Cell Symposium: Microbiome & Host Health - Lisbon 2013

My colleagues from Statens Serum Institut and I are heading to Lisbon, Portugal, tomorrow morning to attend the Cell Symposium on Microbiome and Host Health (link may be really busy now).

We are bringing a poster displaying some of our work related to our GUT18S project: A Novel Approach For Eukaryotic Phylogenetic Interrogation Of Clinical Samples Using Next Generation Sequencing Of SSU rRNA Genes; a pdf version of the poster can be downloaded here.

The GUT18S work is partly funded by the Marie Curie Actions (FP7) program.

YouTube Video on Blastocystis Subtyping

For those who want to venture into Blastocystis subtyping - the easy way - I've recorded and uploaded a video on YouTube fyi.




For even more information, please visit a selection of relevant blog posts here.

More on 'Bugs as Drugs'

This morning, I was doing a lazy ramble through my favourite blogs and found a post by Carl Zimmer on 'Bugs as Drugs' - primarily on probiotics. And I just came to realise that there is a very interesting tendency these years of using bugs as drugs in a variety of fields.

We are all very much aware of the worries about the increase in antibiotic resistance in bacterial and other pathogens. Moreover, it appears that sometimes antibiotic treatment leads to imbalance in the intestinal microbiota (dysbiosis); a well-known example is intractable Clostridium difficile infections which can potentially lead to pseudomembranous colitis.

C. difficile infection can lead to pseudomembranous colitis

Earlier this year, an article appeared in the renowned The New England Journal of Medicine on a randomised, controlled treatment study on duodenal infusion of donor faeces for recurrent C. difficile. The researchers found that the infusion of donor faeces was significantly more effective for the treatment of recurrent C. difficile infection than the use of vancomycin, the drug usually recommended in this situation. In fact 15/16 patients had resolution of C. difficile-associated diarrhoea upon first or second infusion; however, it might be worthwhile 'shopping around' for the right donor.

And so, how are these faecal transplants developed and administered? Well, it appears that donors are volunteers who have been through a selection process based on a questionnaire on risk factors of infectious diseases. Then donor faeces is screened for parasites (including Blastocystis and Dientamoeba - yes, it warms my heart to see this so explicitly spelled out in the paper... but I wonder which methods were used - it doesn't say!) and enteropathogenic bacteria. Moreover, blood samples from donors are screened for e.g. HIV, hepatitis and antibodies against e.g. Entamoeba histolytica and Strongyloides. Next, a donor pool is created with repeated screening every 4 months. On the day of infusion, faeces is collected by the donor and immediately brought to the hospital, where it is diluted with 500 mL of sterile saline. The solution is stirred, and the supernatant strained and poured in a sterile bottle. Within 6 h after collection of the faecal sample by the donor, the solution is infused through a nasoduodenal tube (2 to 3 mintues per 50 mL). Patients are subsequently monitored for 2 h. Apparently, this is how it works!

Transcriptomic Analysis of Blastocystis ST1!

BLASTing Breaking News!

Probably to support their genomic data, researchers in Andrew Roger's group in Canada have performed transcriptomic analysis of the Nand II strain, which belongs to Blastocystis sp. ST1.

Running from April 29 to May 2 is the SMBE (Society for Molecular Biology and Evolution) satellite meeting on Eukaryotic-Omics; the abstract booklet can be downloaded here. And fellow tweeps, don't let yourselves down by not following #SMBEeuks!

Watching #SMBEeuks tweets ticking in from @druvus @dr_bik and @ctitusbrown feels like attending the actual conference - without the jet lag!
— Chr. Rune Stensvold (@Eukaryotes) April 29, 2013

Until now, we've only known of one complete Blastocystis nuclear genome, namely that of ST7. Now, the release of the ST1 genome may be imminent! In any case, Roger's group have used their transcriptomic data to compare the protein content in ST1 with that in ST7, and it appears from their conference abstract that "the genes encoded by the Nand II strain (ST1) are surprisingly distantly related to ST7 orthologues, sharing on average ~50% identity at the protein level." And more: "Preliminary analyses allowed us to detect ~1000 genes in ST1 that have no homologue in Blastocystis sp. ST7". This means that the extreme genetic diversity that we see across the SSU ribosomal RNA genes is reflected and may be even more pronounced at nuclear genome level.

