Distinct Cerebrospinal Fluid Proteomes Differentiate Post-Treatment Lyme Disease from CFS, 2011, Schutzer et al.

Obermann

Obermann

Senior Member (Voting Rights)
Abstract
Background: Neurologic Post Treatment Lyme disease (nPTLS) and Chronic Fatigue (CFS) are syndromes of unknown etiology. They share features of fatigue and cognitive dysfunction, making it difficult to differentiate them. Unresolved is whether nPTLS is a subset of CFS.

Methods and principal findings: Pooled cerebrospinal fluid (CSF) samples from nPTLS patients, CFS patients, and healthy volunteers were comprehensively analyzed using high-resolution mass spectrometry (MS), coupled with immunoaffinity depletion methods to reduce protein-masking by abundant proteins. Individual patient and healthy control CSF samples were analyzed directly employing a MS-based label-free quantitative proteomics approach. We found that both groups, and individuals within the groups, could be distinguished from each other and normals based on their specific CSF proteins (p<0.01). CFS (n = 43) had 2,783 non-redundant proteins, nPTLS (n = 25) contained 2,768 proteins, and healthy normals had 2,630 proteins. Preliminary pathway analysis demonstrated that the data could be useful for hypothesis generation on the pathogenetic mechanisms underlying these two related syndromes.

Conclusions: nPTLS and CFS have distinguishing CSF protein complements. Each condition has a number of CSF proteins that can be useful in providing candidates for future validation studies and insights on the respective mechanisms of pathogenesis. Distinguishing nPTLS and CFS permits more focused study of each condition, and can lead to novel diagnostics and therapeutic interventions.




How strong is the Schutzer et al. study from 2011?

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I have always thought that the proteomic study on the cerebrospinal fluid by Schutzer et al in 2011 was rock solid. It was selected as one of the top 100 scientific studies in 2011 by Discover Magazine. The Venn diagram (above), where they showed many unique proteins for patients with ME/CFS, was impressive. I just got into an argument with an ME-sceptic professor about that study, and she said that the differences in the Venn diagram weren't significant. After reviewing the supplementary table S1 in the paper, I have to say that I agree. Many proteins that were listed as unique for ME/CFS were positive only for one patient.

Any thoughts on that? @Jonathan Edwards ?

Edit: The samples were pooled, so we don't know how many patients in each group who had the proteins. Table S1 lists the number of unique peptides that were found. However, for the proteins that were unique for a study group, usually only one unique peptide was identified; whereas several tens or hundreds of unique peptides were found for proteins that were detected in all study groups. That does suggest that only few individuals had unique proteins.
 
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Can't speak about the science of the results but the selection criteria for the "CFS Subjects" was Fukuda, and there is no mention of PEM in any form in the study, so there is a big question mark about the cohort that they used.
 
Obermann said:
Any thoughts on that? @Jonathan Edwards ?
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I always thought that study was interesting but I was worried about the statistics. My memory is that they pooled samples and it looked as if they were drawing doubtful conclusions from that. This sort of study that measures lots of things and tries to separate statistically tends to worry me. It is a reasonable thing to do as a preliminary fishing exercise but most of the time does not hold up with repetition.
 
Jonathan Edwards said:
I always thought that study was interesting but I was worried about the statistics. My memory is that they pooled samples and it looked as if they were drawing doubtful conclusions from that. This sort of study that measures lots of things and tries to separate statistically tends to worry me. It is a reasonable thing to do as a preliminary fishing exercise but most of the time does not hold up with repetition.
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Thank you, Jonathan. I have a follow up question. I am often faced with statements such as "there is no scientific basis at all for ME/CFS". I am aware of the fact that many biomedical studies are weak—Schutzer et al was the most recent bad surprise—and I agree that we have no knowledge of aetiology or pathogenesis. However, it may take a long time to find a specific treatment or diagnostic test for ME/CFS. In the mean time, advocates need to find solid arguments that convey what we actually do know and refute the argument that nothing is known at all. The main argument probably is that ME/CFS represents a pattern in onset and trajectory; but I read the article in 2016, "The biological challenge of myalgic encephalomyelitis/chronic fatigue syndrome: a solvable problem", that you wrote with some coauthors. On page 65, you list clues to ongoing mechanisms. I think that it is a good point to start a conversation to say there is little we know for sure, but here are some clues and hints. Do you think that the list still holds? Are there new studies you would like to add, or is it any useful review that you particularly want to recommend?
 
I think what we said in the review is still my view. There have been some genetic and epidemiological studies since then which may point to a genetic component being present but hard to pin down to specific genes but nothing major has changed.

