Do IBS Patients Lack Blastocystis and Dientamoeba??

I feel like sharing data from a poster created by one of my colleagues, Dr Laura Rindom Krogsgaard who works at Køge Sygehus, Denmark. She presented the poster last month at the United European Gastrointestinal (UEG) Week in Berlin.

Laura is currently doing a very interesting survey on IBS and IBS-like symptoms in Danish individuals. Her first publication was on the epidemiology of IBS in Denmark (see literature list below). She performed a web-based survey, using YouGov Zapera, and questionnaires were emailed to a web panel (n = 19,567) representative of the general Danish population aged 18-50 years containing info on gender, age, geography and type of intestinal symptoms (if any). IBS and subtypes were estimated by the Rome III criteria. Of 6,112 responders, 979 (16%) fulfilled the Rome III criteria for IBS and had no organic diagnosis likely to explain their symptoms. IBS subtypes detected included  mixed IBS (36%), IBS with diarrhea (33%), IBS with constipation (18%), and unsubtyped IBS (11%).

At the Laboratory of Parasitology, we helped Laura analyse stool samples from survey participants for parasites. Not surprisingly, Blastocystis and Dientamoeba were by far the most common parasites detected; however, it appeared that individuals with IBS symptoms were less likely to be colonised by these parasites than their controls! Which means that we have a situation reminiscent of that seen in IBD patients, only less pronounced. 

Laura was able to survey symptom developement over 1 year and compare this to the incidence of Blastocystis and Dientamoeba, and none of the parasites (indvidually or in co-infection) were linked to symptom development.

Indeed, Laura's data are in line with the general tendency that we see for Blastocystis (see figure below). Blastocystis appears to be rare in individuals with perturbation of the intestinal microbiota (due to antibiotic treatment, inflammation, infection, diet, etc.), while common in healthy individuals, most of whom are probably characterised by high gut microbial diversity and thereby - apparently - the right substrate/growth conditions for Blastocystis.

Literature:

Krogsgaard LR, Engsbro AL, & Bytzer P (2013). The epidemiology of irritable bowel syndrome in Denmark. A population-based survey in adults ≤50 years of age. Scandinavian Journal of Gastroenterology, 48 (5), 523-9 PMID: 23506174

The entire poster "Dientamoeba fragilis and Blastocystis: Two parasites the irritable bowel might be missing" presented at the UEGweek can be viewed here via SlideShare.

More Bits And Pieces On The Microbiome... Or Maybe Mycobiome...

I promised to include some more stuff from some of the many recent publications in Science and Science Translational Medicine on the intestinal microbiome and its potential role in health and disease, and I've chosen two papers that could have broad public interest; for those who need an introduction to the microbiome, please go here (Wikipedia entry).

Because the microbiome has been more or less exclusively synonymous with the "bacteriome" it's very refreshing to discover a paper on fungal diversity in the gut. Like Blastocystis, and other single-celled parasites, intestinal fungi are also micro-eukaryotes, and we are continuously searching for the role of micro-eukaryotes in health and disease.

In general, very little is known about fungi in the intestine, and most clinicians, even mycologists, hardly bother about the fungi that may be present in our intestine, - I think I can say that without offending anyone! Maybe one of the most interesting things in a clinical respect is the fact that antibodies against the yeast Saccharomyces cerevisiae (see below) is a common finding in patients with Crohn's Disease, but relatively uncommon in patients with ulcerative colitis and healthy individuals.

