Share Your Experience

It is a fact that a lot of people with diarrhoea, IBS and other intestinal symptoms are diagnosed with Blastocystis, and that sometimes drugs are prescibed with the aim to obtain clinical and microbiological improvement. While there is no specific drug against Blastocystis, a lot of different ones (see previous posts) are used in order to try and eradicate the parasite. Since these drugs differ from country to country in terms of availability and since there is no consensus as to which drug(s) to use, it is of great importance that people who have been diagnosed with Blastocystis and who have received treatment share their experience. We need information on what drugs that result in partial or complete alleviation of symptoms (clinical improvement) and that are capable of clearing the parasite from the gut.

Facebook has a forum (Blastocystis sp. (B. hominis and sp.) where there is a very active debate going on just on this. It may be so that you want to share your view/experience there; you can also mail your story to parasitologyonline [at] gmail dot com.

Thanks.

"We Are Them - They Are Us!"

A stimulating talk on the microbiome by Dr Jonathan Eisen at TEDMED 2012. Enjoy!

Do I Get Diagnosed Correctly?

I can tell especially from Facebook discussions that people across the globe wanting to know about their "Blastocystis status" are worried that they are receiving false-negative results from their stool tests, and that many Blastocystis infections go unnoticed. And I think I should maybe try and say a few things on this (please also see a recent blog post on diagnosis, - you'll find it here). I might try and simplify things a bit in order not to make the post too long.

Below, you'll find a tentative representation of the life cycle of Blastocystis. It is taken from CDC, from the otherwise quite useful website DPDx - Laboratory Identification of Parasites of Public Health Concern.

Proposed life cycle of Blastocystis.

 

I don't know how useful it is, but what's important here is the fact that we accidentally ingest cysts of Blastocystis, and we shed cysts that can be passed on to other hosts. The cyst stage is the transmissible stage, and the way the parasite can survive outside the body; we don't know for how long cysts can survive and remain infective. In our intestine and triggered by various stimuli, the cysts excyst, transiting to the non-cyst form, which could be called the trophozoite / "troph" stage, or to use a Blastocystis-specific term, the "vacuolar stage" (many stages have been described for Blastocystis, but I might want to save that for later!). This is possibly the stage in the life cycle where the parasite settles, thrives, multiplies, etc. You can see a picture of vacuolar stages in this blog post. Many protozoa follow this simple life cycle pattern, among them Giardia and most species of Entamoeba. If the stool is diarrhoeic and you are infected by any one or more of these parasites, it may be so that only trophozoites, and, importantly, no cysts, are shed! This has something to do with reduced intestinal transit time and, maybe more importantly, the failure of the colon to resorb water from the stool which means that the trophozoites do not get the usual encystation stimuli. Importantly, trophozoites are in general non-infectious.

There is documentation that once colonised with Blastocystis, you may well carry it with you for years on end, and as already mentioned a couple of times, no single drug or no particular diet appears to be capable of eradicating Blastocystis - this is supported by the notion that Blastocystis prevalence seems to be increasing by age, although spontaneous resolution may not be uncommon, - we don't know much about this. Now, although day-to-day variation in the shedding of Blastocystis has been described, it is my general impression that colonised individuals may shed the parasite with each stool passage, and well-trained lab technicians/parasitologists will be able to pick up Blastocystis in a direct smear (both cysts and trophs may be seen). To do a direct smear you simply just mix a very small portion of the stool with saline or PBS on a slide, put a cover slip over it and do conventional light microscopy at x200 (screening) or x400 (verification). Very light infections may be difficult to detect this way, and if you don't have all the time in the world, a direct smear may not be the first choice.

The "king" of parasitological methods, however, is microscopy of faecal concentrates (Formol Ethyl Acetate Concentration Technique and any variant thereof), which is remarkable in its ability to detect a huge variety of parasites. Especially cysts of protozoa (e.g. Giardia and Entamoeba) and eggs of helminths (e.g. tapeworm, whipworm and roundworm) concentrate well and are identified to genus and species levels based on morphology. The method is not as sensitive as DNA-based methods such as PCR, but as I said, has the advantage of picking up a multitude of parasites and therefore good for screening; PCR methods are targeted towards particular species (types) of parasites. A drawback of the concentration method is that it doesn't allow you to detect trophzoites (i.e. the fragile, non-cystic stage), and, as mentioned, diarrhoeic samples may contain only trophozoites and no cysts...

