Blastocystis and IBD

We recently published what could be seen as a pilot study on inflammatory bowel disease (IBD) and the two most common intestinal parasites, Blastocystis and Dientamoeba fragilis.

The aim of the study was to identify possible differences in the prevalence of infection with Blastocystis and D. fragilis in patients with active and inactive IBD compared to controls.

We included 100 Danish patients with IBD (42 with Crohn's Disease, 41 with ulcerative colitis and 17 with ileal pouch-anal anastomosis) and 96 controls, used state-of-the-art diagnostics for Blastocystis and D. fragilis (PCR) and we saw striking differences in prevalence. While 19% of all healthy individuals had Blastocystis, only 5% of those with IBD had Blastocystis, and of the 42 patients with Crohn's Disease, only 1 had Blastocystis. In contrast, D. fragilis was not more common in healthy individuals than in IBD patients. Also, in patients with ulcerative colitis, Blastocystis was significantly more common in patients with inactive disease compared to patients with active disease.

Absence of Blastocystis in patients with Crohn's Disease and active ulcerative colitis may be due to unfavourable conditions for colonisation and should be explored further in order to investigate whether these potentially unfavourable conditions reflect differences in the composition of the microbiota in these patients, and/or whether this has something to do with host immunity. We are currently confirming the virtual absence of Blastocystis in Crohn's patients in another study based on metagenomic analysis of faecal DNA, and it will be very interesting to analyse the differences in Blastocystis prevalence in view of potential differences in bacterial communities.

The literature on Blastocystis and IBD is relatively limited, and I plan to return, maybe later this year, with a more elaborate post on the topic.

Reference:

Petersen AM, Stensvold CR, Mirsepasi H, Engberg J, Friis-Møller A, Porsbo LJ, Hammerum AM, Nordgaard-Lassen I, Nielsen HV, & Krogfelt KA (2013). Active ulcerative colitis associated with low prevalence of Blastocystis and Dientamoeba fragilis infection. Scandinavian journal of gastroenterology PMID: 23528075

Happy Birthday!

In these very hours, my daughter is turning two years old! This blog was put up one year ago as she was celebrating the conclusion of her first year on this planet, and given all the fun I've had along the way putting up posts on this and that, I'd like to dedicate the blog to her. In return(!), I take the liberty of using some of her artwork for this post which marks the birthday of the Blastocystis Parasite Blog.

Artwork by Raiya Rochelle Traub

It's surprising to me that this blog has had more than 50,000 views in only one year. Due to all the feedback I get, I'm prone to believe that most of the page views reflect factual "blog consumption" (rather than referral spam and bots). Anyway, even if there were only a few people out there who'd stop by every now and then, my efforts would certainly be worthwhile. 
Blastocystis has been known for more than 100 years. But it is only recently that we have found tools to enable accurate distinction of Blastocystis carriers from non-carriers, thanks to DNA-based diagnostic methods. Last year, we published a paper on our new real-time PCR in Journal of Clinical Microbiology, and it seems as if we now finally have the chance to try and use it for screening a larger panel of faecal DNAs from patients with and without intestinal symptoms to get an idea about the factual prevalence of Blastocystis in this type of samples with the added benefit of analysis of colonisation intensity. It's very exciting...

And to those who are involved in Blastocystis subtyping, -  in case you didn't see it, there is a paper out on the comparison of the two principal methods used for subtyping which you might find useful.
I've also added a few lines on barcoding in "Lab Stuff" for those who are new to subtyping and want to practice a bit - please go here.

We are currently trying to strengthen collaborative efforts of different labs across the world and we are facing some very exciting challenges, involving the generation and analysis of data output related to genomics, transcriptomics, metagenomics and possibly proteomics; more about that in "Season II" of the Blastocystis Parasite Blog!

But for now: Happy birthday, Raiya! And Happy Easter everyone!

Suggested reading:

Stensvold CR (2013). Comparison of sequencing (barcode region) and sequence-tagged-site PCR for Blastocystis subtyping. Journal of Clinical Microbiology, 51 (1), 190-4. PMID: 23115257 

Stensvold, C., Ahmed, U., Andersen, L., & Nielsen, H. (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. DOI: 10.1128/JCM.00007-12

LUMINEX xMAP Technology in Parasite Diagnostics

Over the past few years nucleic acid based methods have revolutionised parasite diagnostics in modern clinical microbiology (CM) labs. Real-time PCR is really gaining a foothold in CM labs, but despite the opportunity for plexing, mostly only up to 6 DNA targets can be included in each assay (due to the number of available channels).