I guess my answer to the 'no scientific basis' people is that there is no scientific basis for the strength of hurricanes hitting Florida in any given year, but nobody is doubting that hurricanes hit Florida. Nobody can doubt the existence of an illness known as ME/CFS. We have a huge amount of scientific information about it but mostly negative - it does not seem to be one infection, it does not seem to be due to deconditioning, and so on.
 
Obermann said:
I have always thought that the proteomic study on the cerebrospinal fluid by Schutzer et al in 2011 was rock solid. It was selected as one of the top 100 scientific studies in 2011 by Discover Magazine.
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I qualified for both the ME/CFS and Lyme parts of this study, and was in fact recruited to participate. Somewhere along the line it was decided I could not participate since I did in fact have both diseases.

In a way, for me at least, that pretty much sums up the results.
 
Wild speculation here!
Possibly the genome wide association study (GWAS) Chris Pointing is hoping to carry out would help with this type of study. I.e. identify target genes (which produce proteins?) and pathways. Then repeat this type of protein study i.e. focused on a limited number of proteins and a larger number of people.
Similarly, if the nano-needle does identify people with ME/a subgroup of people with ME then this type of protein study, i.e. focused on a more controlled group, might yield a needle in the haystack - protein indicative of disease.

I think we should consider the application of this technique to other diseases i.e. has it delivered and if so why? E.g. successes using this method may have been in diseases which had a more homogeneous group (selected on the basis of a diagnostic test?), or a more limited range of proteins were examined - since there were clues on where to look (GWAS?).
 
Obermann said:
After reviewing the supplementary table S1 in the paper, I have to say that I agree. Many proteins that were listed as unique for ME/CFS were positive only for one patient.
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Just to point out that there were 43 CFS patients in the pooled sample and only 14 patients in the individual sample group. So not really enough patients in the individual sample group to draw meaningful conclusions, especially if the instrument is near the detection limit with the amount of sample fluid used, could be a reason why we see so many 1's and 0's.

I'm wondering why they used pooled samples, rather than all individual samples (I guess I would need to read the paper). Did it allow them to have a larger sample to give better protein detection thresholds and use multiple instruments?

EDIT : Shouldn't the individual data in the study mostly all detect the same proteins, just in different varying amounts? Looks like a much more sensitive Mass Spec is needed for this type of study.

Aside : Hanson made raw data available (Excel format) in her metabolomic study last Dec. In it you can make an educated guess as to how close readings are to the detection threshold, and better interpret differences between study participants and groups within the study to see if the papers conclusions hold up.
 
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wigglethemouse said:
This was an analysis of cerebrospinal fluid.
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Agreed i.e. I'd forgotten that this was a cerebro spinal fluid study. However, my point is that you may need to look at the contents of exosomes (using this technique - mass spectrometry proteomics) in order to understand what is going on in this disease. This may explain why Whitney Defoe's (+ other severely ill group) results on standard tests look normal - wrong sample type (plasma not exosomes).
 
I suspect that the follow up on this study faltered for lack of funding. Dr. Natelson was certainly making an effort to find funding to move on to the next phase.

In that next phase they were going to test the most suspect of the unique proteins from the pooled results against all the individual members of their respective groups (CFS and Lyme), thus establishing the prevalence of the most promising proteins within those groups. The pooling of the spinal fluid samples in the first place was a technical necessity that allowed them to find those relatively few (out of millions) that were unique to their respective groups. In the next phase, they were going to see how common (or rare) some of those target proteins were within their groups, but they had to know what to look for first, which was the purpose of this study.

I'd imagine that, since you'd probably want to use the same patients, and since the disease can vary over time, you'd want to move on to phase 2 as quickly as possible. I don't know, but I fear it may be too late to do that now.
 
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Before they did anything, they should have performed the same metrics on HC and late stage Lyme (as opposed to early Lyme) and what they call PTLDS to see if there were any differences between LS Lyme and PTLDS. I suspect there would have been no meaningful differences.

If that is the case, then what they were actually measuring was well-qualified late stage Lyme vs ME/CFS. Those results might have yielded a few interesting - and contrasting - inferences.

Personally, I'd want to see what % of ME/CFS patients who tested negative for Borrelia actually demonstrated nominal Bb proteins in their CSF.

Since this was in the US, one thing I'd want to try to capture is what portion of people diagnosed with ME/CFS actually may have a TBD which tested negative for serum antibodies. Even if they test negative on CSF thresholds, I bet netting a couple Bb-specific proteins, for instance (or bartonella or babs) would raise an eyebrow or two.

Then I'd test an ME/CFS cohort that tested negative for Lyme and apply the same constraints to see what portion exhibited some signs of Bb, however faint, and draw conclusions accordingly. Remember, Stonybrook was involved thru Pat Coyle, so they'd know what they were looking for.

Perhaps it could have been a starting point at distinguishing different types of ME/CFS in the US, e.g. infectious agents vs autoantibodies etc.
 
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