Nature Article Uses ME/CFS to Examine The Microgenderome

[Simon]

They've made over 100 comparisons (13 symptoms each for 8 taxa). Yet they don't seem to have corrected their statistics for multiple comparisons - how would you correct for such in a study like this?

Associations between microbial level and ME/CFS symptoms: Spearman’s rank order correlations (rs) were used to investigate sex-interactions between microbial RA and ME/CFS symptom factors (Table S3). Missing data were excluded pairwise from the analyses. Correlations were deemed statistically significant at P < 0.05.

No sign of corrections. I posted a comment to ask if this is correct.

edit: resubmitted comment/question as it wasn't showing.

 

[Richard7]

@alicec

You may be right. My understanding from one of my doctors and various stuff I've read online (ie no scientific papers or careful studies) Is that the DNA test reveals things the other tests don't but they can also miss things that the culture tests pick up.

My doctor has gave up on the medicare covered DNA based test. She she had several patients with the symptoms of yeast overgrowths who came back negative on the DNA test. When she sent them for culured tests the tests came back positive.

I gather part of the issue is thought to be sample size. And this seems to makes sense to me when you consider that they are just taking a swab of a from a heterogenous sample.

But this is just anecdote related to the diagnostic tests available to the public/doctors. If these really are systemic problems, researchers may be able to solve them with different sampling techniques and multiple sampling, for example.

Of course the researchers in this paper are just using a database of analyses that were conducted for patients who not primarily research subjects. If the researchers had had the chance to conduct a project based on research needs alone they may have done it very differently.

 

[Crux]

I


On my recent GI Effects test done in December there were no lactobacillus showing at all but previously on a stool test around 2011 my level was 4+.

I am taking some good probiotics with LAB from Biocare and they seem to suit me.

Pam

Hi Pam,

I remember you've been on some courses of antibiotics, rifaximin, etc. Have you taken others, if so, which?

In 2011, when your results were 4+ of lactobacillus, were you having ME/CFS symptoms?

Hope you don't mind the questions, thanks.

 

[alex3619]

What concerns me about the culture based tests is that they miss the vast majority of species present in the gut. We are not dealing with variation due to sampling error but technical limitations which result in a picture of an almost totally different ecosystem, one which doesn't bear much resemblance to reality.

Presumably because the culture methods are aerobic and distort the outcome. That is to be suspected on general principles. I would prefer very different methods to culturing, but I am not sure DNA methods are ideal either. Maybe we have yet to develop the most reliable technology.

 

[jimells]

the species selected and studied are mostly minor constituents of the gut. They are studied because the technique facilitates their selection rather than because they are necessarily important.

Sounds like looking under the lamp post for dropped keys 'cause that's where the light is best.

 

[bertiedog]

Hi Pam,

I remember you've been on some courses of antibiotics, rifaximin, etc. Have you taken others, if so, which?

In 2011, when your results were 4+ of lactobacillus, were you having ME/CFS symptoms?

Hope you don't mind the questions, thanks.

Firstly I don't mind the questions and also want to say that the Rimfaximin has helped my gut a lot, it is much calmer now and I don't have the bugs partying under my ribs the way they did prior to taking the 10 day course. I do think I would have benefitted from a bit longer course though because there is still a bit of disturbance in my gut which might well be SIBO but I am waiting to see how things go.

I haven't taken antibiotics for Lyme since 2003, did about 9 months but got worse due to side effects of Tinidazole (horrible neuro symptoms and bad yeast) but did well then on Doxy. I have taken regular courses of Azithromycin the past year for throat infections (probably about 4 times in all of 5 days at a time; 500 mg day 1 followed by 4 days 250 mg) and also had one course of Amociclav (think I might have got the name wrong) this past September for an infected wound so I guess that is quite a lot of antibiotics but each time I was taking probiotics but it doesn't look like they worked very well.

I tried resistant starch but it was a disaster for my gut and blood sugar it seemed to feed the SIBO. The Japanese probiotic mentioned as being great here on PR also was a disaster for my gut and fed the SIBO.

