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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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Hi, Simon thanks for report. Are the first results going to be published just about general viral testing or have they actually started stage 2? Stage 2 is what I'm waiting for. I hope they keep some of the patients who are positive in stage 1, at least as controls. If we have immune deficiency, and pick up lots of viruses, eliminating the people with other viruses might get rid of your main cohort. I've never had really high titres myself, but I think we need to look at both groups.
I found Dr. Chia's video very convincing, that we need to look in the tissues. Lerner had also found viruses through biopsies. I think Lipkin said that this throughput method did not work on tissue, too complicated in make up (not if memory got that backward). Someone has been collecting tissues from patients doing standard procedures for years. I think we still have to find some way to get tissues looked at.
 
Hi, Simon thanks for report. Are the first results going to be published just about general viral testing or have they actually started stage 2? Stage 2 is what I'm waiting for. I hope they keep some of the patients who are positive in stage 1, at least as controls. If we have immune deficiency, and pick up lots of viruses, eliminating the people with other viruses might get rid of your main cohort. I've never had really high titres myself, but I think we need to look at both groups.
I found Dr. Chia's video very convincing, that we need to look in the tissues. Lerner had also found viruses through biopsies. I think Lipkin said that this throughput method did not work on tissue, too complicated in make up (not if memory got that backward). Someone has been collecting tissues from patients doing standard procedures for years. I think we still have to find some way to get tissues looked at.
Is there any list of the 18 viruses they looked for?
 
Are the first results going to be published just about general viral testing or have they actually started stage 2? Stage 2 is what I'm waiting for.

I hope they keep some of the patients who are positive in stage 1, at least as controls. If we have immune deficiency, and pick up lots of viruses, eliminating the people with other viruses might get rid of your main cohort.

...I think we still have to find some way to get tissues looked at.
- Afraid I have no more info on what will be published when.

- 20% of those positive in stage 1 get sequenced in stage 2 regardless so if they tend to have more viruses, that should show up. If a large group are taken out by the first stage then that would make the group very interesting indeed, and I'm sure there would be further work on it. From a few initial figures Mady showed, only a small proportions were screened out in stage 1, but it looked like early data so final results could be v different.

- I agree it would be v interesting to look at tissues too, but I suspect this study will be a (pretty-impressive) start, not the final answer.
Is there any list of the 18 viruses they looked for?
I am sure there is, but I don't know what's on it! Though it probably includes not just viruses but bacteria too such as Borrellia bacteria that trigger lyme disease.

Not sure I was able to help a huge amount there, sorry!
 
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.

Thank you so much for volunteering for that, Acer.
 
markers for inflammation and immune dysfunction interest me far more than pathogens. It seems unlikely they'll find a pathogen, although they may find evidence that several pathogens are involved.

I'm not impressed by Klimas talking about what keeps the illness going as she talks about deconditioning and suggests using a heart monitor in a way that would mean I could hardly go upstairs.
 
I'm not impressed by Klimas talking about what keeps the illness going as she talks about deconditioning and suggests using a heart monitor in a way that would mean I could hardly go upstairs.
Well, I feel a lot better if I don't go up or down a flight of stairs for a week or more. So she really might be onto something in that regard. Those little spurts of over-exertion (or overly-raised heartbeat) might be having a relatively mild effect that is resulting in worse symptoms and reduced ability to do other activities.

And her talk of deconditioning seems to put it in a rather peripheral status ... it's something to avoid if possible, but she also clearly states that PEM is not something to push through if we react badly to a certain level of exertion. And she certainly does not suggest that deconditioning has any central involvement in ME/CFS.
 
Breaking news from Invest in ME conference (tweets from Jørgen Jelstad):

1. They MIGHT have something interesting from the pathogen study, but too early to say:
Jørgen Jelstad@DeBortgjemte 55m
#InvestME Progress Hornig-study: much lab work done, but much remaining. Do have candidates -potentially novel- but much too early to say.

2. Looks like they found a difference between CFS and controls in CSF fluid cytokines
Pretty sure these are the Peterson/Simmaron samples, 60 patients but controls inc other illness groups too
#InvestME Hornig: Finds altered patterns of cytokine associative networks in cerebro-spinal fluid in ME/CFS compared to controls..

