This Month In Blastocystis Research (SEP 2013)

This month has been extremely busy, and I've been preoccupied with networking, meetings and conferences.

I want to thank the arrangers of the Scandinavian-Baltic Society for Parasitology (SBSP) meeting (which was this year merged with The 8th European Congress of Tropical Medicine & Tropical Medicine)  for arranging a conference session on 'Intestinal Protists - Diagnostic Tools and Emerging Trends', which I was very honoured to chair. One of the four talks was unfortunately cancelled, but the rest of the talks were centred on Blastocystis, and due to great speakers and a very engaged and experienced audience, it turned out to be an extremely interesting and awarding session, sporting topics such as in-vitro susceptibility testing, subtype distribution in different cohorts, diagnostics, and 'Blastomics' advances. The conference took place in the Tivoli Congress Center in Copenhagen. Excellent facilities, but apparently catering other than tap water, tea + coffee could not be accommodated in the congress budget; but then again, it was a big meeting with more than 1,000 people registered (unconfirmed). Anyway, I look forward to more meetings and symposia focusing on Blastocystis! For more on this, stay tuned!

PubMed-wise there is not really much to include in the 'This Month in Blastocystis Research' series though, and I think I'll skip it this time. However, for those with an insatiable appetite for scientific papers on Blastocystis, I have very good news: The London School of Hygiene & Tropical Medicine Online Research Library, which can be accessed here, apparently offers free download of accepted papers (i.e. the original files accepted by the various journals and not the printed versions). A search on Blastocystis renders about 25 publications (not of all of which are specifically on Blastocystis, though), so there should be plenty to read...

Thanks to our excellent librarian here at the SSI, my entertainment for the weekend will be 'Studies on Human Intestinal Protozoa' published in Acta Medica Scandinavica (Supplementum LXX) in 1935 by Ruth Svensson - her doctoral thesis dedicated to Dr Clifford Dobell...

Fellowships in Blastocystis Research

We are continually looking into the opportunity for funding for research in Blastocystis and we are on the lookout for young researchers with a MSc or PhD degree who want to spend at least a couple of years in Blastocystis research. Right now, taking an omics approach to studying the clinical significance of Blastocystis is extremely relevant of course, given the amount of genetic diversity of the parasite, its apparent association to groups of bacteria/bacterial richness, its varied distribution across different cohorts, and the general availability of ngs technology and data pipelines.

I'm going to focus my next funding application on the integration of metagenomics, metabolomics, comparative genomics, and transcriptomics, and I'm hoping to find one or two persons with track records documenting extensive experience with one or more of these disciplines and who take an interest in parasites/parasitic protists in general and/or in Blastocystis in particular.

Please note that this is NOT a job offer, but merely an invitation to get into some sort of a dialogue. What we can offer is access to samples, strains, technology, and a Blastocystis-centred network.

Please do not answer in the comments section, but contact me directly (mail/phone) for further info + expression of interest. You'll find a link to my contact details in the previous blog post. Thanks.

Yes, we do take orders!

I get an increasing amount of requests for Blastocystis testing (and testing for other parasites as well, for instance Dientamoeba fragilis). Initially, I was happy to do this for free, but now the requests are so regular that I need to add a fee to the tests.

And yes, we do take orders! As the regular reader of this blog would know by now, I run the part of our  Parasitology lab at Statens Serum Institut, Copenhagen, that deals with Blastocystis diagnostics and diagnostics for intestinal parasites in general. I have been developing and optimising molecular Blastocystis diagnostics for years, something which is also witnessed by my scientific production. Please note that we take orders only from health authorities. This means that if you want to have samples tested in our lab, you should contact your GP/specialist/whatever, and have him/her put the order through.

For general screening, I recommend real-time PCR analysis. For evaluation of treatment I recommend adding Blastocystis culture (a positive culture means ongoing Blastocystis infection, while DNA-based tools such as our real-time PCR will detect both dead and live organisms). We also perform subtyping of Blastocystis upon request.

In cases where colleagues want to outsource diagnostic work related to research, we are currently opening up for the possibility of testing large panels of faecal samples (fresh, frozen, or ethanol-preserved) for Blastocystis, Dientamoeba fragilis or other parasites by molecular assays (including DNA extraction) - and - if requested - in combination with traditional microscopy of faecal concentrates.

A selection of our analyses for parasites can be viewed here.