The group also studied genes acquired by lateral gene transfer (LGT; see previous post for more on LGT, also known as horizontal gene transfer), and what they basically found was that ST1 appears to have acquired bacterial genes related mainly to metabolism, while genes acquired from eukaryotes code for proteins related to cellular processes and signaling mechanisms.

Finally, they have discovered genes obtained by LGT that has had importance for Blastocystis' adaptation to parasitism; among these genes that enable resistance to host immune responses.

Roger's group is based at the Dept. of Biochemistry and Molecular Biology, Dalhouise University, Halifax, Nova Scotia in Canada.

'Invasive Blastocystis' in ECCMID 2013

ECCMID - the annual European Congress of Clinical Microbiology and Infectious Diseases (hosted by ESCMID) is currently taking place in Berlin. This year, I'm not attending, but I've been scanning the abstract book for 'Blastocystis', and it appears that an oral presentation was scheduled for yesterday in the "Emerging Infectious Diseases" section:

First of all: it's great to see fellow researchers screening larger (i.e. hundreds) of faecal DNAs by PCR for Blastocystis. I wish more people would do that to produce reliable data on prevalence and subtypes.

Now, as I've already mentioned, there are currently mainly two methods in use for subtyping, barcoding and STS PCR, and recently I evaluated these. To cut a long story short, barcoding is recommended for subtyping, since the STS method, which was used in the study by Tarasova et al. (abstract), appears to miss the majority of ST4 strains (the major genotype), and moreover, no STS primers exist for ST8 and ST9 (or any of the other 8 subtypes identified to date, but which have only been found in animals). So, the subtype data found in this study should be interpreted with this in mind.

Importantly however, I'm not sure whether the authors used the original Yoshikawa STS terminology or the terminology acknowledged in our 2007 consensus.

First, let us assume that consensus terminology is used. Then it's surprising to find ST5 in human samples in the first place, and finding a ST5 prevalence of 45% in a cohort of humans included in a larger study like this is very unlikely based on current evidence of more than 3,000 observations from all over the world, where the overall prevalence of ST5 in humans is <1%. Also, finding so much ST6 is also really striking. Also, if the consensus terminology is used, then I'm a bit puzzled why the authors put emphasis on ST7 not being found, since ST7 is relatively rare in humans.

And so let us assume that consensus terminology was not used, and the original Yoshikawa terminology was used instead. This would translate into STs 4, 6, and 7 not being detected in the CVH group. Which makes sense, since ST6 is extremely rare (at least in Europe), ST7 is only seen on occasion, and, as I said, the majority of ST4 infections are likely to go undetected by the STS method. However, ST4 appears quite common in Europe, and I suspect that it should be quite common in St Petersburg as well. But then there is one thing that comes to my mind: If ST4 infections are common, then there should be a relatively large number of samples detected by PCR which were untypable by PCR...and there is no information on untypable positive samples in the abstract...
But what is more:  STS subtype 5 translates into ST2 in consensus terminology, and similarly STS subtype 6 equals ST5 (yes, it may seem confusing, but we have provided a table in the 2007 consensus paper to make this easy). This means that no matter which of the two terminologies were used, ST5 is seen in abundance in patients with CVH in St Petersburg! Which is a very remarkable observation, and maybe more interesting than the rest of the data, which  I, by the way, find a bit difficult to follow (I expected to learn something about Blastocystis invasion, when I read the title of the abstract, but there is no data or information on invasiveness... and I'm very curious as to how the authors managed to obtain such a high number of samples from 'healthy people'! To evaluate the prevalence of Blastocystis in the control group, demographic data are needed, and a prevalence as low as 5.3% among healthy individuals makes me suspect that this control group consisted of newborns/toddlers who generally have a low prevalence of Blastocystis). Also, since when was ST1 'zoonotic'?