In many countries it is very common for people to be infected by both protozoa and helminths, and in those countries microscopy of faecal concentrates is a relevant diagnostic choice. In Denmark and many Western European countries, the level of parasitism is higher than might be expected (from a hygiene and food safety point of view) but due to only few parasitic species. Paradoxically, the intestinal parasites that people harbour in this part of the world are parasites that do not concentrate well. They are mainly:

1) Blastocystis
2) Dientamoeba fragilis
3) Pinworm (Enterobius vermicularis)

Only troph stages have been described for Dientamoeba fragilis and it may be transmitted by a vector, such as pinworm (look up paper by Röser et al. in the list below for more information); this mode of transmission is not unprecedented (e.g. Histomonas transmission by Heterakis). Eggs of pinworm may be present in faeces, but a more sensitive method is the tape test.

Now, Blastocystis often disintegrates in the faecal concentration process, and while you might be lucky to pick up the parasite in a faecal concentrate, you shouldn't count on it, and hence the method is not reliable, unless the faecal sample was fixed immediatley after being voided. This is key, and also why fixatives are used for the collection of stool samples in many parts of the world - to enable the detection of fragile stages of parasites. There are many fixatives, e.g. SAF (sodium acetate-acetic formalin), PVA (poly-vinyl alcohol) and even plain formalin will do the trick if it's just a matter of preserving the parasite in the sample. If SAF or PVA is used, this allows you to do permanently stained smears of faecal concentrates, and you will be able to pick up not only cysts of protozoa, but even trophozoites. Trichrome and iron-haematoxylin staining are common methods and are sensitive but very time-consuming and may be related to some health hazards as well due to the use of toxic agents. But this way of detecting parasites is like good craftmanship - it requires a lot of expertise, but then you get to look at fascinating structures with intriguing nuclear and cytoplasmatic diagnostic hallmarks. Truly, morphological diagnosis of parasites is an art form! Notably, samples preserved in such fixatives may be useless for molecular analyses.

Iron-haematoxylin stain of trophozoites of Entamoeba coli
(note the "dirty" cytoplasm characteristic of E. coli).
Source: http://www.atlas-protozoa.com

At our lab we supplement microscopy of faecal concentrates with DNA-based detection of parasites. For some clinically significant parasites, we do a routine screen by PCR, since this is more sensitive than microscopy of faecal concentrates and because this is a semi-automated analysis that involves only DNA extraction, PCR and test result interpretation, which are all things that can be taught easily. Major drawbacks of diagnostic PCR is that you cannot really distinguish between viable (patent infection) and dead organisms (infection resolving, e.g. due to treatment). This is why, in the case of Blastocystis, you may want to do a stool culture as well (at least in post-treatment situations), since only viable cells will be able to grow, obviously.

Two diagnostic real-time PCR analyses have been published, one using CYBR Green and one using a TaqMan probe.

Now, it certainly differs from lab to lab as to which method is used for Blastocystis detection. Some labs apparently apply thresholds for number of parasites detected per visual field, and only score a sample positive if more than 5 parasites per visual field have been detected. I see no support for choosing a threshold, since 1) we do not know whether any Blastocystis-related symptoms are exacerbated by parasite intensity, 2) the number of parasites detected in a faecal concentrate may depend on so many things which have nothing to do with the observer (fluctuations in shedding for instance), and 3) the pathogenic potential of Blastocystis may very well depend on subtype.

If Blastocystis was formally acknolwedged as a pathogen, like Giardia, standardisation of methods would have happened by now. Meanwhile, we can only advocate for the use of PCR and culture if accurate diagnosis of Blastocystis is warranted, while permanent staining of fixed faecal samples constitutes a very good alternative in situations where PCR is not an option.

I have the impression that some labs do DNA-based detection of microbes, including protozoa, and that a result such as "taxonomy unknown" is not uncommon. I don't know how these labs have designed their molecular assays, and therefore I cannot comment on the diagnostic quality and relevance of those tests... it also depends on whether labs do any additional testing as well, such as the more traditional parasitological tests. However, we do know that there is a lot of organisms in our intestine, for which no data are available in GenBank, which is why it is sometimes impossible to assign a name to e.g. non-human eukaryotic DNA amplified from a stool sample.