Our parasitology lab is merged with the mycology lab, and therefore we have plenty of opportunity to test the same stool sample for parasites and yeasts (e.g. Candida), if requested. As a new feature, Blastocystis+Dientamoeba+Candida analyses can now be requested in combination as a 'package' with a discount. We are happy to send out test tubes and transport envelopes, but I repeat that charges will apply.

Research-wise, we are currently taking different approaches to detecting and differentiating non-human eukaryotic DNA/RNA in human faecal samples, among these the GUT 18S approach.

For further inquiries and information, please do not hesitate to contact me (contact details can be found here).

Relevant articles on molecular diagnostics for Blastocystis detection and subtyping:

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

Final TM&IH Copenhagen Conference Programme Now Available.

The final programme for 8th European Congress on Tropical Medicine & International Health and the 5th Conference of the Scandinavian-Baltic Society for Parasitology (merged venue) has now appeared and can be downloaded here. Abstract book (including poster abstracts) not yet available, but there will be a few that include Blastocystis. For those attending the conference and interested in Blastocystis, I recommend session 6.3.3. (11 SEP 2013).

Read more here. 

This Month In Blastocystis Research (AUG 2013)

Quite a few papers relevant to Blastocystis research have made it to PubMed over the past month! Therefore, the August version of 'This Month in Blastocystis Research' is more like a list of papers + short descriptions/comments, rather than one or two actual paper reviews.

Dr Aldert Bart and his Dutch colleagues have published a study that confirms data emerging from other parts of Europe. Using microscopy (fixed faecal smears) and PCR, they found an almost 40% prevalence of Blastocystis in returning travelers with symptoms, and a prevalence of 18% in patients referred for other reasons. The distribution of subtypes found in the study population was quite similar to what has been found elsewhere in Europe with ST3 predominating (42%) and the rest of the subtypes attributable to ST1 (22%), ST2 (22%), ST4 (12%), ST6 (1%) and ST7 (1%).

The Tropical Parasitology theme issue on Blastocystis has now gone live. You’ll find a link to the editorial and the three papers included in the symposium here.

In my previous post I referred to a new study from Colombia which includes subtyping of Blastocystis isolates from humans, and a variety of animals, including birds. The paper is interesting for a number of reasons, but first and foremost it confirms the virtual absence of ST4 in humans in S America. Moreover, the study included 70 Blastocystis positive samples from asymptomatic carriers, 40 positive samples from patients with diarrhoea, and 15 positive samples form patients with IBS. Remarkably, all samples from healthy carriers were typed as ST1, those from patients with diarrhoea belonged to ST2, and those from IBS patients to ST3. Such a clear-cut distribution of subtypes across cohorts is unprecedented and of course warrants confirmation and further investigation. In Europe, ST4 is very common in humans, while it appears rare in humans in many other parts of the world. ST4 also appears rare among non-human primates (NHPs), our closest living relatives, and while NPHs and humans otherwise tend to share the same major subtypes (ST1, ST2, and ST3), this suggests that while subtypes 1, 2 and 3 have probably co-evolved with primates, ST4 has only recently entered the primate population with a preference for humans! I have hinted at this many times by now, but I find it extremely interesting which is why I keep repeating it.

There is a paper out by Santos and Rivera from the Philippines comparing microscopy of direct faecal smear with culture and PCR for detection of Blastocystis. They ended up concluding that culture was the best diagnostic modality, but it should be noted that the PCR used in the study targets a 1.8 kbp product (complete SSU rRNA gene!), and much smaller products are usually targeted in diagnostic PCR assays. The Blastocystis real-time PCR developed by me and my colleagues targets a sequence stretch of ~120 bp, securing optimum test sensitivity. The results of the Philippine study should be interpreted with this in mind.

Li et al., have published data on experimental infection of ST1 in Sprague-Dawley rats. Animals belonging to this species appeared susceptible to a ST1 strain isolated from a diarrhoeic patient that had been kept in culture and for which induction of cysts had been performed with a view to infecting the rats. The study confirms that Blastocystis is mainly a parasite of the coecum and colon. The authors found evidence of Blastocystis invasion into the lamina propria in one of the animals, and signs of inflammation in all animals challenged. While it is great to see that experimental models can be sustained and that encystation can be induced in vitro, at least two important factors must be kept in mind to fully comprehend the study: Although cysts were isolated by gradient centrifugation prior to inoculation, it is unlikely that all bacteria have been removed from cyst suspensions; in other words, the cyst preparation is not likely to be 'sterile', and any effect of the potentially accompanying bacterial flora is difficult to determine. Moreover, rats may not be natural hosts of ST1 (very few data available on the topic!), and so, the pathology caused in the rats may be an unlikely finding in humans, who are indeed natural hosts of ST1 and may have developed a high degree of tolerance to this subtype.

