Can Guidelines Harm Patients?

2 05 2012

ResearchBlogging.orgRecently I saw an intriguing “personal view” in the BMJ written by Grant Hutchison entitled: “Can Guidelines Harm Patients Too?” Hutchison is a consultant anesthetist with -as he calls it- chronic guideline fatigue syndrome. Hutchison underwent an acute exacerbation of his “condition” with the arrival of another set of guidelines in his email inbox. Hutchison:

On reviewing the level of evidence provided for the various recommendations being offered, I was struck by the fact that no relevant clinical trials had been carried out in the population of interest. Eleven out of 25 of the recommendations made were supported only by the lowest levels of published evidence (case reports and case series, or inference from studies not directly applicable to the relevant population). A further seven out of 25 were derived only from the expert opinion of members of the guidelines committee, in the absence of any guidance to be gleaned from the published literature.

Hutchison’s personal experience is supported by evidence from two articles [2,3].

One paper published in the JAMA 2009 [2] concludes that ACC/AHA (American College of Cardiology and the American Heart Association) clinical practice guidelines are largely developed from lower levels of evidence or expert opinion and that the proportion of recommendations for which there is no conclusive evidence is growing. Only 314 recommendations of 2711 (median, 11%) are classified as level of evidence A , thus recommendation based on evidence from multiple randomized trials or meta-analyses.  The majority of recommendations (1246/2711; median, 48%) are level of evidence C, thus based  on expert opinion, case studies, or standards of care. Strikingly only 245 of 1305 class I recommendations are based on the highest level A evidence (median, 19%).

Another paper, published in Ann Intern Med 2011 [3], reaches similar conclusions analyzing the Infectious Diseases Society of America (IDSA) Practice Guidelines. Of the 4218 individual recommendations found, only 14% were supported by the strongest (level I) quality of evidence; more than half were based on level III evidence only. Like the ACC/AHH guidelines only a small part (23%) of the strongest IDSA recommendations, were based on level I evidence (in this case ≥1 randomized controlled trial, see below). And, here too, the new recommendations were mostly based on level II and III evidence.

Although there is little to argue about Hutchison’s observations, I do not agree with his conclusions.

In his view guidelines are equivalent to a bullet pointed list or flow diagram, allowing busy practitioners to move on from practice based on mere anecdote and opinion. It therefore seems contradictory that half of the EBM-guidelines are based on little more than anecdote (case series, extrapolation from other populations) and opinion. He then argues that guidelines, like other therapeutic interventions, should be considered in terms of balance between benefit and risk and that the risk  associated with the dissemination of poorly founded guidelines must also be considered. One of those risks is that doctors will just tend to adhere to the guidelines, and may even change their own (adequate) practice  in the absence of any scientific evidence against it. If a patient is harmed despite punctilious adherence to the guideline-rules,  “it is easy to be seduced into assuming that the bad outcome was therefore unavoidable”. But perhaps harm was done by following the guideline….

First of all, overall evidence shows that adherence to guidelines can improve patient outcome and provide more cost effective care (Naveed Mustfa in a comment refers to [4]).

Hutchinson’s piece is opinion-based and rather driven by (understandable) gut feelings and implicit assumptions, that also surround EBM in general.

  1. First there is the assumption that guidelines are a fixed set of rules, like a protocol, and that there is no room for preferences (both of the doctor and the patient), interpretations and experience. In the same way as EBM is often degraded to “cookbook medicine”, EBM guidelines are turned into mere bullet pointed lists made by a bunch of experts that just want to impose their opinions as truth.
  2. The second assumption (shared by many) is that evidence based medicine is synonymous with “randomized controlled trials”. In analogy, only those EBM guideline recommendations “count” that are based on RCT’s or meta-analyses.

Before I continue, I would strongly advice all readers (and certainly all EBM and guideline-skeptics) to read this excellent and clearly written BJM-editorial by David Sackett et al. that deals with misconceptions, myths and prejudices surrounding EBM : Evidence based medicine: what it is and what it isn’t [5].

Sackett et al define EBM as “the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients” [5]. Sackett emphasizes that “Good doctors use both individual clinical expertise and the best available external evidence, and neither alone is enough. Without clinical expertise, practice risks becoming tyrannised by evidence, for even excellent external evidence may be inapplicable to or inappropriate for an individual patient. Without current best evidence, practice risks becoming rapidly out of date, to the detriment of patients.”

Guidelines are meant to give recommendations based on the best available evidence. Guidelines should not be a set of rules, set in stone. Ideally, guidelines have gathered evidence in a transparent way and make it easier for the clinicians to grasp the evidence for a certain procedure in a certain situation … and to see the gaps.

Contrary to what many people think, EBM is not restricted to randomized trials and meta-analyses. It involves tracking down the best external evidence there is. As I explained in #NotSoFunny #16 – Ridiculing RCTs & EBM, evidence is not an all-or-nothing thing: RCT’s (if well performed) are the most robust, but if not available we have to rely on “lower” evidence (from cohort to case-control to case series or expert opinion even).
On the other hand RCT’s are often not even suitable to answer questions in other domains than therapy (etiology/harm, prognosis, diagnosis): per definition the level of evidence for these kind of questions inevitably will be low*. Also, for some interventions RCT’s are not appropriate, feasible or too costly to perform (cesarean vs vaginal birth; experimental therapies, rare diseases, see also [3]).

It is also good to realize that guidance, based on numerous randomized controlled trials is probably not or limited applicable to groups of patients who are seldom included in a RCT: the cognitively impaired, the patient with multiple comorbidities [6], the old patient [6], children and (often) women.

Finally not all RCTs are created equal (various forms of bias; surrogate outcomes; small sample sizes, short follow-up), and thus should not all represent the same high level of evidence.*

Thus in my opinion, low levels of evidence are not per definition problematic. Even if they are the basis for strong recommendations. As long as it is clear how the recommendations were reached and as long as these are well underpinned (by whatever evidence or motivation). One could see the exposed gaps in evidence as a positive thing as it may highlight the need for clinical research in certain fields.

There is one BIG BUT: my assumption is that guidelines are “just” recommendations based on exhaustive and objective reviews of existing evidence. No more, no less. This means that the clinician must have the freedom to deviate from the recommendations, based on his own expertise and/or the situation and/or the patient’s preferences. The more, when the evidence on which these strong recommendations are based is ‘scant’. Sackett already warned for the possible hijacking of EBM by purchasers and managers (and may I add health insurances and governmental agencies) to cut the costs of health care and to impose “rules”.

I therefore think it is odd that the ACC/AHA guidelines prescribe that Class I recommendations SHOULD be performed/administered even if they are based on level C recommendations (see Figure).

I also find it odd that different guidelines have a different nomenclature. The ACC/AHA have Class I, IIa, IIb and III recommendations and level A, B, C evidence where level A evidence represents sufficient evidence from multiple randomized trials and meta-analyses, whereas the strength of recommendations in the IDSA guidelines includes levels A through C (OR D/E recommendations against use) and quality of evidence ranges from level I through III , where I indicates evidence from (just) 1 properly randomized controlled trial. As explained in [3] this system was introduced to evaluate the effectiveness of preventive health care interventions in Canada (for which RCTs are apt).

Finally, guidelines and guideline makers should probably be more open for input/feedback from people who apply these guidelines.

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*the new GRADE (Grading of Recommendations Assessment, Development, and Evaluation) scoring system taking into account good quality observational studies as well may offer a potential solution.

Another possibly relevant post at this blog: The Best Study Design for … Dummies

Taken from a summary of an ACC/AHA guideline at http://guideline.gov/
Click to enlarge.

References

  1. Hutchison, G. (2012). Guidelines can harm patients too BMJ, 344 (apr18 1) DOI: 10.1136/bmj.e2685
  2. Tricoci P, Allen JM, Kramer JM, Califf RM, & Smith SC Jr (2009). Scientific evidence underlying the ACC/AHA clinical practice guidelines. JAMA : the journal of the American Medical Association, 301 (8), 831-41 PMID: 19244190
  3. Lee, D., & Vielemeyer, O. (2011). Analysis of Overall Level of Evidence Behind Infectious Diseases Society of America Practice Guidelines Archives of Internal Medicine, 171 (1), 18-22 DOI: 10.1001/archinternmed.2010.482
  4. Menéndez R, Reyes S, Martínez R, de la Cuadra P, Manuel Vallés J, & Vallterra J (2007). Economic evaluation of adherence to treatment guidelines in nonintensive care pneumonia. The European respiratory journal : official journal of the European Society for Clinical Respiratory Physiology, 29 (4), 751-6 PMID: 17005580
  5. Sackett, D., Rosenberg, W., Gray, J., Haynes, R., & Richardson, W. (1996). Evidence based medicine: what it is and what it isn’t BMJ, 312 (7023), 71-72 DOI: 10.1136/bmj.312.7023.71
  6. Aylett, V. (2010). Do geriatricians need guidelines? BMJ, 341 (sep29 3) DOI: 10.1136/bmj.c5340




Stories [9]: A Healthy Volunteer

20 09 2010

The host of Next Grand Rounds (Pallimed) asked to submit a recent blog post from another blogger in addition to your own post.
I choose “Orthostatics – one more time” from DB Medical rants and a post commenting on that from Musings of a Dinosaur.