I have taken lots of different probiotics, lots of high dose ones from Holland & Barrett but don't think they are very effective but also ones often mentioned on here like Align which didn't seem to have had any effect at all. It was the same with Symprove, a liquid probiotic which is supposed to have done well in tests.

In view of the results of the stool test in December I took one week's intensive course of Biocare one packet daily containing 70 billion acidopholous and bifida bacteria as well as FOS and followed on with their powdered version. Have had nearly a month on that and am moving back to their one daily capsule of 25 billion plus FOS as I find capsules are better for me. I can tell when I have taken their probiotics and also when I have taken VSL (which I haven't for a year) as there is a reaction in my gut whereas nothing happens on the other probiotics I have tried which makes me think they don't suit my micro biome. Not sure if I mentioned it but I had low diversity on the stool test and was advised to add my fibre to have been taking psyllium 3 times daily plus more ground flaxseed plus 1 ryvita crisp bread daily.

When I had the 4+ lactobacillus and also 2+ bifidobacteria if anything I had worse energy than I do now. My energy is only better because I have done over 14 months of Cowden herbals for Lyme and other bacteria. I regularly do between 7000-8000 steps daily to give you some idea of how active I can be. (When I have the throat infections the steps will be down to 3,500-4000 though).

Pam

 

[Crux]

When I had the 4+ lactobacillus and also 2+ bifidobacteria if anything I had worse energy than I do now. My energy is only better because I have done over 14 months of Cowden herbals for Lyme and other bacteria. I regularly do between 7000-8000 steps daily to give you some idea of how active I can be. (When I have the throat infections the steps will be down to 3,500-4000 though).

Thanks for the description with details.

Again, it looks like more accurate testing is needed, but, even at that, how will a healthy microbiome be defined?

It seems that the range for lactobacillus and bifidobacteria might should be more narrow, because, people seem to have negative responses to higher yet 'normal' titres.

 

[Crux]

@alicec ,

Would you please direct me to the specific species that you mentioned?

I'm only seeing genus. These genera seem to be typical of gut testing that I've read about.

Which genera would you consider to be most important to test?

 

[msf]

@alicec

You seem to know a lot about the testing, do you know if culture tests are reproducible/reflect the amount of a particular genera or species in the gut as shown by DNA analysis? That would seem to be the crucial thing with this study (apart from the statistics). If they do, then the findings would still be relevant, if only part of the picture.

 

[alicec]

My doctor has gave up on the medicare covered DNA based test. She she had several patients with the symptoms of yeast overgrowths who came back negative on the DNA test. When she sent them for culured tests the tests came back positive.

We may be talking at cross-purposes here. I assume you are referring here to a multiplex test for various gut parasites (ie PCR amplification of DNA using primers specific for several different organisms) offered by various pathology companies. I am familiar with one done by QML in Queensland but I think they are all fairly similar. This type of testing is very sensitive but it will only detect organisms specific to the primers used and yeast is not included so of course it will not be detected.

Culture based tests select for anything that will grow under the culture conditions used (and survive the usually aerobic unprotected conditions between collection of sample and arrival at laboratory) so might indeed detect yeast. (I do have a problem with what this might mean in light of what we are now learning about the composition of the gut microbiome but that is another issue).

My concerns don't relate to these multi-plex DNA tests for certain specific organisms (I'd include the Genova GI Effects test here which grafts some PCR for a select number of gut bacterial species or genera onto a traditional CDSA). These are just sensitive tests for the particular organisms specified - they tell us nothing about what else might be present.

I am talking about the kinds of tests performed by uBiome and American Gut where they try to sequence all bacterial DNA and hence identify all the organisms present. These two companies (and most of the on-going academic studies) focus on bacterial DNA since bacteria constitute the overwhelming majority of our microbiome.

As a side issue but one that helps us get culture based detection of things like yeast and parasites in proper context, other studies which have tried to sequence all DNA from all sources in the human gut have come up with the following sort of distribution - 5.8% viruses, 0.8% archaea, 0.14% human contamination, 0.5% all other eukaryotes (this includes fungi and parasites), EVERYTHING ELSE BACTERIAL - ie a bit less than 93%.