Anyway, often conference abstract are previews of upcoming articles, and so I expect that there will be a paper out soon from this group, and hopefully these issues will be clarified. The occasional confusion in Blastocystis epidemiology could be reduced to a minimum if everyone got into using barcoding and the Blastocystis 18S subtyping site (and go here for a video introduction to Blastocystis subtyping).

Are some citizens of St Petersburg infected by Blastocystis sp. ST5, a subtype seen primarily in livestock and African apes? Source

References:

Tarasova E, Suvorova M, Sigidaev A, Suvorov A. Blastocystis invasion in patients with chronic viral hepatitis in Saint Petersburg. ECCMID 2013 abstract O338.

Alfellani MA, Stensvold CR, Vidal-Lapiedra A, Onuoha ES, Fagbenro-Beyioku AF, & Clark CG (2013). Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Tropica, 126 (1), 11-8 PMID: 23290980

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 CR, Suresh GK, Tan KS, Thompson RC, Traub RJ, Viscogliosi E, Yoshikawa H, & Clark CG (2007). Terminology for Blastocystis subtypes--a consensus. Trends in Parasitology, 23 (3), 93-6 PMID: 17241816

This Month in Blastocystis Research (APR 2013)

I've been extremely bored all day writing up my evaluation of a (not so interesting) PhD thesis, and I thought I'd spice up my day by introducing a new series of posts on this blog inspired by so many other blogs, namely: This Month in Blastocystis Research! A place for me to go through some of the most recent papers on Blastocystis.

There is paper out by Gould and Boorom who look at the stability of Blastocystis surface antigen over time. They show that detection of Blastocystis by an immunofluorescense assay (IFA) is not hampered after1 year of storage of faecal material in formalin compared to results immediately after the sampling point. Detection of Blastocystis by IFA is something that is not often used (that's my impression, anyway), but makes sense in cases where laboratory analyses can be performed only weeks-months after sample collection (e.g. during field work), in which case samples need to be preserved. We usually, however, recommend storing faecal material in (70%) ethanol (in the relationship 1 part faecal sample to 4 parts of ethanol), where the sample is mixed with the ethanol initially by vortexing the tube (typically a 2 mL Eppendorf tube) for 5-10 min, and subsequently keeping the tubes away from light until further processing. Importantly, in contrast to formalin-fixed stool, ethanol-fixed stool can be made highly suitable for PCR by just washing the samples x3 in PBS prior to DNA extraction. An example of this methodology can be seen in our study of Blastocystis in members of the Tapirapé tribe in Mato Grosso, Brazil (go here for a free download).

I'd wish that Gould and Boorom had validated their findings by running a PCR on the samples too (specificity and sensitivity testing). The IFA assay was also used in a publication from 2010 by Dogruman-Al et al., who found a diagnostic sensitivity of the IFA assay of 86.7% compared to culture; also here, adding PCR would have been relevant to better determine the diagnostic qualities of the IFA assay.

I was lucky to be involved in field work in the Lao PDR in 2003 conducted by regional WHO authorities; preserving and analysing faecal samples for parasites by microscopy (Kato Katz) and - later - PCR was what we did!