Bob Center’s (@medrants) posts was about the value of orthostatic vital sign measurements (I won’t go into any details here), and about who should be doing them, nurses or doctors. In his post, Bob Center also mentioned briefly that students were seeing this as scut work similar as drawing your own bloods and carrying them to the lab.

That reminded me of something that happened when I was working in the lab as a PhD, 20 years ago.

I was working on a chromosomal translocation between chromosome 14 and 18. (see Fig)

The t(14;18) is THE hallmark of follicular lymphoma (lymphoma is a B cell cancer of the lymph nodes).

This chromosomal translocation is caused by a faulty coupling of an immunoglobulin chain to the BCL-2 proto-oncogene during the normal rearrangement process of the immunoglobulins in the pre-B-cells.

This t(14;18) translocation can be detected by genetic techniques, such as PCR.

Using PCR, we found that the t(14:18) translocation was not only present in follicular lymphoma, but also in benign hyperplasia of tonsils and lymph nodes in otherwise healthy persons. Just one out of  1 : 100,000 cells were positive. When I finally succeeded in sequencing the PCR-amplified breakpoints, we could show that each breakpoint was unique and not due to contamination of our positive control (read my posts on XMRV to see why this is important).

So we had a paper. Together with experiments in transgenic mice, our results hinted that t(14;18) translocations is necessary but not sufficient for follicular lymphoma. Enhanced expression of BCL-2 might give make the cells with the translocation “immortal”.

All fine, but hyperplastic tonsils might still form an exception, since they are not completely normal. We reasoned that if the t(14;18) was an accidental mistake in pre B cells it might sometimes be found in normal B cells in the blood too.

But then we needed normal blood from healthy individuals.

At the blood bank we could only get pooled blood at that time. But that wasn’t suitable, because if a translocation was present in one individual it would be diluted with the blood of the others.

So, as was quite common then, we asked our colleagues to donate some blood.

The entire procedure was cumbersome: a technician first had to enrich for T and  B cells, we had to separate the cells by FACS and I would then PCR and sequence them.

The PCR and sequencing techniques had to be adopted, because the frequency of positive cells was lower than in the tonsils and approached the detection limit. ….. That is in most people. But not in all. One of our colleagues had relatively prominent bands, and several breakpoints.

It was explained to him that this meant nothing really. Because we did find similar translocations in every healthy person.

But still, I wouldn’t feel 100% sure, if so many of my blood cells (one out of 1000 or 10.000) contained t(14:18) translocations.

He was one of the first volunteers we tested, but from then on it was decided to test only anonymous persons.

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Does the NHI/FDA Paper Confirm XMRV in CFS? Well, Ditch the MR and Scratch the X… and… you’ve got MLV.

30 08 2010

ResearchBlogging.orgThe long awaited paper that would ‘solve’ the controversies about the presence of Xenotropic Murine Leukemia Virus-related virus (XMRV) in patients with chronic fatigue syndrome (CFS) was finally published in PNAS last week [1]. The study, a joint effort of the NIH and the FDA, was withheld, on request of the authors [2], because it contradicted the results of another study performed by the CDC. Both studies were put on hold.

The CDC study was published in Retrovirology online July 1 [3]. It was the fourth study in succession [4,5,6] and the first US study, that failed to demonstrate XMRV since researchers of the US Whittemore Peterson Institute (WPI) had published their controversial paper regarding the presence of XMRV in CFS [7].

The WPI-study had several flaws, but so had the negative papers: these had tested a less rigorously defined CFS-population, had used old and/or too few samples (discussed in two previous posts here and here).
In a way,  negative studies, failing to reproduce a finding, are less convincing then positive studies.  Thus everyone was eagerly looking forward to the release of the PNAS-paper, especially because the grapevine whispered this study was  to confirm the original WPI findings.

Indeed after publication, both Harvey Alter, the team leader of the NIH/FDA study, and Judy Mikovitz of the WPI emphasized that the PNAS paper essentially confirmed the presence of XMRV in CFS.

But that isn’t true. Not one single XMRV-sequence was found. Instead related MLV-sequences were detected.

Before I go into further details, please have a look at the previous posts if you are not familiar with the technical details , like the PCR-technique. Here (and in a separate spreadsheet) I also describe the experimental differences between the studies.

Now what did Lo et al exactly do? What were their findings? And in what respect do their findings agree or disagree with the WPI-paper?

Like WPI, Lo et al used nested PCR to enable detect XMRV. Nested means that there are two rounds of amplification. Outer primers are used to amplify the DNA between the two primers used (primers are a kind of very specific anchors fitting a small approximately 20 basepair long piece of DNA). Then a second round is performed with primers fitting a short sequence within the amplified sequence or amplicon.

The first amplified gag product is ~730 basepairs long, the second ~410 or ~380 basepairs, depending on the primer sets used:  Lo et al used the same set of outer primers as WPI to amplify the gag gene, but the inner gag primers were either those of WPI (410 bp)  or a in-house-designed primer set (380 bp).

Using the nested PCR approach Lo et al found gag-gene sequences in peripheral blood mononuclear cells (PBMC)  in 86.5% of all tested CFS-patients (32/37)  and in 96% (!) of the rigorously evaluated CFS-patients (24/25) compared with only 6.8% of the healthy volunteer blood donors (3/44). Half of the patients with gag detected in their PBMC, also had detectable gag in their serum (thus not in the cells). Vice versa, all but one patient with gag-sequences in the plasma also had gag-positive PBMC. Thus these findings are consistent.

The gels  (Figs 1 and 2) showing the PCR-products in PBMC don’t look pretty, because there are many aspecific bands amplified from human PBMC. These aspecific bands are lacking when plasma is tested (which lacks PBMC). To get the idea. The researchers are trying to amplify a 730 bp long sequence, using primers that are 23 -25 basepairs long, that need to find the needle in the haystack (only 1 in 1000 to 10,000 PBMC may be infected, 1 PBMC contains appr 6*10^9 basepairs). Only the order of A, C, G and T varies! Thus there is a lot of competition of sequences that have a near-fit, but are more preponderant than the true gag-sequences fitting the primers).

Therefore, detecting a band with the expected size does not suffice to demonstrate the presence of a particular viral sequence. Lo et al verified whether it were true gag-sequences, by sequencing each band with the appropriate size. All the sequenced amplicons appeared true gag-sequences. What makes there finding particularly strong is that the sequences were not always identical. This was one of the objections against the WPI-findings: they only found the same sequence in all patients (apart from some sequencing errors).

Another convincing finding is that the viral sequences could be demonstrated in samples that were taken 2-15 years apart. The more recent sequences had evolved and gained one or more mutations. Exactly what one would expect from a retrovirus. Such findings also make contamination unlikely. The lack of PCR-amplifiable mouse mitochondrial DNA also makes contamination a less likely event (although personally I would be more afraid of contamination by viral amplicons used as a positive control). The negative controls (samples without DNA) were also negative in all cases. The researchers also took all necessary physical precautions to prevent contamination (i.e. the blood samples were prepared at another lab than did the testing, both labs never sequenced similar sequences before).
(people often think of conspiracy wherever the possibility of contamination is mentioned, but this is a real pitfall when amplifying low frequency targets. It took me two years to exclude contamination in my experiments)

To me the data look quite convincing, although we’re still far from concluding that the virus is integrated in the human genome and infectious. And, of course, mere presence of a viral sequence in CFS patients, does not demonstrate a causal relationship. The authors recognize this and try to tackle this in future experiments.

Although the study seems well done, it doesn’t alleviate the confusion raised.

The reason, as said, is that the NIH/FDA researchers didn’t find a single XMRV sequence  in any of the samples!

Instead a variety of related MLV retrovirus sequences were detected.

Sure the two retroviruses belong to a similar “family”. The gag gene sequences share 96.6% homology.

However there are essential differences.