Not sure if this is based on CSF still, or on bloods, so sample size uncertain:
#InvestME Hornig:Show that it is a very different way the immune system as a whole is functioning, or dysfunctioning, in ME comprd to cntrls
 
Breaking news from Invest in ME conference (tweets from Jørgen Jelstad):

1. They MIGHT have something interesting from the pathogen study, but too early to say:


2. Looks like they found a difference between CFS and controls in CSF fluid cytokines
Pretty sure these are the Peterson/Simmaron samples, 60 patients but controls inc other illness groups too


Not sure if this is based on CSF still, or on bloods, so sample size uncertain:


Thanks for the update, Simon! It's all too easy for me to lose track of what's going on in these relatively long-term studies, so it's a huge help to me when someone who's managing to keep up shares their info.:thumbsup:
 
I read this today: http://www.eurekalert.org/pub_releases/2013-06/slu-slu061713.php which basically is a 'new' technique where they sequence all the DNA and RNA in the blood (as Lipkin and co are doing) and then subtract the entire human genetic sequence from the genetic material of a blood sample and identify pathogens based on what remains.
I assumed this was what Lipkin was doing with his next-gen sequencing, but it says at the bottom of this notice that its a new technique that St. Louis hope to patent. Does that mean that Lipkin isn't doing this human DNA subtraction as part of his technique, or is he doing so using some other method?
 
FWIW, I sent emails to Lipkin and Hornig along with a link to one of Chia's research articles. I suggested they speak with Chia, or at least ask Klimas whether she thinks his tissue investigations are worth consideration. Hornig replied that she agrees with the importance of investigating whether microbes are in tissue other than blood, and she said she has regular contact with Klimas about this and other things.
 
I read this today: http://www.eurekalert.org/pub_releases/2013-06/slu-slu061713.php which basically is a 'new' technique where they sequence all the DNA and RNA in the blood (as Lipkin and co are doing) and then subtract the entire human genetic sequence from the genetic material of a blood sample and identify pathogens based on what remains.
I assumed this was what Lipkin was doing with his next-gen sequencing, but it says at the bottom of this notice that its a new technique that St. Louis hope to patent. Does that mean that Lipkin isn't doing this human DNA subtraction as part of his technique, or is he doing so using some other method?
I'm a bit puzzled by this, as its exactly the approach described by Lipkin to discover unknown viruses in the past, and also my understanding of what they will do in this pathogen study. Maybe it's a twist on the technique, but don't think it's a brand new approach at all.
 
We have discovered a technology that allows us to detect new viruses," said Adrian Di Bisceglie, M.D., chairman of the department of internal medicine at Saint Louis University. "We isolate DNA and RNA, amplify the amount of genetic material present in the blood, do ultra-deep sequencing and use an algorithm to search for matches for every known piece of genetic code, both human and for microbes.

Key to the research team's success was the discovery of how to amplify the genetic material in the blood, says study researcher Xiaofeng Fan, M.D., associate professor of internal medicine at Saint Louis University.

I would say the new technique is the amplification of genetic material which probably makes finding things either easier or more reliable with fewer samples
 
I just read this article today.

Thank you Simon for a great article and for your replies to the variety of questions here.

I have a very basic (probably silly) question. When they talk about finding EBV, do they mean and active EBV virus or are they talking about elevated IGG titers?

I am always confused about this because if they do find the active virus then it could explain the symptoms and might rule out ME/CFS.

My doctor, Dr. Enlander checks for antibodies for many pathogens like EBV, CMV, HHV6, M. Pneumonia, Coxsackie B, Parvovirus B19...etc I show high titers for all of these and he believes that it is part of my ME/CFS. When I show these results to my GP, he laughs at it and says we all have antibodies to many pathogens and it doesn't mean anything.

What's up with this?
 
I just read this article today.

Thank you Simon for a great article and for your replies to the variety of questions here.

I have a very basic (probably silly) question. When they talk about finding EBV, do they mean and active EBV virus or are they talking about elevated IGG titers?

I am always confused about this because if they do find the active virus then it could explain the symptoms and might rule out ME/CFS.

My doctor, Dr. Enlander checks for antibodies for many pathogens like EBV, CMV, HHV6, M. Pneumonia, Coxsackie B, Parvovirus B19...etc I show high titers for all of these and he believes that it is part of my ME/CFS. When I show these results to my GP, he laughs at it and says we all have antibodies to many pathogens and it doesn't mean anything.

What's up with this?
Thanks, NeilK

I'm pretty sure they are looking for pathogens directly in the blood (so EBV protein or DNA), rather than elevated IGG titres, either general or specific to EBV. And of course they will be comparing the rate of EBV in patients with those in their carefully matched controls. And I think the EBV resutls will be out later this year - may well have been submitted for publication already.
 
Came across this earlier from Deckoff-Jones. Links to video presentation from Hornig. Don't know if you covered it before in the thread Simon but thought I would throw it out there anyway:

CFS Patient Advocate IiME Conference 2013: Posted on 4 June 2013

"Mady Horning gave a fine talk, echoing the one she gave in Florida in January. That talk can be accessed here. She spoke of the terrain and genetic defects leading to ME/CFS - what variables contribute to getting ME/CFS. In a follow-up question she was asked what we all want to know. What information can she give about the ongoing CFI Lipkin study? She said that 80% of the blood work is done, but that much additional work needs to be done on saliva, feces and urine. She said that they had identified several promising pathogen “candidates” including a “novel pathogen” - but the work was still early and no conclusions can be drawn. I have heard the term “novel pathogen” somewhere before."