Anyhow it was once these types of studies started identifying the bacterial components of the gut that we began to realise that the perspective given by the previous culture-based analysis was completely skewed. Less than 1/10 of constituents were being detected and these were largely minor components.

This is what leads to my concern about what culture based tests really mean. I can find no compelling reason to place much faith in them at all.

 

[alicec]

Presumably because the culture methods are aerobic and distort the outcome.

Exactly. Also there is the problem of the time lapse between collection of sample and laboratory analysis. Most gut species don't survive the exposure to oxygen. Also many organisms are just not culturable under any known conditions.

The Bioscreen Australia does try to protect anaerobes but really only succeeds in preserving the more aero-tolerant end of the spectrum (more than the standard tests which do little to protect anaerobes, but still not enough for most gut anaerobes).

I would prefer very different methods to culturing, but I am not sure DNA methods are ideal either

The DNA based methods are still relatively new and have had teething problems but huge progress is being made. What other kind of testing do you envisage?

 

[alicec]

Sounds like looking under the lamp post for dropped keys 'cause that's where the light is best.

A very good description

 

[alicec]

Would you please direct me to the specific species that you mentioned?

Sorry I used the term loosely. Only genera are mentioned.

These genera seem to be typical of gut testing that I've read about.

Culture-based gut testing yes. These tests give the impression that Lactobacillus and Bifidobacterium are the most important gut constituents and that a few other species/genera, might be imbalanced (these might include E. coli, Streptococcus, Enterococcus plus some potential pathogens like Klebsiella, Enterobacter etc).

All of these genera are minor gut constituents, some very minor. The much vaunted Lactobacillus is a very minor gut constituent - maybe 0.01% - (much more important in the mouth, vagina and maybe small intestine) and almost all species are transients. Very few actually colonise the gut. Bifidobacterium is more numerous - around 1.4%, but small compared with the two most numerous on average Bacteroides (21.8%) and Faecalibacterium (9.9%) .

In the paper they do look at a few more genera than usual, but consider the phylum Firmicutes which is being studied. The genera selected are Clostridium, Streptococcus, Lactobacillus and Enterococcus.

In the uBiome community, these genera constitute, on average, 0.8%, 1%, maybe 0.01% and 0.8% respectively of the total microbiome (I am familiar with these rather the American Gut analysis though I don't think there are any great differences). But what about the far more numerous Firmicutes such as Faecalibacterium at 9.9%, Blautia at 7.9%, Ruminococcus at 5.9%, Roseburia at 4.7% (there are many more before we reach Streptococcus at 1%)? The technique can't measure these strict anaerobes and so they are ignored.

Now I'm not saying that the most numerous are necessarily the most important. There is plenty of evidence that some minor constituents play a very important role, it's just that I can't place much faith in a technique that isn't even aware of the existence of most of the ecosystem being studied.

Which genera would you consider to be most important to test?

That is a very difficult question to answer. Technology has allowed us to accumulate much more data than we can really understand, so it is easy to do a uBiome or American Gut test but very difficult to know what the results mean. It is a bit like 23 and me analysis of SNPs. Many people jumped on the bandwagon and started offering analysis of an individual's SNPs but most of the so-called knowledge circulating on the internet is nonsense. One has to work really hard and read a lot of research as well as review opinion sceptically to come to conclusions about SNPs and then for most of them we just don't know.

With a uBiome analysis there is even less known, though this is changing. It is not so much individual genera that are important though some certainly seem to be. As well there seems to be a lot of redundancy in the system so that various genera can fulfil the same or similar functions, so patterns of change are important. I have been doing them regularly over more than 12 months and only now feel I am getting a handle on what is going on in my gut, but I still only have a broad picture.

With the benefit of hindsight I don't believe that the culture-based tests I have done showed anything useful in light of what is really going on in my gut as shown by DNA analysis.

 

[Richard7]

@alicec

Thanks for the explanation.