One is that XMRV is a  Xenotropic virus, hence the X: which means it can no longer enter mouse cells (MR= murine (mouse) related) but can infect cells of other species, including humans. (to be more precise it has both xenotropic and polytropic characteristics). According to the phylogenetic tree Lo et al  constructed,  the viral sequences they found are more diverse and best matches the so-called polytropic MLV viruses (able to infect both mouse and non-mouse cells infected). (see the PNAS commentary by Valerie Courgnaud et al for an explanation)

The main question, this paper raises is why they didn’t find XMRV, like WPI did.

Sure, Mikovitz —who is “delighted” by the results—now hurries to say that in the meantime, her group has found more diversity in the virus as well [8]. Or as a critical CFS patient writes on his blog:

In my opinion, the second study is neither a confirmation for, nor a replication of the first. The second study only confirms that WPI is on to something and that there might be an association between a type of retroviruses and ME/CFS.
For 10 months all we’ve heard was “it’s XMRV”. If you didn’t find
XMRV you were doing something wrong: wrong selection criteria, wrong methods, or wrong attitude. Now comes this new paper which doesn’t find XMRV either and it’s heralded as the long awaited replication and confirmation study. Well, it isn’t! Nice piece of spin by Annette Whittemore and Judy Mikovits from the WPI as you can see in the videos below (… ). WPI may count their blessings that the NIH/FDA/Harvard team looked at other MLVs and found them or otherwise it could have been game over. Well, probably not, but how many negative studies can you take?

Assuming the NIH/FDA findings are true, then the key question is not why most experiments were completely negative (there may many reasons why, for one thing they only tested XMRV), but why Lo didn’t find any XMRV amongst the positive CFS patients, and WPI didn’t find any MLV in their positive patient samples.

Two US cohorts of CFS patients with mutually exclusive presence of either XMRV or MLV, whereas the rest of the world finds nothing?? I don’t believe it. One would at least expect overlap.

My guess is that it must be something in the conditions used. Perhaps the set of primers.

As said, Lo used the same external primers as WPI, but varied the internal primers. Sometimes they used those of WPI (GAG-I-F/GAG-I-R ; F=forward, R=reverse) yielding a ~410 basepair product, sometimes their own primers (NP116/NP117), yielding a ~380 basepair product. In the Materials and Methods section  Lo et al write The NP116/NP117 was an in-house–designed primer set based on the highly conserved sequences found in different MLV-like viruses and XMRVs”.
In the supplement they are more specific:

…. (GAG-I-F/GAG-I-R (intended to be more XMRV specific) or the primer set NP116/NP117 (highly conserved sequences for XMRV and MLV).

Is it possible that the conditions that WPI used were not so suitable for finding MLV?

Lets look at Fig. S1 (partly depicted below), showing the multiple sequence alignment of 746 gag nucleotides (nt) amplified from 21 CFS patient samples (3 types) and one blood donor (BD22) [first 4 rows] and their comparison with known MLV (middle rows) and XMRV (last rows) sequences. There is nothing remarkable with the area of the reverse primer (not shown). The external forward primer (–>) fits all sequences (dots mean identical nucleotides). Just next to this primer are 15 nt deletions specific for XMRV (—-), but that isn’t hurdle for the external primers. The internal primers (–>) overlap, but the WPI-internal primer starts earlier, in the region with heterogeneity: here there are two mismatches between MLV- and XMRV-like viruses. In this region the CFS type MLV (nt 196) starts with TTTCA, whereas XMRV sequences all have TCTCG. And yes, the WPI-primers starts as follows: TCTCG. Thus there is a complete match with XMRV, but a 2 bp mismatch with MLV. Such a mismatch might easily explain why WPI (not using very optimal PCR conditions) didn’t find any low frequency MLV-sequences. The specific inner primer designed by the group of Lo and Alter, do fit both sequences, so differences in this region don’t explain the failure of Lo et al to detect XMRV. Perhaps MLV is more abundant and easier to detect?

But wait a minute. BD22, a variant detected in normal donor blood does have the XMRV variant sequence in this particular (very small) region. This sequence and the two other sequenced normal donor MLV variants differ form the patient variants, although -according to Lo- both patient and healthy donor variants differ more from XMRV then from each other (Figs 4 and 2s). Using the eyeball test I do see many similarities between XMRV and BD22 though (not only in the above region).

The media pay no attention to these differences between patient and healthy control viral sequences, and the different primer sets used. Did no one actually read the paper?

Whether theses differences are relevant, depends on whether identical conditions were used for each type of sample. It worries me that Lo says he sometimes uses the WPI inner primer sets and sometimes the other specific set. When is sometimes? It is striking that Fig 1 shows the results from CFS patients done with the specific primers and Fig 2 the results from normal donor blood done with the WPI-primers. Why? Is this the reason they picked up a sequence that fit the WPI-primers (starting with TCTCG)?

I don’t like it. I want to know how many times tested samples were positive or negative with either primer set. I not only want to see the PCR results of CFS-plasma (positive in half of the PBMC+ cases), but also of the control plasma. And I want a mix of the patient, normal samples, positive and negative controls on one gel. Everyone doing PCR knows that the signal can differ per PCR and per gel. Furthermore, the second PCR round gives way too much aspecific bands, whereas usually you get rid of those under optimal conditions.

Another confusing finding is a statement at the FDA site:

Additionally, the CDC laboratory provided 82 samples from their published negative study to FDA, who tested the samples blindly.  Initial analysis shows that the FDA test results are generally consistent with CDC, with no XMRV-positive results in the CFS samples CDC provided (34 samples were tested, 31 were negative, 3 were indeterminate).

What does this mean? Which inner primers did the FDA use? With the WPI inner primers MLV sequences might just not be found (although there might be other reasons as well, as the less stringent patient criteria).

And what to think of the earlier WPI findings? They did find “XMRV” sequences while no one else did.

I have always been skeptic (see here and here), because:

  • no mention of sensitivity in their paper
  • No mention of a positive control. The negative controls were just vials without added DNA.
  • No variation in the sequences detected, a statement that they retracted after the present NIH/FDA publication. What a coincidence.
  • Although the PCR is near the detection limit, only first round products are shown. These are stronger then you would expect them to be after one round.
  • The latter two points are suggestive of contamination. No extra test were undertaken to exclude this.
  • Surprisingly in an open letters/news items (Feb 18), they disclose that culturing PBMC’s is necessary to obtain a positive signal.  They refer to the original Science paper, but this paper doesn’t mention the need for culturing at all.
  • In an other open letter* Annette Whittemore, director of the WPI,writes to Dr McClure, virologist of tone of the negative papers that WPI researchers had detected XMRV in patient samples from both Dr. Kerr’s and Dr. van Kuppeveld’s cohorts. So if we must believe Annette, the negative samples weren’t negative
  • At this stage of controversy, the test is sold as “a reliable diagnostic tool“ by a firm with strong ties to WPI. In one such mail Mikovits says: “First of all the current diagnostic testing will define with essentially 100% accuracy! XMRV infected patients”.

Their PR machine, ever changing “findings” and anti-scientific attitude are worrying. Read more about at erv here.

What we can conclude from this all. I don’t know. I presume that WPI did find “something”, but weren’t cautious, critical and accurate enough in their drive to move forward (hence their often changing statements). I presume that the four negative findings relate to the nature of their samples or the use of the WPI inner primers or both. I assume that the NIH/CDC findings are real, although the actual positive rates might vary depending on conditions used (I would love to see all actual data).

Virologist “erv”is less positive, about the quality of the findings and their implications. In one of her comments (17) she responds:

No. An exogenous mouse ERV in humans makes no sense. But thats what their tree says. Mouse ERV is even more incredible than XMRV. Might be able to figure this out more if they upload their sequences to genbank. I realize they tried very hard not to contaminate their samples with mouse cells. That doesnt mean mouse DNA isnt in any of their store-bought reagents. There are H2O lanes in the mitochondral gels, but not the MLV gels (Fig 1, Fig 2). Why? Positive and negative controls go on every gel, end of story. First lesson every rotating student in our lab learns.

Finding mere virus-like sequences in CFS-patients is not enough. We need more data, more carefully gathered and presented. Not only in CFS patients and controls, but in cohorts of patients with different diseases and controls under controlled conditions. This will tell something about the specificity of the finding for CFS. We also need more information about XMRV infectivity and serology.

We also need to find out what being normal healthy and MLV+ means.

The research on XMRV/MLV seems to progress with one step forward, two steps back.

With the CFS patients, I truly hope that we are walking in the right direction.

