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Lipkin and Hornig go hunting for ME/CFS pathogens

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by Simon McGrath


For me, the star attraction of Nancy Klimas' recent CFS/GWI conference was always going to be Professor Mady Hornig and her talk.

Hornig might not be well known by ME/CFS patients - yet - but her boss is: Ian Lipkin, who so skillfully handled the XMRV 'dediscovery' study (which she worked on too). Despite disproving a link with XMRV, Professor Lipkin made clear his belief that ME/CFS was a serious disease that had not received the serious attention it deserved. Even more important - given his stellar record as a scientist - was his commitment to playing a serious role in trying to solve the illness. He's asked Mady Hornig to lead their hunt for the cause of ME/CFS, so I was hoping for an update on how exactly they were going about the work and when results were likely.

As well as that, we had a presentation of science that made my eyes pop, as Mady Hornig took us through some of her incredible work in other illnesses where brain and immune system take centre stage too. If she brings these kind of approaches to ME/CFS then I think we will benefit enormously - more on this next week.

First, though, to the world's largest ever ME/CFS biomedical study: the hunt by Hornig, Lipkin and colleagues for a virus or other pathogen that may cause our illness.


Huge initial studies look for pathogens or tell-tale signs of infection

Work has already started with around 400 patients plus controls, including 200 patients + controls from the CFI pathogen study (Professor Hornig is Principal Investigator for CFI's Pathogen Discovery and Pathogenesis). If that's not enough, they are hoping to get funding for up to a further 400 patients and controls. Assuming my maths are right, that makes a mind-boggling maximum of 1,600 subjects: 800 patients and 800 controls. This is a big step up for ME/CFS research.

The major part of the study is looking for pathogens - viruses, bacteria or protozoans both known and unknown. But they are also looking for protein/immune abnormalites in patients, and they even plan to take a look at gene expression too to see if that throws up any clues.

The pathogen hunt has three steps:
  1. Screen for a panel of 18 specific pathogens already implicated in ME/CFS, such as EBV.
  2. If no such pathogens are detected, they move to the heavy-duty phase, basically sequencing all DNA/RNA in the blood, which should identify both known and unknown viruses. This technique has been successfully used by Ian Lipkin in the past to discover new viruses.
  3. To be thorough (and these people are nothing if not thorough) any 'finds' from the first two steps will be confirmed by a smaller-scale but more accurate technique.


Finally, once they have confirmed candidate pathogens - assuming they find ones that have a statistically significant link with ME/CFS - they will develop new tests specifically for these pathogens.

As a bonus, the heavy-duty sequencing techniques in stage 2 makes it fairly easily to look at gene expression too. Previous attempts to identify unusual patterns of gene expression in ME/CFS patients have found differences, but these have not stood up to the test of replication. Hopefully the much larger cohorts used here will produce more reliable findings, and show which genes go awry with with ME/CFS.


Protein signature for ME/CFS?

The team will also try to define 'host profiles', looking for a unique protein signature associated with the illness. If the study does find a robust CFS signature, it could be used for diagnosis - and a validated diagnostic test is almost the Holy Grail of ME/CFS research. The signature could also be used to measure treatment progress.

Yet more high-tech approaches

They are using a fancy technique called 'multiplexed immunoassay' to look at over 50 specific proteins that are markers of immune/inflammatory changes, or oxidative stress. Again, the idea is to home in on plausible candidates for disruption in ME/CFS. For a smaller sub-sample, they will look more widely for any abnormality in protein profiles using "Target proteomics Mass Spectroscopy" - Nature's "2012 Method of the Year", no less. This approach could throw up proteins no one has yet considered playing a role in ME/CFS, giving new clues to the causes of the illness.

Another good reason to look for protein signatures is to detect general signs of infection. One possible scenario raised by Hornig is that lots of different bugs can trigger ME/CFS, and if that's the case then each individual pathogen might not show up as statistically significant. (This is to do with statistical power, but you really don't want to know). Finding the fingerprints of infection or inflammation instead might prove a general link between infection and ME/CFS, even if no individual pathogens can be pinned down for sure.


First results due later this year

The first result for these studies will be submitted to journals in the next few months, with more papers submitted later in the year. By the time the year is out we should have a fairly good idea if there is a strong link between pathogens and ME/CFS.

