intensity greater than 4 on an 11-point (0 to 10) numerical rating scale and had CRPS for 6 to 30 months that was refractory to standard treatment.

They found that a single, low-dose infusion of intravenous immunoglobin (IVIG) provided significant pain relief in nearly 50 percent of patients treated. IVIG is a blood product that contains immunoglobulin G extracts from the plasma of more than 1,000 blood donors. It is used to treat inflammatory and autoimmune diseases, immune deficiencies, and acute infections. In this study, the pain relief lasted on average five weeks.

The editors of the Annuals make the point I want to stress:

Because the study is so small, it is difficult to know whether these results apply to most other patients with CRPS. Also, the small number of patients increases the possibility that chance may affect the results.

For now, the cornerstone in the treatment of RSD / CRPS remains normal use of the affected part as much as possible. This is done through education, pain control, and physical therapy. (photo credit)

For more information check this eMedicine article and this website (RSD Foundation). You will also find a nice video animation on the RSD Foundation site that shows how an injury might trigger RSD / CRPS.

SOURCES:

Intravenous Immunoglobulin Treatment of the Complex Regional Pain Syndrome: A Randomized Trial; Ann Intern Med. 2010;152:152-158; Goebel A, Baranowski A, Maurer K, Ghial A, McCabe C, Ambler G

The Voices of Tourette’s Syndrome

By TARA PARKER-POPE
Stuart Isett for The New York Times Bernadette Witty, a 41-year-old mother from Washington State, is one of seven people with Tourette’s syndrome featured in the latest installment of Patient Voices.

Tourette’s syndrome is an odd and little understood disorder, characterized by sudden jerking movements and uncontrollable tics and vocalizations.

It’s estimated that three of 1,000 children in the United States have Tourette’s. Although the cause of Tourette’s is unknown, studies suggest it is related to abnormalities in the brain circuitry and the neurotransmitters that help nerve cells communicate.

For a fascinating look at the people behind the disorder, listen to the latest Patient Voices feature from the producer Karen Barrow. You’ll meet:

Louis Centanni, 24, of Deptford, N.J.: Mr. Centanni, a comedian and actor, notes that his tics disappear when he is acting and focusing on a character. He says he is comfortable being “the funny guy with Tourette’s.” “I always love making people laugh. How can you not laugh at this disorder?” he says. “It doesn’t cut my lifespan. I’m not sick because of it. It just makes it really hard to shave.”

Bernadette Witty, 41, Bainbridge Island, Wash.: Ms. Witty says that walking in open spaces and clutter can exacerbate her tics. “This is what I have and what I live with,” she says. “You learn to take what you get.”

Cameron Crane, 16, of of Prairie Village, Kan.: Mr. Crane says his tics make it tough to concentrate at school, and he often gets headaches and a sore neck as a result of his tics. He’s particularly bothered when people stare. “It gets really annoying, and you feel like an outcast,” he says.

Marc Elliot, 24, of St. Louis: Mr. Elliot compares Tourette’s to having an “itch” that forces him to say inappropriate things. “It’s incredibly painful not to say it,” he explains. He also talks about dating with Tourette’s, and tics that cause him to say another girl’s name or “you’re fat.” “With girlfriends, you need to find someone who is really understanding,” he says. “You do have to find someone who can laugh at it.”

Stephen Michael McCall, 32, of Virginia Beach: Mr. McCall tried to hide his Tourette’s much of his life. Now a minister, he has decided to talk about Tourette’s to help others. ”It’s like I’m making up for all the time when I was 15 and 14 and I could have encouraged someone who was 6 or 7,” he said. “I’m making up for that time now.”

Brad Cohen, 36, of Atlanta: A teacher, Mr. Cohen explains his disorder to students by comparing it to their need to blink on a regular basis. He tells them that in addition to blinking, his brain instructs him to make funny faces and noises. “I think the biggest misconception about Tourette’s syndrome is that it’s a cursing disease,” he says. “That’s not the case. I don’t say bad words, or at least I can’t really blame it on Tourette’s syndrome.”

Kellie Haines, 35, of Vancouver: Ms. Haines, a ventriloquist, said a tic caused her to mimic people, and she learned to do voices without moving her lips. One of her puppets, Magrau the Bird, also has Tourette’s, which she uses to educate children about the disorder.

Click on this link to listen to all the voices of Tourette’s syndrome

Tests for Down Syndrome are Now Safer

Safer tests to detect the presence of Down syndrome in the fetus during pregnancy are now available after health services in New Zealand started a new screening program.

Women who visit a midwife or a doctor during their pregnancy will be offered this blood test, which is free. They will also be asked to undergo an ultrasound, which costs $30 to $40.

The new test will be offered because older tests were found to compromise normal pregnancies as well.

Peter Stone, the professor of maternal fetal medicine at Auckland University said that "more normal pregnancies may be adversely affected by amniocentesis than the numbers of Down syndrome fetuses detected".

The risk of Down syndrome increases after a woman crosses the age of 35, so older women will be most benefited from using the new test.


Source-Medindia
RAS