Adding to the endless row of cross-sectional prevalence papers, there is an article out just now by Abdulsalam et al. (2013) on Prevalence, predictors and clinical significance of Blastocystis sp. in Sebha, Libya (free for download here). The study used culture (Jones' medium) as diagnostic modality and confirmed the existence of frequent asymptomatic carriage. The authors used questionnaire info and multivariate statistical analysis to identify risk factors for Blastocystis carriage among 380 individuals aged 1-75, and what I find really interesting is that they found that participants aged > 18 years were much more prone to having Blastocystis than participants < 18 years (P < 0.001). This is something that we see in Denmark too, and I'm currently trying to collect "sufficient proof"! Whether this is an age accumulation effect due to the chronicity of colonisation remains to be investigated. The authors also found that carriers were more likely to experience symptoms than those who were not carriers (P < 0.001), mainly abdominal pain (P < 0.001), but notably not diarrhoea (P = 0.117).
It's a pity that molecular data was not included the study from Libya. Incidentally, our group recently published subtype data from Sebha, Libya, and it appears that Blastocystis found in humans in Libya mainly belongs to ST1, whereas ST3 is often the most common subtype in most other countries, and what is more: ST4 appears virtually absent in Libya and the rest of Africa... But let's see: The investigators might have more data up their sleeve waiting to be published...

May I also again draw your attention to our recent paper on Blastocystis in non-human primates, in which we find that despite the fact that there is a great overlap of subtypes in human and non-human primates, it appears that ST1 and ST3 strains found in non-human primates differ genetically from those found in humans, indicating cryptic host specificity. We have additional data supporting the theory that Blastocystis in humans is a result of human-to-human transmission (anthroponotic) rather than animal-to-human (zoonotic) transmission. Which is really interesting, since the theory of zoonotic transmission of Blastocystis has been heavily (I dare not say purported, so I'll say) propagated. Having said that, I think we still need to look much deeper into barcoding of Blastocystis from pets and other synanthropic animals before we can make more poignant conclusions.

And, finally, yet another add for our recent review on Recent Development in Blastocystis Research!

Please note that I'm happy to take suggestions for future posts, and I'd also like to encourage guest blogging!

Suggested reading:

Abdulsalam AM, Ithoi I, Al-Mekhlafi HM, Khan AH, Ahmed A, Surin J, & Mak JW (2013). Prevalence, predictors and clinical significance of Blastocystis sp. in Sebha, Libya. Parasites & Vectors, 6 PMID: 23566585

Alfellani MA, Jacob AS, Perea NO, Krecek RC, Taner-Mulla D, Verweij JJ, Levecke B, Tannich E, Clark CG, & Stensvold CR (2013). Diversity and distribution of Blastocystis sp. subtypes in non-human primates. Parasitology, 1-6 PMID: 23561720

Alfellani MA, Stensvold CR, Vidal-Lapiedra A, Onuoha ES, Fagbenro-Beyioku AF, & Clark CG (2013). Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Tropica, 126 (1), 11-8 PMID: 23290980

Clark CG, van der Giezen M, Alfellani MA, & Stensvold CR (2013). Recent developments in Blastocystis research. Advances in Parasitology, 82, 1-32 PMID: 23548084

Dogruman-Al F, Simsek Z, Boorom K, Ekici E, Sahin M, Tuncer C, Kustimur S, & Altinbas A (2010). Comparison of methods for detection of Blastocystis infection in routinely submitted stool samples, and also in IBS/IBD Patients in Ankara, Turkey. PloS One, 5 (11) PMID: 21124983 

Gould R, & Boorom K (2013). Blastocystis surface antigen is stable in chemically preserved stool samples for at least 1 year. Parasitology research PMID: 23609598

Malheiros AF, Stensvold CR, Clark CG, Braga GB, & Shaw JJ (2011). Short report: Molecular characterization of Blastocystis obtained from members of the indigenous Tapirapé ethnic group from the Brazilian Amazon region, Brazil. The American Journal of Tropical Medicine and Hygiene, 85 (6), 1050-3 PMID: 22144442

Recent Developments in Blastocystis Research

I would like to draw your attention to an open access link to our 2013 review on Recent Developments in Blastocystis Research; please go here to read/download the paper.