Professor Hornig concluded by saying that they may not find an infectious agent, it may be an immune change. It could even be something else. But if you look at the scale and cutting-edge nature of their work then, even if it does turn out not to be a pathogen, these are very good people to have on the case.


Aiming to look for genetic factors


Heading upstream to the source

In the Q&A section after her talk, someone asked if Mady Hornig and her team also plan to look for any genes that are associated with ME/CFS. It turns out they don't have enough samples to look at the whole genome, so instead they would like to use a targeted approach that Hornig calls 'swimming upstream'.

This involves identifying unusual patterns in proteins and/or gene expression using results from this study, then working backwards to identify which gene could be involved in producing these protein changes. It's an approach the group has used successfully before, and requires a smaller sample size for robust results.

A wider look at the whole genome might be also possible at a later date using meta-analysis. This would combine their data with data from other studies - giving sufficient 'statistical power' for robust results.

The studies I've covered above are hugely impressive, but what grabbed me even more was the rest of Mady Hornig's presentation, where she discusses some of the extraordinary work she's done on other hard-to-understand illnesses. She's very keen on bringing similarly innovative approaches to ME/CFS (assuming the current work doesn't solve the illness!) - as I describe in my next article, Mady Hornig: How do you solve a problem like CFS?.




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Reasonable points, and Mady Hornig and Ian Lipkin have designed the studies to take account of this, at least to some extent.

Yes - they do seem aware of the potential problems. And they seem good. Hopefully that's enough! CFS just seems like such a difficult diagnosis to do useful research on (to me anyway).
 
Hi Simon, thanks for this. I just read cort johnson's piece on Hornig's talk on the simmaron website. He wrote a lot on how Horning suggests an overgrowth of bad gut bacteria could cause immune, stress and Cns problems.

I couldn't really connect the dots. Was she saying that one possible cause of the immune dysfunction can be traced back to the bad bacteria in the gut? Is the gut just one avenue she is considering? Was there the implication that the bad gut bacteria could be theroot cause or a consequence of immune dysfunction? Many thanks
 
Bit more on cohorts:
Are they going to be looking at CNS fluid in the studies? Or just blood?
So far, just bloods, but Mady Hornig enthused about Dan Peterson's Cerebro Spinal Fluid samples and hopes to get funding for these too. Peterson has 60 extremely-well defined ME/CFS patients, plus an equal number of healthy controls or 'comparators' i.e. patients with specific illnesses eg MS.
I hope the cohort is well-defined too.
I think they are. Here's a quote from CFI's Scott Carlson about their cohort in a CFS Central Interview:
The CFI-sponsored cohort recruitment protocol uses the updated CDC definition and the Canadian Consensus Criteria to identify well-characterized subjects. We also noted the new definition presented at the recent IACFSME conference [International ME Criteria].
So that's half of the pathogen samples in this study. I'm fairly sure (but not 100%) that most of the remainder are from the NIH XMRV study (that Lipkin & Hornig ran) which used Canadian Consensus Criteria only.
Re cohorts, when studies say they use both Canadian and CDC criteria I never understand how this works. Does this mean the patients have to fulfil both case definitions? Doesn't the Canadian supercede the CDC as there are more symptoms in the Canadian criteria over and above the CDC definition? Am I missing something? Thanks
 
What's the significance of gene expression and where can I read more about it? I've heard someone mention that methylation affects gene expression which I'm curious about since I'm following a methylation protocol. It's also been referenced in Life Extension magazine in relation to calorie restriction and mimetics. They seem to think it's important, but I don't understand any of it.
As I'm sure you know, genes encode proteins - but not all genes are 'turned on', many are dormant, and those that are "on" can lead to production of proteins at different levels from very low levels to very high. Gene expression studie looks at the pattern of protein production in healthy patients and controls to see if there are any differences. Eg are some proteins present in CFS patients but not in controls, or vice versa? Or, more likely, are some genes expressed at very different levels between patients and controls? Any differences can give clues as to what;s going wrong in patients. Bit more from Wikipedia.
I didn't see in the article who is funding these studies. Is it the CFI? I imagine they would be very expensive!
CFI will be funding the CFI cohort of 200, for both the pathogen study and proteins studies, I think. Someone else will be funding the rest of the patients, and Mady Hornig is trying to get more funding from elsewhere to increase the study size. I think a lot of scientists' time goes into securing funding rather than doing science.