Now, Iliev et al. (2012) start out by confirming the fact that fungi are indeed common commensals and thus a part of our normal intestinal flora. They then showed that colitis chemically induced in mice led to circulating antibodies against S. cerevisiae, which suggested that fungal antigens commonly found in the gut might be responsible for the induction of these antifungal antibodies during colitis.
The innate immune receptor Dectin-1 appears to have a key role in fungal recognition and combating. Therefore the authors wanted to further explore the role of this receptor by studying mice with and without Dectin-1. They found that Dectin-1 deficiency led to increased susceptibility to chemically induced colitis, including weight loss, tissue destruction and cell infiltration by inflammatory cells, etc. Moreoever, evidence was found of fungal invasion of inflamed tissue in the Dectin-1 knockout mice and taken together, their data suggest that Dectin-1 deficiency leads to altered immunity to commensal gut fungi.
To cut a long story short, results from these experiments in mice led the investigators to search for mutations in CLEC1A (the human Dectin-1 gene) in patients with ulcerative colitis, and they found that mutations were significantly more common in patients with severe ulcerative colitis (patients requiring colectomy) than in those with a less aggressive disease progression. This suggests that not only bacteria but also intestinal fungi interact with the intestinal immune system and may thereby influence health and disease. If this can be confirmed by others, this is an example of how biomarkers can predict the disposition towards/progression of disease and the results may have profound consequences for diagnostic strategies (e.g. screening for mutations in the Dectin-1 gene) and therapeutic management of patients with severe ulcerative colitis. Maybe it would have been interesting to know about such mutations in patients with Crohn's Disease as well...

Next, the investigators took to identifying what types of fungi were actually present in the colon of these mice. What may be a little bit controversial is the fact that the authors - by amplification and deep sequencing of  ITS1-2 (genetic marker commonly used to identify and taxonomically group fungi) - appear to have found not only species representing a staggering 50 well-annotated fungal genera in the mouse microbiome, but an additional 100 "novel and/or un-annotated fungi" as well - this does sound like a lot, but somehow the reader is calmed down a bit, when the authors later tell us that 97.3% of all fungi detected in the mouse faeces belonged to only 10 species, with 65.2% of the fungal sequences belonging to Candida tropicalis. So, whether the 100 novel fungi are indeed fungi colonising the intestinal tract is unknown, but they may very well represent fungi "on transit", so to say, acquired from food, drink or environment maybe... we know that fungi are ubiquitous - we inhale fungi every day for instance, and when deep sequencing is applied, it may be possible to trace even fungi only present in very small quantities; also ITS-2 analysis does not tell us whether the sequences are from "intact/live" (i.e. colonising) fungi or from degraded fungi (i.e. ingested); a classic example is Saccharomyces cerevisiae (Brewer's or Baker's yeast), which we may often acquire from food and drink, but which may also colonise (settle and proliferate) our intestines. Contamination of the faecal samples from fungi present in the environment and during processing is also a possibility (one of the reasons why PCR-based diagnostics for fungal infections is a tricky task...). Well so, all of these new species/genera may not necessarily represent the "mouse mycobiome". However, the authors found only few of the fungi in the food that was fed to the mice, so this still may remain a bit of a mystery... it would have been interesting to know whether the fungi detected were yeasts or molds, for instance, and very little information can be extracted from the supplementary material (phylogenetic analysis) accompanying the paper. Anyway, it's all very stimulating and further studies will assist in exploring fungal diversity and, hopefully, the diversity of micro-eukaryotes in general.

Saccharomyces cerevisiae is used in food and drink, but may also colonise our guts.

The next paper is one of many recent papers heralding the implementation of microbiome-based therapies in future personalised and precision medicine, possibly relevant to diseases such as inflammatory bowel disease, obesity and diabetes. Microbiome manipulation, so to say, is key to this concept and includes controlled diet, pre- and probiotic interventions, bariatric surgery (e.g. gastric bypass), faecal transplants (see my recent blog post on feacal bacteriotherapy), helminth therapy (yes!) or ecological engineering. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, and these may be known to many as lactobacilli or bifidobacteria (or simply "yoghurt"!) that protect us against harmful bacteria by inhibiting their growth and by helping reduce cholesterol levels, synthesise vitamins and sustain immune responses. Prebiotics are non-digestible dietary sugar molecules (oligosaccharides) that can enhance the activity of for instance lactobacilli and bifidobacteria. While the potential benefits of pre- and probitics have been known for many years, it is only with current available technology that we are starting to get a mechanistic understanding of their impact on our bodies.