Are dogs, wolves, and other canids natural hosts of Blastocystis?

When visiting Australia earlier this month, I had the pleasure of meeting Wenqi Wang and Tawin Inpankaew, both PhD students working at School of Veterinary Science, The University of Queensland Gatton Campus and supervised by Dr Rebecca Traub. One of the foci of this group is to study Blastocystis in animals, for instance in households where animals are kept as pets. Recently, a paper emerged from this group on diversity of Blastocystis subtypes in dogs in different geographical settings, hence domestic/pound dogs from Brisbane, Australia, semi-domesticated dogs from a village in Cambodia, and stray dogs from Mumbai and other Indian cities. Using sensitive PCR methods they observed that almost one fourth of the Indian dogs were infected, while dogs in the Cambodian village and in Queensland remained largely uninfected. Coprophagy and access to Blastocystis-positive stool from different hosts may account for the relatively high prevalence in stray dogs in India, although one might assume that the prevalence would then be even much higher? The team used nested PCR in their study and found four different subtypes in the Indian dogs, including ST1, ST4, ST5 and ST6. Whether all of their data collectively indicate that dogs are not natural hosts of Blastocystis is a matter of debate and remains to be more thoroughly investigated. Indeed, prevalence and subtype data from studies of samples from wild life canids (dingos, jackals, wolves, coyotes, but also foxes and raccoon dogs) would shed further light on this topic.

Finally, for those interested in how Blastocystis deals with oxidative stress and related metabolic issues, there is a paper out on iron-sulphur cluster biogenesis in protozoan parasites by Ali and Nozaki citing works by Tsaousis (2012), Denoeud (2011), Long (2011), and Stechmann (2008).

Literature:

Ali V, & Nozaki T (2013). Iron-sulphur clusters, their biosynthesis, and biological functions in protozoan parasites. Advances in Parasitology, 83, 1-92 PMID: 23876871

Bart A, Wentink-Bonnema EM, Gilis H, Verhaar N, Wassenaar CJ, van Vugt M, Goorhuis A, van Gool T. Diagnosis and subtype analysis of Blastocystis sp. in patients in a hospital setting in the Netherlands. BMC Infectious Diseases, 13:289.

Li J, Deng T, Li X, Cao G, Li X, & Yan Y (2013). A rat model to study Blastocytis subtype 1 infections. Parasitology Research PMID: 23892480 DOI: 10.1007/s00436-013-3536-7

Parija SC (2013). Blastocystis: Status of its pathogenicity. Tropical Parasitology, 3 (1) PMID: 23961433

Parija SC, & Jeremiah S (2013). Blastocystis: Taxonomy, biology and virulence. Tropical Parasitology, 3 (1), 17-25 PMID: 23961437 

Ramírez JD, Sánchez LV, Bautista DC, Corredor AF, Flórez AC, & Stensvold CR (2013). Blastocystis subtypes detected in humans and animals from Colombia. Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases PMID: 23886615

Sekar U, & Shanthi M (2013). Blastocystis: Consensus of treatment and controversies. Tropical Parasitology, 3 (1), 35-9 PMID: 23961439

Stensvold CR (2013). Blastocystis: Genetic diversity and molecular methods for diagnosis and epidemiology. Tropical Parasitology, 3 (1), 26-34 PMID: 23961438  

Wang W, Cuttell L, Bielefeldt-Ohmann H, Inpankaew T, Owen H, & Traub RJ (2013). Diversity of Blastocystis subtypes in dogs in different geographical settings. Parasites & Vectors, 6 PMID: 23883734

Birds of America

Yesterday evening, I was watching another compelling BBC production, broadcast on Danish television: Earthflight, North America. In quite a unique way, the viewers got the rare opportunity to see through the eyes of birds such as eagles, geese, and pelicans and follow birds as they were migrating, escaping, hunting for prey, etc. It made me think of the 19th century masterpiece 'Birds of America' by John James Audubon, which can be viewed in the National History Museum in London. The book features 435 stunning hand-coloured plates that show birds life-size, in natural positions and in their natural habitat.

One of the things that I find interesting - and quite unexplored - is Blastocystis in birds. By 'unexplored' I mean that relatively little sampling has been done, and so the number of observations of Blastocystis in birds is still limited compared to other types of hosts. However, there is a brand new paper out in 'Infection, Genetics and Evolution' which includes observations on Blastocystis in birds (of America!).