* More than 1 billion people may harbour Blastocystis.
* Blastocystis is found mainly in the large intestine.
* 95% of humans colonised by Blastocystis have one of the following subtypes: ST1, ST2, ST3, ST4.
* DNA-based detection combined with culture ensures accurate detection of Blastocystis in stool samples and enables subtyping and viability assessment.


Further reading:

Poirier P, Wawrzyniak I, Albert A, El Alaoui H, Delbac F, & Livrelli V (2011). Development and evaluation of a real-time PCR assay for detection and quantification of blastocystis parasites in human stool samples: prospective study of patients with hematological malignancies. Journal of clinical microbiology, 49 (3), 975-83 PMID: 21177897

Röser D, Nejsum P, Carlsgart AJ, Nielsen HV, & Stensvold CR (2013). DNA of Dientamoeba fragilis detected within surface-sterilized eggs of Enterobius vermicularis. Experimental parasitology, 133 (1), 57-61 PMID: 23116599

Scanlan PD, & Marchesi JR (2008). Micro-eukaryotic diversity of the human distal gut microbiota: qualitative assessment using culture-dependent and -independent analysis of faeces. The ISME journal, 2 (12), 1183-93 PMID: 18670396

Stensvold CR, Ahmed UN, Andersen LO, & Nielsen HV (2012). Development and Evaluation of a Genus-Specific, Probe-Based, Internal-Process-Controlled Real-Time PCR Assay for Sensitive and Specific Detection of Blastocystis spp. Journal of clinical microbiology, 50 (6), 1847-51 PMID: 22422846

Stensvold CR, Arendrup MC, Jespersgaard C, Mølbak K, & Nielsen HV (2007). Detecting Blastocystis using parasitologic and DNA-based methods: a comparative study. Diagnostic microbiology and infectious disease, 59 (3), 303-7 PMID: 17913433

Stensvold CR, & Nielsen HV (2012). Comparison of microscopy and PCR for detection of intestinal parasites in Danish patients supports an incentive for molecular screening platforms. Journal of clinical microbiology, 50 (2), 540-1 PMID: 22090410

On Blastocystis and Animal Models

I was recently encouraged by one of my readers to do a blog post on Blastocystis and animal experimental models. This is not exactly my core competence, which probably boils down to the fact that animal models have only been scarcely used in Blastocystis research for reasons that I will try to account for below.

Animal models (mice, rats, guinea pigs) have often been used to study interactions between hosts and microbes as well as the effect of chemotherapeutic interventions. Therefore, one might assume that animal models are an obvious way of potentially establishing a link between Blastocystis and pathology. But currently, the rationale for carrying out some types of Blastocystis experiments on, say, mice or rats is limited. Why? Well, first and foremost because of at least three major issues.

1) Lack of correlation between in vitro and in vivo evidence. Experimental infections of laboratory mice (Elwakil and Hewedi, 2010) resulted in tissue invasion - something never reported in humans. Another study showed increased oxidative stress in Blastocystis infected rats (Chandramathi et al., 2010), again something not linked to human colonisation. Studies that provided evidence for induction of cytokines, contact mediated apoptosis, and barrier disruption all used axenic Blastocystis and in vitro mammalian cell cultures with no evidence that these effect occur in vivo.

2) Host specificity. Blastocystis exhibits extreme genetic diversity and multiple, genetically very different variants (species, subtypes) exist. These subtypes exhibit moderate host specificity. This means that some subtypes are common in one type of host, whereas other subtypes are common in other types of hosts. For instance, ST5 is very common in pigs, but we rarely see it in humans. ST4 is common in rodents, and in some human populations (mainly Europe it seems), but otherwise extremely uncommon. And so on. This means that some subtypes may be difficult to establish in experimental animals. It also means that any pathology detected in the animal, may not be “reproducible” in another host, - maybe due to the fact that this host has adapted to this particular subtype or even strain. Blastocystis is common in a huge variety of animals, and different animals may have adapted do different subtypes. It is not unlikely that this is due to co-evolution, and therefore it may not turn out to be a big surprise if Blastocystis per se is not usually directly associated with disease. It may still be so, however, that for humans, some subtypes or strains may be associated with disease, preliminary data point in this direction.

3) Study design. Another issue is the use of appropriate controls – for example, experimental infection of animals with Blastocystis from cultures growing with bacteria need to have the appropriate controls - namely infection with the accompanying bacterial flora alone – before it can be concluded that Blastocystis is responsible for any effects seen. It is extremely difficult to axenise (i.e. make sterile) Blastocystis strains, so they will always be accompanied by some bacterial species. Hence, any effect noticed after challenge with a Blastocystis strain will be difficult to interpret, - is it due to Blastocystis or to accompanying bacterial strains? (If you want to see what Blastocystis look like in culture, go to my previous blog post here.)