And yes, I don't think they will be cheap. I remember watching a Lipkin lecture talking about pathogen discovery (not for CFS) where in the Q&A he was asked about applying these techniques to CFS. "I'd love to" he said, "if someone gave me $1million" - though I think the money was for the science not his fees :). Not long after there was talk that a New York philanthropist was in contact with Lipkin, and not that much later the CFI was formed and Lipkin was lined up for the pathogen study. I don't know if that's a conicidence, but I did wonder if the philanthropist was watching that lecture too.
 
sorry, struggling a bit to keep up here!
I would have thought Dr Chia's patients would have been of interest - stomach biopsies??
Interesting point: Mady Hornig did talk about the potential role of the gut. Also, she worked a possible link between the measles virus in the gut and children with autism and gut problems so she's taken that approach before.
Re cohorts, when studies say they use both Canadian and CDC criteria I never understand how this works. Does this mean the patients have to fulfil both case definitions? Doesn't the Canadian supercede the CDC as there are more symptoms in the Canadian criteria over and above the CDC definition? Am I missing something? Thanks
The Canadian Criteria are generally narrower than the CDC ones - CFI specificed that patients had to meet both criteria and my guess is they met Canadian they met CDC anyway - but if they only met Canadian they would not have been included.
Hi Simon, thanks for this. I just read cort johnson's piece on Hornig's talk on the simmaron website. He wrote a lot on how Horning suggests an overgrowth of bad gut bacteria could cause immune, stress and Cns problems.

I couldn't really connect the dots. Was she saying that one possible cause of the immune dysfunction can be traced back to the bad bacteria in the gut? Is the gut just one avenue she is considering? Was there the implication that the bad gut bacteria could be theroot cause or a consequence of immune dysfunction? Many thanks
Yes, she was saying that. Cort is ahead of me - I just wrote about her new study, and will write about her other work next week - while Cort got it all into the one article.
 
If Im understanding this study right... those who are easily found to have things like EBV wont then be going throu the High thoughput PCR senquencing? (and also hence the results of their gene expressions wont be included?). :(

Its the EBV group I'd like to see very rigoreously studied as that is linked to so many ME/CFS cases... maybe those are even more likely to be carrying other things too then the group which wasnt obviously carrying something. Some stand out things may be missed by not looking deeper at this group in the same way as the other group is being looked at.
 
Are they going to be looking at CNS fluid in the studies? Or just blood?

I participated in this study. The "Columbia" studies are in 3 parts. The first was blood samples for the XMRV study. The second phase was looking for all known and novel pathogens. It was several months ago, but my recollection is that they took blood, tears, rectal swab, and I think one more (either nasal or throat swab - my memory is fuzzy so I don't recall at the moment). The third study was/is supposed to use spinal fluid. At the time I gave my samples for part 2, they were still working out the details for part 3. They may well have sorted it out now.
 
Thanks for that info Simon.

When reading about this study, I often get a fear that the likely heterogeneous nature of CFS will lead to a misleading negative result.

While I have no idea what they will ultimately find, I know that they were very particular about selection for these studies. I have been a patient with my CFS doctor for over 5 years and it wasn't at all guaranteed that I would be selected when I applied. I know they were very specific about controls as well. I was lucky in that they could find someone around my age/sex/location otherwise I wouldn't have been able to participate despite having qualified due to my illness and labs. My impression is that they are very focused on making sure these studies are done correctly.
 
I participated in this study because my doctor is one of the sites. The "Columbia" studies are in 3 parts. The first was blood samples for the XMRV study. The second phase was looking for all known and novel pathogens. It was several months ago, but my recollection is that they took blood, tears, rectal swab, and one more (either nasal or throat swab).
This is super interesting, I'd never heard of pathogens being looked for in rectal or throat swabs before. Thx acer.
 
This is super interesting, I'd never heard of pathogens being looked for in rectal or throat swabs before. Thx acer.

Yeah neither had I, but I am glad that they did multiple samples other than just blood. It increases the chance of finding something. Like others have said, I really hope this thing continues to gain momentum and that someone will do another tissue study like Dr. Chia did. That was smart of him.
 