Note

The title from this post was taken from: http://www.veteranstoday.com/2010/08/20/xmrv-renamed-to-hgrv/

References

  1. Lo SC, Pripuzova N, Li B, Komaroff AL, Hung GC, Wang R, & Alter HJ (2010). Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors. Proceedings of the National Academy of Sciences of the United States of America PMID: 20798047
  2. Schekman R (2010). Patients, patience, and the publication process. Proceedings of the National Academy of Sciences of the United States of America PMID: 20798042
  3. Switzer WM, Jia H, Hohn O, Zheng H, Tang S, Shankar A, Bannert N, Simmons G, Hendry RM, Falkenberg VR, Reeves WC, & Heneine W (2010). Absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States. Retrovirology, 7 PMID: 20594299
  4. 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
  5. Groom, H., Boucherit, V., Makinson, K., Randal, E., Baptista, S., Hagan, S., Gow, J., Mattes, F., Breuer, J., Kerr, J., Stoye, J., & Bishop, K. (2010). Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome Retrovirology, 7 (1) DOI: 10.1186/1742-4690-7-10
  6. van Kuppeveld, F., Jong, A., Lanke, K., Verhaegh, G., Melchers, W., Swanink, C., Bleijenberg, G., Netea, M., Galama, J., & van der Meer, J. (2010). Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort BMJ, 340 (feb25 1) DOI: 10.1136/bmj.c1018
  7. 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
  8. Enserink M (2010). Chronic fatigue syndrome. New XMRV paper looks good, skeptics admit–yet doubts linger. Science (New York, N.Y.), 329 (5995) PMID: 20798285

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What One Short Night’s Sleep does to your Glucose Metabolism

11 05 2010

ResearchBlogging.orgAs a blogger I regularly sleep 3-5 hours just to finish a post. I know that this has its effects on how I feel the next day. I also know short nights don’t promote my clear-headedness and I also recognize short-term effects on  memory, cognitive functions, reaction time and mood (irritability), as depicted in the picture below. But I had no idea of any effect on heart disease, obesity and risk of diabetes type 2.

Indeed, short sleep duration is consistently associated with the development of obesity and diabetes in observational studies (see several recent systematic reviews, 3-5). However, as explained before, an observational design cannot establish causality. For instance, diabetes type 2 may be the consequence of other lifestyle aspects of people who spend little time sleeping, or sleep problems might be a consequence rather than a cause of diabetogenic changes.

Diabetes is basically a condition characterized by difficulties processing carbohydrates (sugars, glucose). Type 2 diabetes has a slow onset. First there is a gradual defect in the body’s ability to use insulin. This is called insulin resistance. Insulin is a pancreatic hormone that increases glucose utilization in skeletal muscle and fat tissue and suppresses glucose production by the liver, thereby lowering blood glucose levels.  Over time, damage may occur to the insulin-producing cells in the pancreas (type 2 diabetes),  which may ultimately progress to the point where the pancreas doesn’t make enough insulin and injections are needed. (source: about.com).

Since it is such a slow process one would not expect insulin resistance to change overnight. And certainly not by just partial sleep deprivation of 4-5 hrs of sleep.

Still, this is the outcome of a study, performed by the PhD student Esther Donga. Esther belongs to the study group of Romijn who also studied the previously summarized effects of previous cortisol excess on cognitive functions in Cushing’s disease .

Donga et al. have studied the effects of one night of sleep restriction on insulin sensitivity in 9 healthy lean individuals [1] and in 7 patients with type 1 diabetes [2]. The outcomes were practically the same, but since the results in healthy individuals (having no problems with glucose metabolism, weight or sleep) are most remarkable, I will confine myself to the study in healthy people.

The study design is relatively simple. Five men and four healthy women (mean age 45 years) with a lean body weight and normal  sleep pattern participated in the study. They were not using medication affecting sleep or glucose metabolism and were asked to adhere to their normal lifestyle pattern during the study.

There were 3 study days, separated by intervals of at least 3 weeks. The volunteers were admitted to the clinical research center the night before each study day to become accustomed to sleeping there. They fasted throughout these nights and spent 8.5 h in bed.  The subjects were randomly assigned to sleep deprivation on either the second or third occasion. Then they were only allowed to sleep from 1 am to 4 am to secure equal compression of both non-REM and REM sleep stages.

(skip blue paragraphs if you are not interested in the details)

Effects on insulin sensitivity were determined on the day after the second and third night (one normal and one short night sleep) by the gold standard for quantifying insulin resistance: the hyperinsulinemic euglycemic clamp method. This method uses catheters to infuse insulin and glucose into the bloodstream. Insulin is infused to get a steady state of insulin in the blood and the insulin sensitivity is determined by measuring the amount of glucose necessary to compensate for an increased insulin level without causing hypoglycemia (low blood sugar). (see Figure below, and a more elaborate description at Diabetesmanager (pbworks).

Prior to beginning the hyperinsulinemic period, basal blood samples were taken and labeled [6,6-2H2]glucose was infused  for assessment of glucose kinetics in the basal state. At different time-points concentrations of glucose, insulin, and plasma nonesterified fatty acids (NEFA) were measured.

The sleep stages were differently affected  by the curtailed sleep duration: the proportion of the stage III and stage II sleep were greater (P < 0.007), respectively smaller (P < 0.006) in the sleep deprived night.

Partial sleep deprivation did not alter basal levels of glucose, nonesterified fatty acids (NEFA), insulin, glucagon, or cortisol measured the following morning, nor did it affect basal endogenous glucose production.

However, during the CLAMP-procedure there were significant alterations on the following parameters:

  • Endogenous glucose production – increase of approximately 22% (p< 0.017), indicating hepatic insulin resistance.
  • Rate of Glucose Disposal – decrease by approximately 20% (p< 0.009), indicating decreased peripheral insulin sensitivity.
  • Glucose infusion rate – approximately 25% lower after the night of reduced sleep duration (p< 0.001). This is in agreement with the above findings: less extra glucose needed to maintain plasma glucose levels.
  • NEFA – increased by 19% (p< 0.005), indicating decreased insulin sensitivity of lipolysis (breakdown of triglyceride lipids– into free fatty acids).

The main novelty of the present study is the finding that one single night of shortened sleep is sufficient to reduce insulin sensitivity (of different metabolic pathways) in healthy men and women.

This is in agreement with the evidence of observational studies showing an association between sleep deprivation and obesity/insulin resistance/diabetes (3-5). It also extends results from previous experimental studies (summarized in the paper), that document the effects on glucose-resistance after multiple nights of sleep reduction (of 4h) or total sleep deprivation.

The authors speculate that the negative effects of multiple nights of partial sleep restriction on glucose tolerance can be reproduced, at least in part, by only a single night of sleep deprivation.

And the media conclude:

  • just one night of short sleep duration can induce insulin resistance, a component of type 2 diabetes (Science Daily)
  • healthy people who had just one night of short sleep can show signs of insulin resistance, a condition that often precedes Type 2 diabetes. (Medical News Today)
  • even a single of night of sleep deprivation can cause the body to show signs of insulin resistance, a warning sign of diabetes (CBS-news)
  • And this was of course the message that catched my eye in the first place: “Gee, one night of bad sleep, can already disturb your glucose metabolism in such a way that you arrive at the first stage of diabetes: insulin resistance!…Help!”

    First “insulin resistance” calls up another association than “partial insulin resistance” or a “somewhat lower insulin sensitivity” (as demonstrated in this study).  We interpret insulin resistance as a disorder that will eventually lead to diabetes, but perhaps adaptations in insulin sensitivity are just a normal phenomenon, a way to cope with normal fluctuations in exercise, diet and sleep. Or a consequence of other adaptive processes, like changes  in the activity of the autonomous nervous system in response to a short sleep duration.

    Just as blood lipids will be high after a lavish dinner, or even after a piece of chocolate. And just as blood-cortisol will raise in case of exercise, inflammation or stress. That is normal homeostasis. In this way the body adapts to changing conditions.

    Similarly -and it is a mere coincidence that I saw the post of Neuroskeptic about this study today- an increase of blood cortisol levels in children when ‘dropped’ at daycare, doesn’t mean that this small increase in cortisol is bad for them. And it certainly doesn’t mean that you should avoid putting toddlers in daycare as Oliver James concludes, because “high cortisol has been shown many times to be a correlate of all manner of problems”. As neuroskeptic explains:

    Our bodies release cortisol to mobilize us for pretty much any kind of action. Physical exercise, which of course is good for you in pretty much every possible way, cause cortisol release. This is why cortisol spikes every day when you wake up: it helps give you the energy to get out of bed and brush your teeth. Maybe the kids in daycare were just more likely to be doing stuff than before they enrolled.

    Extremely high levels of cortisol over a long period certainly do cause plenty of symptoms including memory and mood problems, probably linked to changes in the hippocampus. And moderately elevated levels are correlated with depression etc, although it’s not clear that they cause it. But a rise from 0.3 to 0.4 is much lower than the kind of values we’re talking about there.