The article picks up on host-gut microbiota metabolic interactions and the so-called "host-microbe metabolic axes", which include pathways and interactions responsible for gut permeability, formation of blood vessels (angiogenesis) in the gut mucosa, ion transports, sulfation ability of xenobiotics, and many other things; sulfation ability is a key component in metabolising of drugs, for instance. Differences in our individual abilities to sulfate certain compounds give us at least one explanation as to why different people may respond differently two drugs treatment (see previous posts), and our ability to metabolise a common drug such as acetaminophen (paracetamol) can apparently be predicted form our preinterventional excretion of the microbial co-metabolite 4-cresyl sulfate; other gut microbial contributions that can alter the absorption, metabolism, and safety of drugs have been demonstrated recently.

Gastric bypass (Roux-en-Y) is a surgical procedure carried out to delay and reduce the absorption of calories and includes bypassing a large part of the stomach and a part of the small intestine by a procedure known as "stapling". Roux-en-Y appears to be associated with major and stable changes in the microbiota and in many microbially generated compounds, all of which are key components in the host-microbe metabolic axes. "This suggests that the microbiota is an essential part of the "gearbox" that connects the physical effects of bariatric surgery to the resulting beneficial effects."

Gut ecology changes with age, and current investigations seek to define the rationale of and potential for manipulating the microbiome of older people, for instance with pre- and probiotics, to secure higher microbiome diversity (high microbiome diversity appears to be beneficial) and resilience to antibiotics-induced changes in gut flora.

For those of you who nearly choked on "helminth therapy" - I may put up a post in the future on how helminths (and maybe other intestinal eukaryotes such as amoebae?) apparently play a role in the presentation and regulation of diseases such as asthma and inflammatory bowel diseases...

The cells of our intestinal microbiome outnumber our own body cells by 10 to 1. Within the next decade or so we will be able to extract a lot of information about how the bacteria and other "bugs" in our guts influence and contribute to health and disease. Importantly, we may have to realise now more than ever that "germs and bugs" and their actions and interactions can hold the key to a healthy life in ways that we wouldn't think were possible only a few years ago. This means that we should acknowledge that some bacteria and parasites may be a sign of a healthy intestinal environment / a healthy gut function, and that consumption of drugs such as antibiotics may produce shifts in our microbiota that may not necessarily be beneficial.

References:

Iliev ID, Funari VA, Taylor KD, Nguyen Q, Reyes CN, Strom SP, Brown J, Becker CA, Fleshner PR, Dubinsky M, Rotter JI, Wang HL, McGovern DP, Brown GD, & Underhill DM (2012). Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. Science (New York, N.Y.), 336 (6086), 1314-7 PMID: 22674328
 

Holmes E, Kinross J, Gibson GR, Burcelin R, Jia W, Pettersson S, & Nicholson JK (2012). Therapeutic modulation of microbiota-host metabolic interactions. Science translational medicine, 4 (137) PMID: 22674556

The Circular Problem of Blastocystis

After submitting stool samples for microbiological analyses, many people with intestinal symptoms are informed by their GPs that they have Blastocystis, and that the clinical significance of this parasite is unknown (which is not entirely wrong). However, some GPs may want to try to eradicate Blastocystis in the absence of other potential causes of the symptoms, prescribing drugs such as Protostat/Flagyl (Metronidazole). During and after treatment, many patients will experience temporary alleviation only "to get back to where they started" after a couple of weeks or so. And often, they will also remain positive for Blastocystis (sometimes Blastocystis may be very difficult to detect during the course of treatment and immediately after treatment, which may be due to a transitory decrease in parasite load for direct and indirect reasons; see below). Anyway, this is the classical scenario.

The problem with Blastocystis is a circular one: There is currently no single 100% successful treatment, and when people with symptoms + Blastocystis cannot get rid of their parasites and thereby get the chance to report on symptom status after permanently cleared infection (+/-clinical improvement), it is - to put it mild - extremely challenging to collect information and data that can assist us in drawing conclusions. It doesn't make it any better that we know that a lot of people have Blastocystis without knowing and without having symptoms.We therefore shouldn't blame health care professionals for being in the dark.