When I look a little deeper (or listen to someone with deeper knowledge) a lot of these models seem threadbare.

I spent some years seeing a doctor who worked with bioscreen to try to make my microbiome match the normal pattern. When we finally got something that looked about right I was much worse than I had been with the overgrowths, but I did not make a connection between these two events.

Since I have managed to get a bit better (seemingly by working on diet and dealing with SIBO) it has occurred to me that I was possibly pursuing the wrong goal.

So much of medicine, or the stuff one reads as an outsider, seems a kind of cargo cult. One sees the behaviours or biomarkers of a healthy person and tries to copy them with the hope of becoming a healthy person too.

And yes if we can only see a small part of the diversity “weed seed feed” becomes kill a bunch of stuff you never knew was there, seed with stuff that may not be relevant, feed a lot of stuff that you do not know is there.

We can see something about the shape of the airport, the shape of the planes and the behaviour of the people who work there, but have no idea of what is really going on.

And this is timely, I have been flailing about trying to work out what direction to try to move in to try to get to a better more manageable level of CFS, or better; I would certainly accept better.

And I was thinking of, and saving for, another round of faecal analysis to work out what was going on in the lower gut in the hope that I would be able to completely sort out my gut, and that this would lead to greater improvements.

Hmmm, I think perhaps not.

I wasn't thinking of having one but, your point about the anaerobes really makes me wonder about the validity of faecal transplants.

Maybe the best solution is to ... no I'm all out.

 

[Richard7]

Oh, I started writing my last post before you wrote your last post.

Are you finding that the ubiome test gives you actionable information or just information that is interesting?

(I like interesting, I love interesting on those occasions when I can think clearly enough to understand it, but I have to choose where I spend my energy and money.)

 

[alicec]

do you know if culture tests are reproducible/reflect the amount of a particular genera or species in the gut as shown by DNA analysis?

Not as far as I can see. I'll give an example. Towards the end of 2014 I did a Genova GI Effects and my first uBiome test at the same time. According to the culture part of the Genova test, I was +4 for Bifidobacterium and E.coli, 2+ for alpha haemolytic Streptococcus, Klebsiella oxytoca, Bacillus and 1+ for Enterobacter; Lactobacillus was not detected.

The PCR part of the test did not detect Bifidobacterium, Lactobacillus was very low (1st quintile) and E. coli was fairly high (4th quintile). The others were not tested.

On the uBiome test, only Streptococcus was detected - the entire genus constituting 0.05% of my gut bacterial DNA of which only a small part was likely to be the species detected by culture.

Can these results be reconciled? Well maybe in part but for the most part no.

From having done a number of uBiome tests, I've come to realise that at very low levels of DNA (around 0.01%) it is a bit of a lottery as to whether a particular genus turns up in a test - it might be there at very low levels but missed by the sampling technique. In other words if a genus is absent in a uBiome test it could still be there but it would be at extremely low level.

So a genus which is present at very low levels but is readily culturable might be detected by a culture technique but not by uBiome. However the attempts at quantitation by culture are just meaningless - it is a property of the culturability of the particular organism selected and has nothing to do with concentration in the gut.

In my case, I suspect the Bifidobacterium detected in the Genova culture test was really a residue of a probiotic species (I did stop probiotics about a week before the test but some hardy organisms might have survived). By definition a probiotic species would be very amenable to culture.

As for the others detected by culture, well I can't see how they could possibly be of significance since almost one year and 4 uBiome tests later, none of them have ever turned up, apart from Streptococcus which hovered around 0.5 - 0.1 % and has only recently started to increase to more normal values (0.68, cf av 1%). They might be present, but remain at extremely low levels.

The increase in Streptococcus (which is a normal gut constituent) has been accompanied by a stable occurrence of Bifidobacterium, originally at 0.02%, more recently 0.16%. This latter result is still only about 1/10 of average but I am confident that at this level of DNA, the result is real and Bifido has finally been established in my gut. (I am waiting on more recent results which should confirm this).