    So the same may be true for a small temporary decrease in glucose sensitivity. Of course insulin resistance can be a bad thing, if blood sugars stay elevated. And it is conceivable that bad sleep habits contribute to this (certainly when combined with the use of much alcohol and eating junk food).

    What is remarkable (and not discussed by the authors) is that the changes in sensitivity were only “obvious” (by eyeballing) in 3-4 volunteers in all 4 tests. Was the insulin resistance unaffected in the same persons in all 4 tests or was the variation just randomly distributed? This could mean that not all persons are equally sensitive.

    It should be noted that the authors themselves remain rather reserved about the consequences of their findings for normal individuals. They conclude “This physiological observation may be of relevance for variations in glucoregulation in patients with type 1 and type 2 diabetes” and suggest that  “interventions aimed at optimization of sleep duration may be beneficial in stabilizing glucose levels in patients with diabetes.”
    Of course, their second article in diabetic persons[2], rather warrants this conclusion. Their specific advise is not directly relevant to healthy individuals.

    Credits

    References

    1. Donga E, van Dijk M, van Dijk JG, Biermasz NR, Lammers GJ, van Kralingen KW, Corssmit EP, & Romijn JA (2010). A Single Night of Partial Sleep Deprivation Induces Insulin Resistance in Multiple Metabolic Pathways in Healthy Subjects. The Journal of clinical endocrinology and metabolism PMID: 20371664
    2. Donga E, van Dijk M, van Dijk JG, Biermasz NR, Lammers GJ, van Kralingen K, Hoogma RP, Corssmit EP, & Romijn JA (2010). Partial sleep restriction decreases insulin sensitivity in type 1 diabetes. Diabetes care PMID: 2035738
    3. Nielsen LS, Danielsen KV, & Sørensen TI (2010). Short sleep duration as a possible cause of obesity: critical analysis of the epidemiological evidence. Obesity reviews : an official journal of the International Association for the Study of Obesity PMID: 20345429
    4. Monasta L, Batty GD, Cattaneo A, Lutje V, Ronfani L, van Lenthe FJ, & Brug J (2010). Early-life determinants of overweight and obesity: a review of systematic reviews. Obesity reviews : an official journal of the International Association for the Study of Obesity PMID: 20331509
    5. Cappuccio FP, D’Elia L, Strazzullo P, & Miller MA (2010). Quantity and quality of sleep and incidence of type 2 diabetes: a systematic review and meta-analysis. Diabetes care, 33 (2), 414-20 PMID: 19910503
    The subjects were studied on 3 d, separated by intervals of at
    least 3 wk. Subjects kept a detailed diary of their diet and physical
    activity for 3 d before each study day and were asked to maintain
    a standardized schedule of bedtimes and mealtimes in accordance
    with their usual habits. They were admitted to our clinical
    research center the night before each study day, and spent 8.5 h
    in bed from 2300 to 0730 h on all three occasions. Subjects fasted
    throughout these nights from 2200 h. The first study day was
    included to let the subjects become accustomed to sleeping in our
    clinical research center. Subjects were randomly assigned to sleep
    deprivation on either the second (n4) or third (n5) occasion.
    During the night of sleep restriction, subjects spent 8.5 h in
    bed but were only allowed to sleep from 0100 to 0500 h. They
    were allowed to read or watch movies in an upward position
    during the awake hours, and their wakefulness was monitored
    and assured if necessary.
    The rationale for essentially broken sleep deprivation from
    2300 to 0100 h and from 0500 to 0730 h, as opposed to sleep
    deprivation from 2300 to 0300 h or from 0300 to 0730 h, was
    that in both conditions, the time in bed was centered at the same
    time, i.e. approximately 0300 h. Slow-wave sleep (i.e. stage III of
    non-REM sleep) is thought to play the most important role in
    metabolic, hormonal, and neurophysiological changes during
    sleep. Slow-wave sleep mainly occurs during the first part of the
    night, whereas REM sleep predominantly occurs during the latter
    part of the night (12). We used broken sleep deprivation to
    achieve a more equal compression of both non-REM and REM
    sleep stages. Moreover, we used the same experimental conditions
    for partial sleep deprivation as previously used in other
    studies (7, 13) to enable comparison of the results.




    Three Studies Now Refute the Presence of XMRV in Chronic Fatigue Syndrome (CFS)

    27 04 2010

    ResearchBlogging.org.“Removing the doubt is part of the cure” (RedLabs)

    Two months ago I wrote about two contradictory studies on the presence of the novel XMRV retrovirus in blood of patients with Chronic Fatigue Syndrome (CFS).

    The first study, published in autumn last year by investigators of the Whittemore Peterson Institute (WPI) in the USA [1], claimed to find XMRV virus in peripheral blood mononuclear cells (PBMC) of patients with CFS. They used PCR and several other techniques.

    A second study, performed in the UK [2] failed to show any XMRV-virus in peripheral blood of CFS patients.

    Now there are two other negative studies, one from the UK [3] and one from the Netherlands [4].

    Does this mean that XMRV is NOT present in CFS patients?

    No, different results may still be due do to different experimental conditions and patient characteristics.

    The discrepancies between the studies discussed in the previous post remain, but there are new insights, that I would like to share.*

    1. Conflict of Interest, bias

    Most CFS patients seem “to go for” WPI, because WPI, established by the family of a chronic fatigue patient, has a commitment to CFS. CFS patients feel that many psychiatrists, including authors of the negative papers [2-4] dismiss CFS as something “between the ears”.  This explains the negative attitude against these “psych-healers” on ME-forums (i.e. the Belgium forum MECVS.net and http://www.forums.aboutmecfs.org/). MECVS even has a section “faulty/wrong” papers, i.e. about the “failure” of psychiatrists to demonstrate  XMRV!

    Since a viral (biological) cause would not fit in the philosophy of these psychiatrists, they might just not do their best to find the virus. Or even worse…

    Dr. Mikovits, co-author of the first paper [1] and Director of Research at WPI, even responded to the first UK study as follows (ERV and Prohealth):

    “You can’t claim to replicate a study if you don’t do a single thing that we did in our study,” …
    “They skewed their experimental design in order to not find XMRV in the blood.” (emphasis mine)

    Mikovits also suggested that insurance companies in the UK are behind attempts to sully their findings (ERV).

    These kind of personal attacks are “not done” in Science. And certainly not via this route.

    Furthermore, WPI has its own bias.

    For one thing WPI is dependent on CFS and other neuro-immune patients for its existence.

    WPI has generated numerous press releases and doesn’t seem to use the normal scientific channels. Mikovits presented a 1 hr Q&A session about XMRV and CFS (in a stage where nothing has been proven yet). She will also present data about XMRV at an autism meeting. There is a lot of PR going on.

    Furthermore there is an intimate link  between WPI and VIP Dx, both housed in Reno. Vip DX is licensed by WPI to provide the XMRV-test. Vipdx.com links to the same site as redlabsusa.com, for Vip Dx is the new name of the former RedLabs.

    Interestingly Lombardi (the first author of the paper) co-founded Redlabs USA Inc. and  served as the Director of Operations at Redlabs, Harvey Whittemore owns 100% of VIP Dx, and was the company President until this year and  Mikovits is the Vice President of VIP Dx. (ME-forum). They didn’t disclose this in the Science paper.

    TEST_RQN_Feb_2010

    Vip/Dx offers a plethora of tests, and is the only RedLab -branch that performs the WPI-PCR test, now replaced by the “sensitive” culture test (see below). At this stage of controversy, the test is sold as “a reliable diagnostic tool“(according to prohealth). Surely their motto “Removing the doubt is part of the cure” appeals to patients. But how can doubt be removed if the association of XMRV with CFS has not been confirmed, the diagnostic tests offered have yet not been truly validated (see below), as long as a causal relationship between XMRV and CFS has not been proven and/or when XMRV does not seem that specific for CFS: it has also been found in people with prostate cancer, autism,  atypical multiple sclerosis, fibromyalgia, lymphoma)(WSJ).

    Meanwhile CFS/ME websites are abuzz with queries about how to obtain tests -also in Europe- …and antiretroviral drugs. Sites like Prohealth seem to advocate for WPI. There is even a commercial XMRV site (who runs it is unclear)

    Project leader Mikovits, and the WPI as a whole, seem to have many contacts with CSF patients, also by mail. In one such mail she says (emphasis and [exclamations] mine):

    “First of all the current diagnostic testing will define with essentially 100% accuracy! XMRV infected patients”. [Bligh me!]….
    We are testing the hypothesis that XMRV is to CFS as HIV is to AIDS. There are many people with HIV who don’t have AIDS (because they are getting treatment). But by definition if you have ME you must have XMRV. [doh?]
    [….] There is so much that we don’t know about the virus. Recall that the first isolation of HIV was from a single AIDS patient published in late 1982 and it was not until 2 years later that it was associated with AIDS with the kind of evidence that we put into that first paper. Only a few short years later there were effective therapies. […]. Please don’t hesitate to email me directly if you or anyone in the group has questions/concerns. To be clear..I do think even if you tested negative now that you are likely still infected with XMRV or its closest cousin..