People who do not know a lot about Blastocystis (and who does?) might take to the Internet to get more information, including how to deal with the infection. Not all the advice given on the Internet may be useful and little of it will be based on scientific evidence. Some people may be desperate to try and clear any parasite that they have been diagnosed with, without realising that some parasites might actually be a sign of a healthy gut ecological system and be of potential benefit in terms of maintaining a healthy immune system; we don't know much about this yet. Or maybe the use of antibiotics will damage the general intestinal flora and cause more or more severe symptoms than would the persistence of the parasitic infection! We don't know, but as hinted at in previous posts, our new technologies will assist us in obtaining the information that we have been looking for so long.

So, how do we move on from here? There is no doubt that scientific studies are key. Pilot data are available showing that at least one of the genetic variants (subtypes) of Blastocystis is more common in patients with symptoms than in the background population, but this still needs confirmation.

The genetic diversity of Blastocystis found in humans is huge. If the genetic diversity of Blastocystis was visible, different subtypes of Blastocystis would probably be as different as these marble balls!

We need substantial funding for carrying out large-scale studies aiming to confirm these data. Once epidemiological association has been sufficiently demonstrated, the next step is to find out whether those strains/subtypes associated with disease are characterised by having effector proteins not seen or - maybe more convincingly - not expressed in strains/subtypes seen in healthy individuals. If we have proof of both epidemiological association and expression of virulence genes, then next step could  include a randomised control treatment (RCT) study in order to identify the drug(s) that lead to microbiological and/or clinical improvement, i.e. parasite eradication and alleviation of symptoms, respectively.

It may be so that different subtypes of Blastocystis respond to different antibiotics. And if successful treatment is dependent on other factors as well such as complex microbial interspecies interactions, it may be perplexing to realise, that different individuals may respond differently to any given treatment. As Pepper and Rosenfield suggest in their paper about microbiome multistability: A key consequence of multistability is that different instances of the same type of system, such as different individual gut microbiomes, may show very different responses to the same perturbation.

And so, how does this relate to Blastocystis treatment? Well, since none of the treatments used for treating Blastocystis are specific for this parasite (metronidazole for instance is a broad-spectrum antibiotic used to eradicate a range of anaerobic bacteria, including Clostridium), there will probably be a mixture of direct and indirect effects on Blastocystis upon treatment. The direct effect on Blastocystis will depend on its susceptibility to the antibiotic, while the indirect effect will depend on the bacterial flora and how it responds during treatment. Hence, drugs may directly affect Blastocystis and/or perturb the intestinal flora to an extent which makes it an unsuitable habitat for Blastocystis. We hope soon to be able to investigate the interaction between Blastocystis and gut bacteria by metagenomic approaches. It should be kept in mind though that metronidazole is absorbed from the proximal part of the intestine, while Blastocystis is a parasite of the colon; hence, it may very well be so that metronidazole does not reach Blastocystis in its niche. When treating intestinal amoebiasis, metronidazole is given together with a luminal drug to ensure targeting both invasive and the luminal Entamoeba histolytica.

So, while we should keep pursuing the role of Blastocystis in disease, we should also try to explore whether there are some good sides to Blastocystis colonisation and whether we can learn to see the parasite as a proxy for something (clinical condition, enterotype, etc.). I have expanded a bit on this in my recent paper "Thinking Blastocystis Out Of The Box", available in the journal Trends in Parasitology. To this end, learning about the bacterial communities that may influence Blastocystis growth and establishment may improve our ability to understand Blastocystis in an ecological context.