Here's the abstract:

Blastocystis is a common parasite of the human large intestine but has an uncertain role in disease. In this review, we appraise the published evidence addressing this and its weaknesses. Genetic diversity studies have led to the identification of numerous subtypes (STs) within the genus Blastocystis and, recently, methods for studying variation within STs have been developed, with implications for our understanding of host specificity. The geographic distribution of STs is summarised and the impact this may have on investigations into the role of the organism in disease is discussed. Finally, we describe the organelle and nuclear genome characteristics and look to future developments in the field.

Blastocystis Hits The 1,000 Entry Mark In PubMed

Yesterday, the number of Blastocystis entries in PubMed reached 1,000! PubMed is a public resource comprising more than 22 million citations for biomedical literature from MEDLINE, life science journals, and online books.

In comparison, there are currently 7,641 entries on Entamoeba, 6,630 on Cryptosporidium and 235 entries on Dientamoeba.

I plan to introduce the "Hall of Fame in Blastocystis Research" in a future post, but already now I can reveal that the researcher with most Blastocystis-related publications is Dr Hisao Yoshikawa according to Web of Science (WoS), which currently returns 895 hits on a search on Blastocystis; Dr Yoshikawa has at least 43 publications on Blastocystis alone (WoS), and at least 37 Blastocystis-specific peer-reviewed journal articles (PubMed) since 1987.

Blastocystis in Non-Human Primates

If my recent blog post "Blastocystis aux Enfers" could be described as "Blastocystis meets Dante Alighieri", then this post might come across as "Blastocystis meets Sir David Attenborough" (with all due respect to both of these gentlemen!).

Non-human primates (NHPs) include apes (hominoids), Old World monkeys (cercopithecoids), New World Monkeys (ceboids) and prosimians such as lemurs. I have been so fortunate to be involved in a study of Blastocystis in NHPs; a study which was led by Dr Alfellani with several co-investigators, and which has just appeared online in the journal Parasitology (click here to be diverted to the the website - first view article section).

The study is the first of its kind aiming to provide a substantial insight into the host specificity of Blastocystis in NHPs and included subtype observations for 441 captive and free-living animals representing no less than 30 genera; most of the data were generated during the study, while sporadic observations from similar studies were also included.

It was a huge study with a lot of interesting information, and I will try and summarise some of the points here.

Apes such as bonobos, chimps, gorillas and orangutans were colonised by some of the most common subtypes in humans, namely ST1, ST2, and ST3, accounting for about 77% of the cases. Contrary to humans though, ST5 also appeared rather common, accounting for about almost 14% of the cases, and some of the gibbons studied had ST8. Interestingly, a chimp and a gibbon were found to be hosts of a new subtype, ST15.

Old World monkeys were studied to an even larger extent, and again, ST1, ST3 and ST2 predominated, accounting for about 95% of all cases of single subtype infection. Here ST5 was also seen (2%) but only in langurs/lutungs and vervet monkeys. Interestingly, ST8 was seen only in 1/226 cases. ST13 was found by colleagues in Tanzanian colobus monkeys (Petrasova et al., 2011), and 8% of the 226 cases represented mixed/unknown subtype infections.

Woolly monkey (Lagothrix lagotricha) (Source)

New World monkeys included in the study were mainly represented by woolly monkeys, and these were colonised first and foremost by ST8 (49%), but ST3, ST2, ST1 were also found. So was a single case of ST4, which in general appears to be surprisingly rare among NHPs.

A few observations on lemurs were included, and such animals appear to host a vast variety of subtypes with no particular predilection, hence ST1, ST2, ST4, ST8, ST10 and ST15.

Ring-tailed lemur (Lemur catta) (Source).

The most striking differences between humans and NHPs in terms of colonisation by Blastocystis subtypes is that humans are very rarely colonised by ST5, while this subtype appears common in apes and Old World monkeys. ST8 was seen only in arboreal apes and in woolly and howler monkeys, which are also tree-dwellers, and it is tempting to think that ST8 is found mainly in tree-dwelling NHPs; to my knowledge, ST8 has not been found in non-primate hosts, except for once in a bird. Human colonisation by ST8 has been demonstrated only very rarely, for instance in a Danish woman returning from holiday in Indonesia and in animal keepers. Conversely, ST4 is seen extremely rarely in NHPs, while very common in humans in some parts of the world, apparently especially in Europe. These clear discrepancies in subtype distribution in humans and NHPs may boil down to host specificity and/or apparent geographically restricted range of some subtypes.