    Kind regards, Judy

    These tests costs patients money, because even Medicare will only reimburse 15% of the PCR-test till now. VIP Dx does donate anything above costs to XMRV research, but isn’t this an indirect way to support the WPI-research? Why do patients have to pay for tests that have not proven to be diagnostic? The test is only in the experimental phase.

    I ask you: would such an attitude be tolerated from a regular pharmaceutical company?

    Patients

    Another discrepancy between the WPI and the other studies is that only the WPI use the Fukuda and Canadian criteria to diagnose CFS patients. The Canadian  criteria are much more rigid than those used in the European studies. This could explain why WPI has more positives than the other studies, but it can’t fully explain that WPI shows 96% positives (their recent claim) against 0% in the other studies. For at least some of the European patients should fulfill the more rigid criteria.

    Regional Differences

    Patients of the positive and negative studies also differ with respect to the region they come from (US and Europe). Indeed, XMRV has previously been detected in prostate cancer cells from American patients, but not from German and Irish patients.

    However, the latter two reasons may not be crucial if the statement in the open letter* from Annette Whittemore, director of the WPI, to Dr McClure**, the virologist of the second paper [2], is true:

    We would also like to report that WPI researchers have previously detected XMRV in patient samples from both Dr. Kerr’s and Dr. van Kuppeveld’s cohorts prior to the completion of their own studies, as they requested. We have email communication that confirms both doctors were aware of these findings before publishing their negative papers.(……)
    One might begin to suspect that the discrepancy between our findings of XMRV in our patient population and patients outside of the United States, from several separate laboratories, are in part due to technical aspects of the testing procedures.

    Assuming that this is true we will now concentrate on the differences in the PCR -procedures and results.

    PCR

    All publications have used PCR to test the presence of XMRV in blood: XMRV is present in such low amounts that you can’t detect the RNA without amplifying it first.

    PCR allows the detection of a single or few copies of target DNA/RNA per milligram DNA input, theoretically 1 target DNA copy in 105 to 106 cells. (RNA is first reverse transcribed to DNA). If the target is not frequent, the amplified DNA is only visible after Southern blotting (a radioactive probe “with a perfect fit to” the amplified sequence) or after a second PCR round (so called nested PCR). In this second round a set of primers is used internal to the first set of primers. So a weak signal is converted in a strong and visible one.

    All groups have applied nested PCR. The last two studies have also used a sensitive real time PCR, which is more of a quantitative assay and less prone to contamination.

    Twenty years ago, I had similar experiences as the WPI. I saw very vague PCR bands that had all characteristics of a tumor-specific sequence in  normal individuals, which was contrary to prevailing beliefs and hard to prove. This had all to do with a target frequency near to the detection limit and with the high chance of contamination with positive controls. I had to enrich tonsils and purified B cells to get a signal and sequence the found PCR products to prove we had no contamination. Data were soon confirmed by others. By the way our finding of a tumor specific sequence in normal individuals didn’t mean that everyone develops lymphoma (oh analogy)

    Now if you want to proof you’re right when you discovered something new you better do it good.

    Whether a PCR assay at or near the detection limit of PCR is successful depends on:

    • the sensitivity of the PCR
      • Every scientific paper should show the detection limit of the PCR: what can the PCR detect? Is 1 virus particle enough or need there be 100 copies of the virus before it is detected? Preferably the positive control should be diluted in negative cells. This is called spiking. Testing a positive control diluted in water doesn’t reflect the true sensitivity. It is much easier for primers to find one single small piece of target DNA in water than to find that piece of DNA swimming in a pool of DNA from 105 cells. 
    • the specificity of the PCR.
      • You can get aspecific bands if the primers recognize other than the intended sequences. Suppose you have one target sequence competing with a lot of similar sequences, then even a less perfect match in the normal genome has every chance to get amplified. Therefore you should have a negative control of cells not containing the virus (i.e. placental DNA), not only water. This resembles the PCR conditions of your test samples.
    • Contamination
      • this should be prevented by rigorous spatial separation of  sample preparation, PCR reaction assembly, PCR execution, and post-PCR analysis. There should be many negative controls. Control samples should be processed the same way as the experimental samples and should preferably be handled blinded.
    • The quality and properties of your sample.
      • If XMRV is mainly present in PBMC, separation of PBMC by Ficoll separation (from other cells and serum) could make the difference between a positive and a negative signal. Furthermore,  whole blood and other body fluids often contain inhibitors, that may lead to a much lower sensitivity. Purification steps are recommended and presence of inhibitors should be checked by spiking and amplification of control sequences.

    Below the results per article. I have also made an overview of the results in a Google spreadsheet.

    WPI
    The PCR conditions are badly reported in the WPI paper, published in Science[1]. As a matter of fact I wonder how it ever came trough the review.

    • Unlike XMRV-positive prostate cancer cells, XMRV infection status did not not correlate with the RNASEL genotype.
    • The sensitivity of the PCR is not shown (nor discussed).
    • No positive control is mentioned. The negative controls were just vials without added DNA.
    • Although the PCR is near the detection limit, only first round products are shown (without confirmation of the identity of the product). The positive bands are really strong, whereas you expect them to be weak (near the detection limit after two rounds). This is suggestive of contamination.
    • PBMC have been used as a source and that is fine, but one of WPI’s open letters/news items (Feb 18), in response to the first UK study, says the following:
      • point 7. Perhaps the most important issue to focus on is the low level of XMRV in the blood. XMRV is present in such a small percentage of white blood cells that it is highly unlikely that either UK study’s PCR method could detect it using the methods described. Careful reading of the Science paper shows that increasing the amount of the virus by growing the white blood cells is usually required rather than using white blood cells directly purified from the body. When using PCR alone, the Science authors found that four samples needed to be taken at different times from the same patient in order for XMRV to be detected by PCR in freshly isolated white blood cells.(emphasis mine)
    • But carefully reading the methods,  mentioned in the “supporting material” I only read:
      • The PBMC (approximately 2 x 107 cells) were centrifuged at 500x g for 7 min and either stored as unactivated cells in 90% FBS and 10% DMSO at -80 ºC for further culture and analysis or resuspended in TRIzol (…) and stored at -80 ºC for DNA and RNA extraction and analysis. (emphasis mine)

      Either …. or. Seems clear to me that the PBMC were not cultured for PCR, at least not in the experiments described in the science paper.

      How can one accuse other scientists of not “duplicating” the results if the methods are so poorly described and the authors don’t adhere to it themselves??

    • Strikingly only those PCR-reactions are shown, performed by the Cleveland Clinic (using one round), not the actual PCR-data performed by WPI. That is really odd.
    • It is also not clear whether the results obtained by the various tests were consistent.
      Suzanne D. Vernon, PhD, Scientific Director of the CFIDS Association of America (charitable organization dedicated to CFS) has digged deeper into the topic. This is what she wrote [9]:
      Of the 101 CFS subjects reported in the paper, results for the various assays are shown for only 32 CFS subjects. Of the 32 CFS subjects whose results for any of the tests are displayed, 12 CFS subjects were positive for XMRV on more than one assay. The other 20 CFS subjects were documented as positive by just one testing method. Using information from a public presentation at the federal CFS Advisory Committee, four of the 12 CFS subjects (WPI 1118, 1150, 1199 and 1125) included in the Science paper were also reported to have cancer – either lymphoma, mantle cell lymphoma or myelodysplasia. The presentation reported that 17 WPI repository CFS subjects with cancer had tested positive for XMRV. So how well are these CFS cases characterized, really?

    The Erlwein study was published within 3 months after the first article. It is simpler in design and was reviewed in less then 3 days. They used whole blood instead of PBMC and performed nested PCR using another set of primers. This doesn’t matter a lot, if the PCR is sensitive. However, the sensitivity of the assay is not shown and the PCR bands of the positive control look very weak, even after the second round (think they mad a mistake in the legend as well: lane 9 is not a positive control but a base pair ladder, I presume). It also looked like they used a “molecular plasmid control in water”, but in the comments on the PLoS ONE paper, one of the authors states that the positive control WAS spiked into patient DNA.(Qetzel commenting to Pipeline Corante) Using this PCR none of the 186 CSF samples was positive.