You see, I was invited in on a study by colleagues in Colombia who had access to DNA from quite a few faecal samples from a number of host species, including feral birds, and what we found confirms the quite unambiguous trend seen so far: Birds - no matter where on this planet - appear to be colonised mainly by ST6 and ST7. As a matter of fact, in the present study only ST6 was seen in almost 50 Colombian passerine birds of varying species, most of which I believe are limited in geographical distribution to the Americas: Passer domesticus, Thraupis episcopus, Oryzoborus maximiliani, Sicalis flaveola, and Petrochelidon pyrrhonota. Moreover, only one allele of ST6, allele 122, was identified. Notably, the prevalence of Blastocystis in the sampled bird population was 90%. I believe that this is the first official report on Blastocystis in passeriformes. Other major groups of birds previously sampled include galliformes, anseriformes, and ratites (Stensvold et al., 2009; Alfellani et al., 2013).

Other subtypes have been reported in birds (Alfellani et al., 2013), but due to the very low number of samplings these subtypes may be more or less co-incidental/abberant findings. Of note, some samples from birds have been untypable. I have a slight recollection of detecting ST3 in Icelandic rock ptarmigans (in mixed ST infection) collected by Dr Karl Skírnission, but that certainly needs confirmation.

Bird contact/bird droppings - a significant source of Blastocystis in humans? Me feeding some 'Birds of Australia'. Photo by Dr Rebecca J Traub.

ST6 is very rarely seen in humans in Europe. In other parts of the world, for instance in Egypt and some Asian countries, ST6 appears relatively common, but we do not know much about 'bird subtypes' in those particular regions. Also, the situation in the US and Canada is more or less completely unknown (Blastocystis subtyping is something that appears not to attract research groups in North America apart from the one led by Dr Ron Fayer in Beltsville, Maryland).

ST7 is occasionally seen in humans in countries such as Sweden and Denmark. But in my - still limited - experience, individuals infected by these subtypes are not necessarily prone to 'suffer more' from intestinal symptoms than those who do not have these subtypes. While human cases of ST6 (and ST7) may represent cases of zoonotic transmission, it is far to early to draw any conclusions on this. It would be important to compare ST6 and ST7 18S alleles from humans and birds. MLST typing systems for these two subtypes are not yet available, but 18S analysis in itself may prove valuable for molecular epidemiological analyses as in the case of other subtypes (Stensvold et al., 2012).

Walton Ford: "Falling Bough" (Source). You will also see the now extinct Passenger Pigeon in 'Birds of America'.

References:

Ramírez JD, Sánchez LV, Bautista DC, Corredor AF, Flórez AC, & Stensvold CR (2013). Blastocystis subtypes detected in humans and animals from Colombia. Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases PMID: 23886615

Alfellani MA, Taner-Mulla D, Jacob AS, Imeede CA, Yoshikawa H, Stensvold CR, & Clark CG (2013). Genetic diversity of Blastocystis in livestock and zoo animals. Protist, 164 (4), 497-509 PMID: 23770574

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

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

My 'Thoughts on Blastocystis' now as eBook in Amazon!

I edited and assembled quite a few blog posts and published them as the eBook Thoughts on Blastocystis in Amazon! 

Buying it will set you back only about one pound, and even if you're completely broke, you may be able to borrow it through the Kindle Owners' Lending Library service. How cool is that?!

Anyway, the book summarises a lot of facts, thoughts, hypotheses and new research data on Blastocystis with a personal take here and there and also with parallels to other areas of clinical microbiology, gastroenterology and microbiomology. I hope you'll enjoy it!

Please also note that our review 'Recent Developments in Blastocystis Research' published in Advances in Parasitology Vol. 82 is also available for purchase in Amazon.

Literature:

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

ICOP XIV in Vancouver 28 July to 2 August 2013

The International Congress of Protistology (ICOP) takes place every four years, and so the 14th ICOP takes place from the 28th of July to the 2nd of August in Vancouver, Canada.

Most single-celled parasites infecting humans are known as 'protozoa', but Blastocystis does not belong to this group of organisms; meanwhile, protists comprise both protozoa along with a multitude of other very diverse species, including the Stramenopiles, to which Blastocystis belong. Protists include both uni- and multi-cellular eukaryotic organisms and are distinguished from animals, fungi and plants by a simpler cellular organisation.