LUMINEX xMAP technology used for detection of specific nucleic acids (Dunbar, 2006) bypasses this limit, and up to 100 DNA targets can be included in one single assay in a 96-well plate format. You can read about the technology here.

Immunology in the Gut Mucosa

Nice video!

Extremophilic Eukaryotes

My recent post Blastocystis aux Enfers was my "literary take" on biological adaptation of intestinal parasitic protists, using Blastocystis as an example. As a parasitologist you'd come across many peculiar and shrewd biological adaptations and life cycles, and I hope to be able to give some examples in a future post. Actually, there is a parasite which is quite common in humans, maybe even just as common as Blastocystis, which is also single-celled, but which may have a much more complicated life cycle than Blastocystis, namely Dientamoeba fragilis; a colleague of mine is currently doing his PhD on Dientamoeba and he has collected multiple sources of evidence to confirm the hypothesis that this parasite is transmitted by a vector, namely pinworm, probably along the same way that Histomonas meleagridis – the cause of blackhead disease in especially turkeys – is transmitted by heterakids (which again are transmitted by parathenic hosts such as earthworms, which get eaten by turkeys, chickens, etc.). Anyway, I’ll probably get back to Dientamoeba, once his data are out.

Meanwhile, Blastocystis comes out of a very heterogeneous group of organisms called Stramenopiles, many of which are algae. Algae are photosynthetic organisms found in habitats as diverse as glacial ice and hot springs.One of these algae is named Galdieria sulphuraria, which is a remarkable unicellular eukaryote inhabiting hostile environments such as volcanic hot sulfur springs where it is responsible for about 90% of the biomass; indeed this certainly qualifies as "Galdieria aux enfers"!

Blastocystis video

Just saw this on YouTube and had to share it. This is Blastocystis (and other microorganisms) viewed through a microscopy (light microscopy). Note that this is Blastocystis from a chicken, but Blastocystis from humans looks the same; at least I don't know how to tell the difference. I wonder whether this is from a culture or a completely fresh egestion... looks more like a culture to me. Note how the Blastocystis looks almost like fat cells...

The video comes with some nice music as well!

Open Access papers in Nature Reviews on functional dyspepsia

"Functional dyspepsia is one of the most common functional gastrointestinal disorders worldwide. Although the condition does not affect life expectancy, it can have a marked influence on quality of life, and is associated with a high economic burden; an estimated US$1 billion per year is spent on the management of functional dyspepsia in the USA alone. This comprehensive Focus issue from Nature Reviews Gastroenterology & Hepatology contains seven Reviews that have been specially commissioned to cover key themes in functional dyspepsia. Experts from around the world provide up-to-date overviews of the most important topics in the field, including the influence of dietary, lifestyle and psychosocial factors, relevance of Helicobacter pylori infection, overlap with GERD, changes in gastrointestinal tract structure and function, symptom pattern and validity of the Rome III criteria, as well as current and emerging treatment options."

For the bunch of papers, please go here.

Blastocystis aux Enfers

We tremble at the thought of being devoured by a ferocious animal, - of ending our days in a narrow, suffocating slimy tube covered in acidic, nauseating glaze! Remarkably, for some eukaryotic beings, this is the only way forward if they want to carry on with their lives! Intestinal protists such as Blastocystis are in a state of hibernation when outside our bodies and the only thing that may rouse these Sleeping Beauties to action is the passage through low pH enzyme ponds. They thrive, grow and raise their progeny only in the swampy Tartarus of our large intestines; they bequeath to their offspring the affinity for this gloomy, filthy slew; this murky, densely populated, polluted channel, and when the pool of poo becomes all too arid, they know it’s time to buckle up, shut down, and prepare themselves for the great unknown which can potentially mean death to them if eventually they are not lucky enough to be gulped down by another suitable host.