    Groom and van Kuppeveld studies
    The two other studies use an excellent PCR approach[3,4]. Both used PBMC, van Kuppeveld used older cryoperserved PBMC. They first tried the primers of Lombardi using a similar nested PCR, but since the sensitivity was low they changed to a real time PCR with other optimized primers. They determined the sensitivity of the PCR by serially diluting a plasmid into PBMC DNA from a healthy donor. The limit of sensitivity equates to 16 and 10 XMRV-gene copies in the UK and the Dutch study respectively. They have appropriate negative controls and controls for the integrity of the material (GAPDH, spiking normal control cDNAs in negative DNA to exclude sample mediated PCR inhibition[1], phocine distemper virus[2]), therefore also excluding that cryopreserved PBMC were not suitable for amplification.

    The results look excellent, but none of the PCR-samples were positive using these sensitive techniques. A limitation of the Dutch study is the that numbers of patients and controls were small (32 CSF, 43 controls)

    Summary and Conclusion

    In a recent publication in Science, Lombardi and co-authors from the WPI reported the detection of XMRV-related, a novel retrovirus that was first identified in prostate cancer samples.

    Their main finding, presence of XMRV in peripheral blood cells could not be replicated by 3 other studies, even under sensitive PCR conditions.

    The original Science study has severe flaws, discussed above. For one thing WPI doesn’t seem to adhere to the PCR to test XMRV any longer.

    It is still possible that XMRV is present in amounts at or near the detection limit. But it is equally possible that the finding is an artifact (the paper being so inaccurate and incomplete). And even if XMRV was reproducible present in CFS patients, causality is still not proven and it is way too far to offer patients “diagnostic tests” and retroviral treatment.

    Perhaps the most worrisome part of it all is the non-scientific attitude of WPI-employees towards colleague-scientists, their continuous communication via press releases. And the way they try to directly reach patients, who -i can’t blame them-, are fed up with people not taking them serious and who are longing for a better diagnosis and most of all a better treatment. But this is not the way.

    Credits

    *Many thanks to Tate (CSF-patient) for alerting me to the last Dutch publication, Q&A’s of WPI and the findings of Mrs Vernon.
    – Ficoll blood separation. Photo [CC] http://www.flickr.com/photos/42299655@N00/3013136882/
    – Nested PCR: ivpresearch.org

    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. Groom, H., Boucherit, V., Makinson, K., Randal, E., Baptista, S., Hagan, S., Gow, J., Mattes, F., Breuer, J., Kerr, J., Stoye, J., & Bishop, K. (2010). Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome Retrovirology, 7 (1) DOI: 10.1186/1742-4690-7-10
    4. van Kuppeveld, F., Jong, A., Lanke, K., Verhaegh, G., Melchers, W., Swanink, C., Bleijenberg, G., Netea, M., Galama, J., & van der Meer, J. (2010). Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort BMJ, 340 (feb25 1) DOI: 10.1136/bmj.c1018
    5. McClure, M., & Wessely, S. (2010). Chronic fatigue syndrome and human retrovirus XMRV BMJ, 340 (feb25 1) DOI: 10.1136/bmj.c1099
    6. http://scienceblogs.com/erv/2010/01/xmrv_and_chronic_fatigue_syndr_5.php
    7. http://scienceblogs.com/erv/2010/01/xmrv_and_chronic_fatigue_syndr_6.php
    8. http://scienceblogs.com/erv/2010/03/xmrv_and_chronic_fatigue_syndr_11.php
    9. http://www.cfids.org/xmrv/022510study.asp
    Sensitivity of PCR screening for XMRV in PBMC DNA. VP62 plasmid was serially diluted 1:10 into PBMC DNA from a healthy donor and tested by Taqman PCR with env 6173 primers and probe. The final amount of VP62 DNA in the reaction was A, 2.3 × 10-2 ng, B, 2.3 × 10-3 ng, C, 2.3 × 10-4 ng, D, 2.3 × 10-5 ng, E, 2.3 × 10-6 ng, F, 2.3 × 10-7 ng or G, 2.3 × 10-8 ng. The limit of sensitivity was 2.3 × 10-7 ng (shown by trace F) which equates to 16 molecules of VP62 XMRV clone.




    The Doctors & the Patient.

    25 04 2010

    Once there was a woman with secondary Addison’s disease. She took her replacement medicine (cortisol, thyrax, growth hormone, DHEAs) as she should and everything was reasonably under control.

    However, gradually she began to feel weak. She often disliked food, sometimes to such an extent that she had an urge to vomit. After a month or so, she began to feel dizzy, especially when standing up. Then her heart suddenly banged very quickly against her chest. It felt frightening.

    Her work wasn’t going well in this period. Feeling weak and not being able to sleep, she could not concentrate and often felt like she could just cry or lay  down on the floor, because she was so extremely, extremely tired.
    But it was a busy and important period. She should write a research proposal, finish a paper, and present a poster at a congress. Furthermore her paper was chosen for a press conference, that was recorded live.

    Nobody took her serious, no one thought that she might be ill. She did recognize some symptoms that could be manifestations of an Addison crisis (weakness, dislike of food). However there was no reason for a crisis: she took all her medicine and had no flu or fever, thus there was no need to raise the cortisol dose.

    Before she went on vacation she just wanted to check with her endocrinologist, but the endo was on vacation too and according to the policy of the hospital she had to be referred by her GP. Her GP also had vacation, so she went to the locum. He reserved his lunch time for her, which was really nice.

    Because this doctor wasn’t familiar with the disease, he looked it up in a book and decided to determine whether she had orthostatic hypotension (drop in pressure and dizziness when standing up), because this is typical for an Addison crisis (she learned then). She had to lay down and stand up several times. He failed to demonstrate orthostatic hypotension (but she felt it was taking him too long to measure the blood pressure after standing up; she didn’t say anything though).

    Then this GP phoned the attending endocrinologist at the hospital. This was one of the endocrinologists who had been in charge of her during the years. Usually she got a new doctor each year.
    From the conversation she could infer that this endocrinologist didn’t think there was really much wrong. Afterwards the GP urged her to take her medicine (as if she didn’t take enough) and to take more if necessary. He gave her a form for blood tests, but urged her only to do the tests when it was REALLY REALLY REALLY necessary…..

    She felt bad, like a show-off. She excused herself, she was sorry to have bothered him. She never did the blood test, but took some extra cortisol though.

    She went on holidays (why would she stay home?), but from there it only went worse. She couldn’t sleep at all. She had gradually lost up to 8-10 kilos in 2-3 months time (about 15% of her body weight). She was dizzy and nauseous and regularly had migraine .

    Back home she went to her own GP. He didn’t examine her, but sighed: “This diseases is too complicated. I think you better see your endocrinologist….”

    So she phoned her (own) endocrinologist and she was invited to come after the regular consultations. The doctor did a physical exam and noticed orthostatic hypotension. Blood tests were done, which revealed a low blood sodium (125), not as dramatically low as during her first crisis (106), but still abnormal (<140). The next days the sodium dropped further and the creatinine got outside the normal range, so the endo proposed to hospitalize her, saying: “this is not an emergency, but i don’t trust it, because this is not like you. It is not like you at all. I don’t want to wait for the weekend.”

    Her endocrinologist had to convince the internist to make a bed available for admission.

    During the intake, the two internists behaved very skeptical. Even though the lab results pointed at a too low cortisol level (later confirmed by the lab). One of them kept repeating that a cup of salty soup would pick her up (lack of salt mainly plays a role in patients with primary Addison’s disease, who also lack aldosterone). He would even say that after she recovered by her cortisol infusion. The recovery was very quick. The other internists said that it was a miraculous metamorphosis.

    Well that female patient? That was me, six years ago.

    And all doctors were males except the doctor who finally hospitalized me.

    Yes of all doctors I had seen as an Addison patient over the years, she was the only woman. Many of the male practitioners were excellent doctors, but none of them would have hospitalized me because his intuition told him “something was not o.k.” Maybe I’m wrong and it is just by chance that this happened to be a female doctor. But somehow I feel that, on average (!) female doctors listen a little bit more to their heart, and male doctors a little bit more to their brains.
    It is only intuition, but then I’m a woman. 😉
    My doctor was not interested in a career as the other doctors, she didn’t like the competition, she didn’t like the hierarchy and she didn’t care for a publication in top journals. Her patients came first.
    She has now moved to another (non-academic) hospital and I have a new doctor again. A man, indeed.