For those who are interested in this, may I suggest some further reading (including papers on (unpredictable) antibiotics-associated changes in gut flora and individualised responses to perturbations in the gut microbiome and a couple of studies on Blastocystis subtypes where links to disease phenotypes have been identified):

Pepper, J., & Rosenfeld, S. (2012). The emerging medical ecology of the human gut microbiome Trends in Ecology & Evolution, 27 (7), 381-384 DOI: 10.1016/j.tree.2012.03.002

Dethlefsen, L., & Relman, D. (2010). Colloquium Paper: Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation Proceedings of the National Academy of Sciences, 108 (Supplement_1), 4554-4561 DOI: 10.1073/pnas.1000087107

Stensvold, C., Christiansen, D., Olsen, K., & Nielsen, H. (2011). Blastocystis sp. Subtype 4 is Common in Danish Blastocystis-Positive Patients Presenting with Acute Diarrhea American Journal of Tropical Medicine and Hygiene, 84 (6), 883-885 DOI: 10.4269/ajtmh.2011.11-0005

Domínguez-Márquez, M., Guna, R., Muñoz, C., Gómez-Muñoz, M., & Borrás, R. (2009). High prevalence of subtype 4 among isolates of Blastocystis hominis from symptomatic patients of a health district of Valencia (Spain) Parasitology Research, 105 (4), 949-955 DOI: 10.1007/s00436-009-1485-y

Stensvold, C., (2012). Thinking Blastocystis Out Of The Box Trends in Parasitology DOI: 10.1016/j.pt.2012.05.004

On Faecal Bacteriotherapy

For those of you who read my most recent blog post and who went on to read Carl Zimmer's article in The New York Times about gut flora transplantation on a woman suffering from chronic Clostridium difficile diarrhoea: The concept of faecal bacteriotherapy is maybe not that new. Allegedly, it dates back to Pliny the Elder and others, who prescribed orally ingested faeces to cure maladies! Stools were, however, incinerated first, and only the ashes ingested.

Pliny the Elder and others with him allegedly recommended  ingesting the ashes of faeces to cure disease.

In less ancient times - in 1989 to be more precise - Tvede and Rask-Madsen from Copenhagen, Denmark (Statens Serum Institut and The Danish State Hospital) reported on bacteriotherapy for chronic relapsing C. difficile diarrhoea in six patients. They hypothesised that absence of Bacteroides results in chronic relapsing C. difficile diarrhoea, and that its presence may prevent colonisation by C. difficile. In the current issue of Microbe Magazine, Young and Aronoff describe some of the mechanisms that may be involved in our indigenous gut flora's ability to prevent the colonisation of potentially pathogenic bacteria such as C. difficile. These include: (1) occupying space (physically preventing contact by newly arrived microbes with the host), (2) directly impairing the growth or germination of C. diffıcile, (3) withholding nutrients or germinants from C. diffıcile, and (4) shaping the host adaptive and innate immune responses.

Hence, the concept of dysbiosis and the ideas of manipulating the gut flora in order to "restore order" have been going on for a long time. Metagenomics, however, will assist us in exploring exactly what is happening in much more detail and in a much broader and standardised context than previously possible. We will be able to predict shifts in the structure, function and interaction of microbial communities - hopefully including micro-eukaryotes such as fungi (the "mycobiome") and common protists such as Blastocystis and Dientamoeba (maybe we can call it the "protistome"?), - and any influence of diet, pro- and antibiotics.

And fortunately, the focus on metagenomics continues: While CMI just launched a themed issue on metagenomics advances (see previous blog post), even Science and Science Translational Medicine now dedicated an entire joint issue to "The Gut Microbiota", and I hope to be able to address one or two of these papers soon. Until then, here's a bit of suggested reading:

'Bugs as Drugs'

Tvede, M., & Rask-Madsen, J. (1989). Bacteriotherapy for chronic relapsing Clostridium difficile diarrhoea in six patients The Lancet, 333 (8648), 1156-1160 DOI: 10.1016/S0140-6736(89)92749-9

Young and Aronoff (2012). Clostridium difficile linked to disrupted gut microbiota. Microbe Magazine (ASM): http://goo.gl/FIZmC 

Mueller, K., Ash, C., Pennisi, E., & Smith, O. (2012). The Gut Microbiota Science, 336 (6086), 1245-1245 DOI: 10.1126/science.336.6086.1245