Source

And yet, despite their remarkable modesty and humble requirements these little buggers are being bullied by their inhospitable human hosts; we’d throw anything at them to force them out, organic and inorganic compounds meant to arrest or even kill them. But the whelps of Blastocystis appear extremely resilient, which may hold the key to part of their success; they stay afloat on the Styx of our bowels. In order to eschew Flagyl, perhaps they bribed Phlegyas?

I think it's sometimes useful to put things into a completely different perspective. In any event, from an evolutionary biology standpoint it is highly interesting that a genus which is genetically related to water molds such as those causing potato blight and sudden oak death, has so successfully adapted to a parasitic, anaerobic life style, capable of protractedly colonising a plethora of very diverse host species including members of primates, other mammals, birds, reptiles, amphibians and arthropods and thereby evading innate and adaptive immune defenses from such a diverse range of hosts. One could be inclined to say: Well done! But which is it? Parasitism? Commensalism? Mutalism? Symbiosis? And what will happen to Blastocystis in the future? Will this successful crusader eventually succumb to our avid but maybe imprudent war strategies? And if so, what will happen to us after removing such a common player from our intestinal ecosystems?

Bubbly Blasto!

Yesterday, I was checking up on a fresh Blastocystis culture. I loaded 20 µL of the culture "sediment" on to a glass slide, placed the cover slip on top and examined it by light microscopy. While examining the slide, I observed a multitude of dividing cells, indicating vigorous growth and a thriving strain, and once again I was struck by the appearance of dividing Blastocystis. This is basically what they may look like:

Like soap bubbles really, only a lot smaller obviously (mikrons), and somewhat opaque! You'll see them in different sizes and the way they divide looks just like this. Apparently some sort of random budding or multiple fission. You'll see little more than this bubbly structure, which means that there are very few morphological hallmarks to describe. A few nuclei may be discernible along the cytoplasmatic rim, but that's about it when you use light microscopy. Ultrastructural and biochemical analysis is required if you hope to be able to describe some of the processes involved in reproduction.

We often say that Blastocystis organisms representing different subtypes are morphologically indistinguishable; what this actually means is that we do not have the tools to differentiate them morphologically. There may actually be great variation between strains in terms of for instance how they grow in vivo and in vitro and maybe also how they reproduce. Vacuolar forms are the most common form seen in xenic cultures, but other morphotypes are sometimes observed, for instance the granular stage, which, in my experience, is typically seen in cultures that are not “well looked after”, i.e. where medium is not being replaced about twice a week. Dunn and colleagues. (1989) observed that the granular stage could arise from vacuolar stages in cultures where the concentration of horse serum was increased.

I have previously stated that there is no evidence for phagocytosis in Blastocystis. Actually, Dunn et al. (1989) captured what they thought to be bacterial engulfment by ultra-structural analysis, and they also observed bacteria-engulfing pseudopodia in amoeboid stages, in which degraded bacteria were observed. I don't think that I've ever come across this amoeboid stage, but it has been described by quite a few researchers.

Anyway, let's hope for another kind of bubbles this Friday night!

Suggested reading:

Dunn LA, Boreham PF, & Stenzel DJ (1989). Ultrastructural variation of Blastocystis hominis stocks in culture. International Journal for Parasitology, 19 (1), 43-56 PMID: 2707962

Waiting For The Human Intestinal Eukaryotome

We were lucky enough to have a paper accepted for publication in the ISME Journal (Nature Publishing Group) in which we call for data on the "human intestinal eukaryotome".

In the paper, we start out:

"Recent developments in Next Generation Sequencing (NGS) technologies have allowed culture-independent and deep molecular analysis of the microbial diversity in faecal samples, and have provided new insights into the bacterial composition of the distal gut microbiota. Studies of the microbiome in different patient groups using metagenomics or 16S rRNA gene sequencing are increasing our knowledge of how the microbiota influences health and disease. The majority of recent advances in our understanding of human microbiota structure and dynamic changes in disease were made through phylogenetic interrogation of small subunit (SSU) rRNA (Paliy and Agans 2012). However, until recently such studies have generally failed to include data on common eukaryotic, endobiotic organisms such as single-celled parasites and yeasts ('micro-eukaryotes'). This deficiency may strongly bias the interpretation of results and ignoring an entire kingdom of organisms is a major limitation of human microbiome studies."