    ——————-

    This post is submitted to the upcoming Grand Rounds at Chronic Babe, with the theme Babes:
    Your post might be about what it’s like to have women patients. Or to be a woman patient, or a woman who’s a health care provider. It could be about trends in health care for women, or how awesome women are, or how annoying they are. It could be about a particular patient who was very ladylike, or someone who totally wasn’t. It’s your choice. Whatever you send, just please make sure it’s related to the Babes in some way, or else I won’t be including you. This is Grand Rounds, ChronicBabe-style, so it’s Ladies Choice!





    Irreversible Effects of Previous Cortisol Excess on Cognitive Functions in Cushing’s Disease

    10 04 2010

    ResearchBlogging.orgApril 8th is Cushing’s Awareness Day. This day has been chosen as a day of awareness as it is the birthday of Dr. Harvey Cushing, a neurosurgeon, who discovered this illness.

    Cushing’s disease is a rare hormone disease caused by prolonged exposure to high levels of the stress hormone cortisol in the blood, whereas Addison’s disease is caused by the opposite: the lack of cortisol. For more background information on both see this previous post. Ramona Bates MD, of Suture for a Living, has written an excellent review (in plain language) about Cushing’s Disease on occasion of Cushing Awareness Day at EmaxHealth.

    From this you can learn that Cushing’s disease can be due to the patient taking cortisol-like glucocorticoids, such as prednisone for asthma (exogenous cause), but can also arise because people’s bodies make too much of cortisol itself.  This may be due to a tumor on the pituitary gland, the adrenal gland, or elsewhere in the body.

    Symptoms of Cushing’s disease are related to the effects of high levels of cortisol or other glucocorticoids on the immune system, the metabolism and  the brain. Symptoms include rapid weight gain, particularly of the trunk and face (central obesity, “moon face” and buffalo neck), thinning of the skin and easy bruising, excessive hair growth, opportunistic infections, osteoporosis and high blood pressure.

    Less emphasized than the clinical features are the often very disabling cognitive deficits and emotional symptoms that accompany Cushing’s disease. Cushing patients may suffer from various psychological disturbances, like insomnia, mood swings, depression and manic depression, and from cognitive decline. Several studies have shown that these glucocorticoid induced changes are accompanied by atrophy of the brain, and in particular of the  hippocampal region, leading to hippocampal volume loss and a profound loss of synapses [2]. This hippocampal loss seems reversible [2], but are neurological and psychological defects also restored? This is far more important to the patient than anatomic changes.

    If we listen to Cushing patients, who are “cured” and have traded Cushing’s disease for Addison’s disease, we notice that they feel better after their high levels of cortisol have normalized, but not fully cured (see two examples of ex-Cushing patients with longlasting if not irreversible health) problems in my previous post here. [added 2010-04-17)
    To realize how this affects daily life, I recommend to read the photo-blog 365 days with Cushing by Robin (also author of Survive the Journey). Quite a few of her posts deal with the continuous weakness (tag muscle atrophy), tiredness (tag fatigue), problems with (short-term) memory (see tag memory)  or both (like here and here).

    Scientifically the question is to which extent ex-Cushing patients score worse than other healthy individuals or chronically ill people and, if so, whether this can be attributed to the previous high levels of glucocorticoids.

    A recent study by endocrinologists (and one neurologists) from the Leiden University Medical Center assessed the cognitive functioning of patients  after long-term cure of their Cushing’s disease (caused by a ACTH producing pituitary adenoma, that induces overproduction of cortisol (hypercortisolism) by the adrenals [1]. Previous studies had contradictory outcomes and/or were too small to draw conclusions.

    The authors first compared a group of 74 Cushing patients (with a previous pituitary tumor) with matched healthy controls (selected by the patients themselves). Matched means that these controls had the same characteristics as the Cushing patients with respect to gender (male/female: 13/61), age (52 yr) and education.
    Cushing patients were on average 13 years in remission and were followed for another 3 years (total 16 yrs follow-up). Cushing’s disease  had been established by clinical signs and symptoms and by appropriate biochemical tests. All patients were treated by transsphenoidal surgery (surgery via the nostrils), if necessary followed by repeat surgery and/or radiotherapy (27%). Cure of Cushing’s disease was defined by normal overnight suppression of plasma cortisol levels after administration of dexamethasone and normal 24-h urinary excretion rates of cortisol. 58% of the patients had at least one form of hypopituitarism (deficiency of one or more hormones) and half of the patients needed hydrocortisone replacement therapy.

    Long after their cure, 62% of the Cushing patients reported memory problems, and 47% reported problems in executive functioning. The Hospital Anxiety and Depression Scale (HADS)-score (10.5)  indicated no clinical depression or anxiety. Patients with long-term cure of Cushing’s disease did not perform worse on measures of global cognitive functioning. However, these patients had several other cognitive impairments, mainly in the memory domain.
    Only a single test result (FAS, measures verbal mental flexibility and fluency) was significantly different between patients with short and long-term remission.

    From direct comparison with healthy controls it is not clear what causes these cognitive alterations in Cushing patients.

    Therefore the cognitive function of Cushing patients was compared to that of patients previously treated for non-functioning pituitary macroadenomas (NFMA).
    NFMA patients were chosen, because they have undergone similar treatments (transsphenoidal surgery (100%), with repeat surgery and/or radiotherapy (44%) as the Cushing patients. They also shared hypopituitarism and the need for hydrocortisone substitution in half of the cases. NFMA patients, however, have never been exposed to prolonged excess of cortisol.

    Cushing patients could not be directly compared to NFMA-patients, because these patient groups differed with regard to age and gender.

    Thus Cushing patients were compared to matched healthy controls and NFMA to another set of healthy controls, matched to these NFMA patients (Male/Female: 30/24  and mean age: 61 yr).

    To compare Cushing patients with NFMA patients the Z-scores* were calculated for each patient group in relation to their appropriate control group. A general linear model was used to compare the Z-scores.

    Overall Cushing patients performed worse than NFMA patients. In the memory domain, patients cured from Cushing’s disease had a significantly lower MQ measured with the Wechsler Memory Scale compared with patients with NFMA in the subscales concentration and visual memory. On the Verbal Learning Test of Rey, patients cured from Cushing’s disease recalled fewer words in the imprinting, the immediate and delayed recall trials. Furthermore, on the Rey Complex Figure, patients with cured Cushing’s disease scored worse on both trials when compared with NFMA patients. In tests measuring executive function, patients cured from Cushing’s disease made fewer correct substitutions on the Letter-Digit Substitution Test and came up with fewer correct patterns on the Figure Fluency Test compared with treated NFMA patients.

    These impairments were not merely related to pituitary disease in general and/or its treatment, because these patients with long-term cure of Cushing’s disease also revealed subtle impairments in cognitive function compared with patients previously treated for NFMA. These are most likely caused by the irreversible effects of previous glucocorticoid excess on the central nervous system (because this is the main difference between the two).

    Sub-analysis indicated that hypopituitarism was associated with mildly impaired executive functioning** and hydrocortisone dependency** and additional radiotherapy were negatively associated with memory and executive functioning, whereas the duration of remission positively influenced memory and executive functioning.

    The main point of criticism, apparently raised during the review process and discussed by the authors, is the presentation of the data without adjustments for multiple comparisons. When more than one test is used, the chance of finding at least one test statistically significant due to chance increases. As the authors point out, however, the positive significant results were not randomly distributed among the different variables. Furthermore, the findings are plausible given the irreversible effects of cortisol excess on the central nervous system in experimental animal and clinical studies.

    Although not addressed in this study, similar cognitive impairments would be expected in patients having continuous overexposure to exogenous glucocorticosteroids, like prednison.

    * Z-scores: The z score for an item, indicates how far and in what direction, that item deviates from its distribution’s mean, expressed in units of its distribution’s standard deviation. The z score transformation is especially useful when seeking to compare the relative standings of items from distributions with different means and/or different standard deviations (see: http://sysurvey.com/tips/statistics/zscore.htm).

    ** This makes me wonder whether Addison patients with panhypopituitarism have lower cognitive functions compared to healthy controls as well.

    Hattip: Hersenschade door stresshormoon lijkt onomkeerbaar (2010/04/08/) (medicalfacts.nl/)

    References

    1. Tiemensma J, Kokshoorn NE, Biermasz NR, Keijser BJ, Wassenaar MJ, Middelkoop HA, Pereira AM, & Romijn JA (2010). Subtle Cognitive Impairments in Patients with Long-Term Cure of Cushing’s Disease. The Journal of clinical endocrinology and metabolism PMID: 20371667
    2. Patil CG, Lad SP, Katznelson L, & Laws ER Jr (2007). Brain atrophy and cognitive deficits in Cushing’s disease. Neurosurgical focus, 23 (3) PMID: 17961025 Freely available PDF, also published at Medscape