I cannot reconcile the attempts at quantitation by PCR with anything. At best I think one could say the PCR part of the Genova test shows that a genus or species could be present but it is not possible to rely on the quantitation.

In short culture based techniques in no way reflect the composition of the gut flora. They may mean something but I don't know what it is.

 

[alicec]

your point about the anaerobes really makes me wonder about the validity of faecal transplants.

The ones that are done in reputable clinics do take this into account (though I don't know enough about the details to know how successfully) but DIY techniques fail dismally.

Are you finding that the ubiome test gives you actionable information or just information that is interesting

Interesting information.

 

[Gingergrrl]

Does anyone know how to get these microbiome or gut tests in the U.S.? The only test I have ever been offered is for SIBO or a generic stool sample test which always comes back "normal."

Does it require a doctor to order and interpret the tests? Are there doctors who know which kinds or probiotics someone might benefit from or is it all still guesswork?

Am asking here b/c everyone seems to know a lot about this here and seems really knowledgeable in this thread. I have been told that Bifido Infantis is probably the only one I would tolerate but I am afraid to add anything new at this point due to a new recurrence of allergic reactions.

Are these the tests offered by Red Labs in Belgium or somewhere different? Thanks for any info and this topic really confuses me in general but if there is a gut link to my other symptoms, I would love to figure it out.

 

[MeSci]

I really appreciate the wealth of information you are posting here, @alicec. It is both extremely interesting and potentially very useful, and I have saved some of it.

It seems to be giving further support to my belief that it is better to use diet/prebiotics to bring the gut microbiome into balance, rather than probiotics. I have believed for some time that the latter may be of limited use, as they consist of such a small range of microorganisms, whereas the gut and other microbiomes are extremely complex.

It seems to me that it is better to just give the gut the right food and let it create its own healthy balance of microorganisms.

Even a faecal transplant has potential downsides, I would guess, as each person's gut microbiome may be exquisitely tuned to their own immune system, and someone else's is less likely to be so.

 

[Sidereal]

It seems to be giving further support to my belief that it is better to use diet/prebiotics to bring the gut microbiome into balance, rather than probiotics.

I'm not overly optimistic about our prospects of being able to fully restore a healthy microbial community with diet & prebiotics only. A study was published in Nature just yesterday (warning: mice) that found that while diet & prebiotics worked in the first generation of animals with dysbiotic guts, in subsequent generations of offspring what was required was FMT + prebiotics (which they are calling MACs - microbiota-accessible carbohydrates). Of course, these animals didn't have ME/CFS. Many here cannot tolerate prebiotics, let alone FMT.

http://www.nature.com/nature/journal/v529/n7585/full/nature16504.html

Diet-induced extinctions in the gut microbiota compound over generations

The gut is home to trillions of microorganisms that have fundamental roles in many aspects of human biology, including immune function and metabolism1, 2. The reduced diversity of the gut microbiota in Western populations compared to that in populations living traditional lifestyles presents the question of which factors have driven microbiota change during modernization. Microbiota-accessible carbohydrates (MACs) found in dietary fibre have a crucial involvement in shaping this microbial ecosystem, and are notably reduced in the Western diet (high in fat and simple carbohydrates, low in fibre) compared with a more traditional diet3. Here we show that changes in the microbiota of mice consuming a low-MAC diet and harbouring a human microbiota are largely reversible within a single generation. However, over several generations, a low-MAC diet results in a progressive loss of diversity, which is not recoverable after the reintroduction of dietary MACs. To restore the microbiota to its original state requires the administration of missing taxa in combination with dietary MAC consumption. Our data illustrate that taxa driven to low abundance when dietary MACs are scarce are inefficiently transferred to the next generation, and are at increased risk of becoming extinct within an isolated population. As more diseases are linked to the Western microbiota and the microbiota is targeted therapeutically, microbiota reprogramming may need to involve strategies that incorporate dietary MACs as well as taxa not currently present in the Western gut.

If the same holds true for humans, it's easy to see why each generation is becoming more obese and sicker. The derangement in the gut microbiome would be more intractable if you are born to a sick mother.