Another striking observation was that cryptic host specificity exists in ST1 and ST3, meaning that ST1 and ST3 strains found in NHPs overall differ genetically from strains found in humans belonging to the same subtypes, adding support to our previous findings.This suggests that humans are generally colonised by other strains than those found in NHPs. It will be interesting to see, whether other types of hosts sharing these subtypes carry distinct, host-specific strains. While MLST is probably the best way of testing for this, a lot of information can be obtained simply by barcoding. Pets, for instance, may share subtypes seen in humans, and so barcoding of "pet blasto" may be one of the very interesting pathways to knowledge.

We found no evidence of those subtypes that we have nicknamed "avian subtypes", namely ST6 and ST7. In some parts of the world, these two subtypes do not appear uncommon in humans; in Denmark and Sweden, for instance, ST7 is seen on quite a few occasions. But, interestingly, both STs are apparently absent in NHPs.

Langurs - the front cover of one of my favourite books showcasing works by the magnificent Walton Ford.

Incidentally, there is a sequence in GenBank from a gorilla (JX159284) which possibly represents a novel subtype, which is related to reptilian Blastocystis, and so it appears that the host spectrum and diversity of Blastocystis in NHPs continues to unfold.

A recent study saw that faecal microbiomes of wild non-human primates co-vary with host species, hence reflecting host phylogeny. This was evidenced by higher intra-species similarity among wild primate species, which may reflect species specificity of the microbiome in addition to dietary influences. This may in part explain the differences in Blastocystis subtypes seen in different NHP host species, but it is also possible that differences in subtypes reflect differences in habitat (and thereby possibly exposure) or geographical differences in subtype distribution. Indeed Homo sapiens is host to a variety of subtypes, and while ST4 is common in Europe, it appears virtually absent in many other parts of the world. Likewise, the differences in the prevalence of ST8 may reflect differences in geographical distribution, habitat and diet (arboreal vs. ground) as well differences in host specificity.

The overall interesting thing here is the schism of exposure vs. host specificity.


Suggested reading:

Alfellani, M., Jacob, A., Perea, N., Krecek, R., Taner-Mulla, D., Verweij, J., Levecke, B., Tannich, E., Clark, C., & Stensvold, C. (2013). Diversity and distribution of Blastocystis sp. subtypes in non-human primates Parasitology, 1-6 DOI: 10.1017/S0031182013000255

Yildirim S, Yeoman CJ, Sipos M, Torralba M, Wilson BA, Goldberg TL, Stumpf RM, Leigh SR, White BA, & Nelson KE (2010). Characterization of the fecal microbiome from non-human wild primates reveals species specific microbial communities. PloS one, 5 (11) PMID: 21103066

Stensvold CR, Arendrup MC, Nielsen HV, Bada A, & Thorsen S (2008). Symptomatic infection with Blastocystis sp. subtype 8 successfully treated with trimethoprim-sulfamethoxazole. Annals of tropical medicine and parasitology, 102 (3), 271-4 PMID: 18348782

Stensvold CR, Alfellani M, & Clark CG (2012). Levels of genetic diversity vary dramatically between Blastocystis subtypes. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 12 (2), 263-73 PMID: 22116021

Technical issues...

I'm experiencing a problem with my post titles being displayed as uncials (sort of nested in each post with blog text wrapped around it), when using Firefox or Chrome (also the headline link function has been disabled). I'm trying to fix it, but for now, I recommend viewing the blog in Internet Explorer rather than eg. Firefox or Chrome. If anyone knows how to fix this, please give me a shout!

Have a great weekend!