So, results from scientific studies using animal experimental models should be interpreted cautiously. In vitro experimental models using enterocyte mono-layers for instance may constitute a more attractive alternative, but the problems of using xenic (i.e. unsterile) strains are evident also here. A great challenge ahead is the development of a standardised method for axenising (sterilising) strains… so far, such a method does not exist.

Our French colleagues recently published the genome of Blastocystis sp. ST7. Functional genomic analysis is key to understanding the extent to which Blastocystis is capable of exerting any direct pathological effect, and will assist us in studying the potential pathogenicity of Blastocystis in the absence of a suitable animal model. Indirect pathological effects may be more difficult to identify and probably require studies of the interaction between the host, the parasite and the rest of the gut microbiota (bacteria). Given our recent technological advances, I believe that a pathway to knowledge lies in the study of Blastocystis in an ecological context. I think that we should get an understanding of: 1) Who are colonized with Blastocystis, 2) From where do we get it, 3) For how long do we have the parasite, and do we establish symptoms in the very beginning, only to adapt to the presence of the parasite later on, 4) does Blastocystis require a particular flora to establish (and are there differences between subtypes (in humans and animals)), 5) could Blastocystis be seen as a proxy for a given gut microbiota (biomarker), and/or does Blastocystis select for a given microbiota phenotype (metatranscriptomic analysis of the intestinal flora accompanying Blastocystis might be useful to study how the bacteria “behave” (i.e. gene expression) in the presence/absence of Blastocystis), 6) are any Blastocystis-induced symptoms related to parasite abundance, etc.; this can be explored in rough detail by using real-time PCR, of which two have been published.

So, while animal models may not be immediately suitable in our quest to study Blastocystis pathogenicity, our “omics” methodologies and data analyses may sooner than we know help us answer many of the questions that we have been pondering for decades.

Having said that, I think that for instance a pig experimental model might be useful in terms of studying the effect of chemotherapeutic intervention. Obvious studies include those aiming to identify drugs capable of eradicating Blastocystis, but it could also be interesting to study the structure and function (gene expression profiling) of the accompanying microbiota before and after intervention.
Since pig feed often contains a range of antibiotics, it could be interesting to test whether pigs on diets +/- antibiotics differ in terms of Blastocystis colonisation... a recent PNAS paper demonstrates a shift in the structure and function of the microbiome in medicated pigs compared to pigs fed a diet void of antibiotics.

Further reading:

Chandramathi S, Suresh KG, Mahmood AA, & Kuppusamy UR (2010). Urinary hyaluronidase activity in rats infected with Blastocystis hominis--evidence for invasion? Parasitology research, 106 (6), 1459-63 PMID: 20358228

Elwakil HS, & Hewedi IH (2010). Pathogenic potential of Blastocystis hominis in laboratory mice. Parasitology research, 107 (3), 685-9 PMID: 20499092

Hussein EM, Hussein AM, Eida MM, & Atwa MM (2008). Pathophysiological variability of different genotypes of human Blastocystis hominis Egyptian isolates in experimentally infected rats. Parasitology research, 102 (5), 853-60 PMID: 18193282 

Iguchi A, Ebisu A, Nagata S, Saitou Y, Yoshikawa H, Iwatani S, & Kimata I (2007). Infectivity of different genotypes of human Blastocystis hominis isolates in chickens and rats. Parasitology international, 56 (2), 107-12 PMID: 17251054

Looft T, Johnson TA, Allen HK, Bayles DO, Alt DP, Stedtfeld RD, Sul WJ, Stedtfeld TM, Chai B, Cole JR, Hashsham SA, Tiedje JM, & Stanton TB (2012). In-feed antibiotic effects on the swine intestinal microbiome. Proceedings of the National Academy of Sciences of the United States of America, 109 (5), 1691-6 PMID: 22307632

Scanlan PD (2012). Blastocystis: past pitfalls and future perspectives. Trends in parasitology PMID: 22738855

Stensvold CR, Alfellani MA, Nørskov-Lauritsen S, Prip K, Victory EL, Maddox C, Nielsen HV, & Clark CG (2009). Subtype distribution of Blastocystis isolates from synanthropic and zoo animals and identification of a new subtype. International journal for parasitology, 39 (4), 473-9 PMID: 18755193

Stensvold CR (2012). Thinking Blastocystis out of the box. Trends in parasitology PMID: 22704911

Yan Y, Su S, Ye J, Lai X, Lai R, Liao H, Chen G, Zhang R, Hou Z, & Luo X (2007). Blastocystis sp. subtype 5: a possibly zoonotic genotype. Parasitology research, 101 (6), 1527-32 PMID: 17665214

Dressed Up For The Party But No Invitation...