I do not think they are going to find anything in the blood. The are looking at the wrong place.
You might be right. But we don't know where something will be found - if we did know, it would be because it had already been found, and no one would need to look! They seem to investigating in a very intelligent and reasonable manner, and I'm glad we've got people with the expertise and funding that are willing to do the research.
 
If Im understanding this study right... those who are easily found to have things like EBV wont then be going throu the High thoughput PCR senquencing? (and also hence the results of their gene expressions wont be included?). :(

Its the EBV group I'd like to see very rigoreously studied as that is linked to so many ME/CFS cases... maybe those are even more likely to be carrying other things too then the group which wasnt obviously carrying something. Some stand out things may be missed by not looking deeper at this group in the same way as the other group is being looked at.
Interesting point. I simplified the process slightly and actually 20% of positives from the initial screen will go into high-throughput phase, but most won't. Still, if a lot of people are positive for EBV then they will probably have enough to detect other pathogens, though perhaps not to detect gene expression differences as that tends to need pretty large samples.

However, these are smart people and if they do find EBV are a substantial group I expect they would want to look at that group in more detail anyway. One of the things that has stood out for me listening to both Lipkin and Hornig is that these are scientists who genuinely want to find out what's going on. They are not looking for ways to show there is no problem. Mady Hornig's work on autism is a good example of this: she looked for and ruled out a link with the measles virus in the gut of children with autism and gut problems - but went on to find a whole load of other abnormalities. More on this in the next blog.
 
I participated in this study. The "Columbia" studies are in 3 parts. The first was blood samples for the XMRV study. The second phase was looking for all known and novel pathogens. It was several months ago, but my recollection is that they took blood, tears, rectal swab, and I think one more (either nasal or throat swab - my memory is fuzzy so I don't recall at the moment). The third study was/is supposed to use spinal fluid. At the time I gave my samples for part 2, they were still working out the details for part 3. They may well have sorted it out now.
Thanks so much for this acer, it's great to get first hand accounts from patients. Sounds like you are in the NIH/XMRV cohort rather than the CFI one, since you were sampled for XMRV too. I hadn't realised they had collected more than blood to date. Mady is defintitely trying to get funding for the spinal fluid work, but hasn't secured it yet, as far as I know.

While I have no idea what they will ultimately find, I know that they were very particular about selection for these studies. I have been a patient with my CFS doctor for over 5 years and it wasn't at all guaranteed that I would be selected when I applied. I know they were very specific about controls as well. I was lucky in that they could find someone around my age/sex/location otherwise I wouldn't have been able to participate despite having qualified due to my illness and labs. My impression is that they are very focused on making sure these studies are done correctly.
More great info. Having such carefully matched controls will particularly help with the gene expression work as it will reduce the amount of 'noise' since age/sex will be controlled for. Noise is the biggest problem in gene expression, so with matched samples and large cohorts they have a much better chance of finding real differences.
 
sorry, struggling a bit to keep up here!

Interesting point: Mady Hornig did talk about the potential role of the gut. Also, she worked a possible link between the measles virus in the gut and children with autism and gut problems so she's taken that approach before.
The Canadian Criteria are generally narrower than the CDC ones - CFI specificed that patients had to meet both criteria and my guess is they met Canadian they met CDC anyway - but if they only met Canadian they would not have been included.
Yes, she was saying that. Cort is ahead of me - I just wrote about her new study, and will write about her other work next week - while Cort got it all into the one article.

Thanks Simon. So have I understood it correctly that Hornig is researching more than one avenue, looking at pathogens in blood such as you have described in this post, but also thinks it may not be viral pathogens involved but gut pathogens so is looking at that too? I look forward to reading your next post on this. Many thanks
 
my recollection is that they took blood, tears, rectal swab, and I think one more (either nasal or throat swab - my memory is fuzzy so I don't recall at the moment).

Tears, wow - I've never heard of tears being collected in any study. What would show up in tears that doesn't show up in other stuff?

Nasal swab sounds like a good plan - chronic sinusitis is common in PWME and there's interest in general in chronic sinusitis being associated with disruption of the normal microbiome of the stuff in your sinuses.