Here's a bit on the personal side: I'm very thankful to the viewers of this blog! I get quite a lot of encouragement from readers and also good suggestions for future blog posts, which is immensely inspiring and helps me to keep up spiritis at times when things are not going my way... like just now: Funding for Blastocystis research is really, - and I mean REALLY difficult to obtain. Lately, I was microns away from notching a €1.34M grant for research in Blastocystis genomics/transcriptomics/phylogenetics/epidemiology - we had the scene all set with great track record and expertise, potential PhD candidates, superb partners and perfect lab infrastructure; I was one of the few runner-ups, but, alas - no luck! Never been so close to something that big and feeling terribly gutted... Well, next up is the EU (again). Also, we're trying to look into the possibility of world experts joining forces, but to find 100% relevant calls for this is extremely challenging. And if the call is not 100% relevant, applying for funding is a complete waste of time...

The Latest News from the Human Microbiome Project

Video podcast from ASM2012. Plenty more at MicrobeWorld. It even comes with a bit of jazz music!

Microbiome Blog Posts

Here's a couple of interesting blog posts on the microbiome, some of which also expand a bit on the hygiene hypothesis:

10 Ways the Human Microbiome Project Could Change the Future of Science and Medicine
"By treating our microbiomes like ecosystems — equipping it with the resources it needs to sort itself out rather than attacking it, guns blazing — some researchers hope to usher in a new way of thinking about our relationship with bacteria and other microorganisms." - Find it here.

Human microbiota and atherosclerosis
Data adding to the infection hypothesis of atherosclerosis; find it here.

Are Your Gut Bacteria Vegetarian?
Examples of how differences in diets may be associated with differences in gut microbiota. Find it here

Gut Flora, Probiotics and Vitamins A + D - Do they influence Allergy and Autoimmunity?
"For over 30 years data has been building to scientifically support the hypothesis that intestinal cohabitants operate in a collective manner with macro and micro food intakes to shape and define our immune systems from an early age." - A post including an updated version of the hygiene hypothesis and a bit on faecal bacteriotherapy as well... go here

The Healthy Human Microbiome
 - from "NIH Research Matters". Very general, but with some other links too, - read it here.

How Bacteria Break Down Human Food
Read about the carbohydrate metabolising abilities of bacteria living in different anatomical sites of your body here.

Dirtying Up Our Diets
More about the possible explanation for the alarming rise in allergic and autoimmune disorders in NY Times,  go here.

More from NY Times, this time by Carl Zimmer:

Our Microbiomes, Ourselves
Using bacteria as living drugs against obesity, autoimmune diseases and intractable GI infections...  find it here.

And - for the more hardcore fanatics - thanks to Jonathan Eisen (@phylogenomics) who writes the blog "The Tree of Life" - here's a collection of many of the recent papers and news stories concerning the human microbiome project (HMP).

And finally - to top it off:

Microbiome analysis helps understand cause of chronic sinus condition, suggests cure
They found that patients with chronic rhinosinusitis (chronic inflammation of the paranasal sinuses) had a depleted nasal microbiome, characterised by a significant reduction in bacterial diversity and an overgrowth of one type of bacteria, Corynebacterium spp. + Lactobacillus depletion. Presented at ASM2012. Read it here.

And for those who think that I have been disgressing lately, - I'll be back with more on Blastocystis in my next post - look out!

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

8th European Congress on Tropical Medicine and International Health

The Danish Society for Tropical Medicine and International Health will be organising the European congress in 2013. We hope to see as many as possible. 

 

You may access the congress home page (as it develops) on www.ectmih2013.dk or via the home page of the Federation of European Societies for Tropical Medicine and International Health (www.festmih.eu). 

The congress will be held in Tivoli Congress Centre on 10-13 September, 2013.

Thanks.

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