The conference abstract book can be downloaded here, and presents a perplexing multitude of very interesting and diverse abstracts. There are four abstracts on Blastocystis alone, and two of them are presented by Dr Roger's group in Halifax, Canada + their international colleagues.

Phylogenomic analyses of large-scale alignments enable the outlining  of evolutionary relationship among major eukaryotic lineages and are highly facilitated by recent technological advances; several abstracts deal with such analyses. Eme et al. (Roger's group) present additional observations from an important phylogenomic study of Blastocystis sp. ST1 reiterating the importance of lateral gene transfer in enabling Blastocystis to adapt to a parasitic life style. Gentekaki et al. (Roger's group) present data on the draft genome of Blastocystis sp. ST1. Until recently, only one Blastocystis genome was available, namely that of ST7. The present data show remarkable differences between the ST1 draft genome and the ST7 genome. While the genome of ST7 comprises 18.8 MB, the genome of ST1 is only 14.0 MB long, and apparently there's  virtually no synteny among the two genomes! Almost 30% of the 5,637 predicted ST1 genes had no homologues in ST7. What is more: 'Orthologous proteins shared by the two genomes are only 51% identical on average. The predicted secreted protein repertoire also differs significantly; ST7 possesses ~300 whereas ST1-NandII has only 129.' Indeed, it appears that Blastocystis comprises some extremely diverse organisms! We are still trying to explore the clinical implications of this...

Alison Jacob, Graham Clark, and I contribute with an abstract on comparative analyses of 8 mitochondrion-like organelle (MLO) genomes from 5 subtypes. Contrary to the nuclear genomes, there is complete synteny and homology between the subtypes at MLO level, although the sequences diverge by up to 25%.

Tamalee Roberts and colleagues present data from analysis of 438 samples from a staggering 38 species in Australia. They found Blastocystis in 18 species, including kangaroos, wallaroos, snow leopard, and ostrich, and obtained subtype data from a total 80 samples.

The genetic universe of  Entamoeba is expanding quickly in these years. Silberman and colleagues (Arkansas, USA) provide data from analysis of Entamoeba from insects such as honeybees, cranefly larvae and multiple cockroach and beetle species. There is no information on any pathogenic properties of insect-infecting Entamoeba however.

The abstract book is also a place to learn that marine diatoms are responsible for about one-fifth of global photosynthesis (Armbrust, Seattle, USA) and that photosynthetic marine algae are responsible for 50% of global CO2 uptake (Worden, Moss Landing, USA).

There is quite a few abstracts on protist diversity and how NGS tools allow us to study this in a more comprehensive and exhaustive way and the need for taxonomic standardisation. Protist-barcoding includes metabarcoding (de Vargas, Roscoff, France) and some of the taxonomic challenges related to this are presented by Dr Pawlowski, Geneva, Switzerland.

Similar to Blastocystis, the trypanosomatids (Trypanosoma and Leishmania) cannot be classified according to morphology and host range, hence, molecular markers are warranted, and there's an abstract by Maslov (California, USA) on the general applicability of 'alternative barcoding', namely the use of Spliced Leader (SL) RNA gene repeats.

There is quite a few abstracts on 'rare ciliates' in harsh environments, and I bring your attention also to a previous blog post on extremophilic eukaryotes.

We also learn that free-living protozoa can tell us more about the origins of anaerobic parasites (Simpson, Halifax, Canada). And there is a group setting up a Plasmodium life cycle to study the metabolic steps critical to the malaria life cycle (McFadden, Melbourne, Australia).

There's a really teasing abstract on analysis of surface water samples from Italy, where Angelici et al. have developed a barcoding-like analysis based on ITS 2 and SSU rRNA genes to enable detection of parasites of clinical and epidemiological interest, but there is no information on how exactly the method was designed, and the authors do not list the parasites that they found... I'm not attending the congress myself, so here's hoping for some twitter updates on this...

One could go on and on, - why don't you have a look inside the abstract book yourself?!

Incidentally, Dr Tai from Vancouver, Canada, promts us to help protists getting into pop culture by wearing t-shirts silkscreened by hand using Ernst Haeckel's diagrams of phytoplankton and light micrographs of parabasalids! Don't know exactly how to get hold of these, but googling 'Ernst Haeckel' and 'phytoplankton' might get you started (go for Google images).

For those interested in protists (and art!), I recommend the blog 'The Ocelloid'. 

Suggested reading:

Denoeud F, Roussel M, Noel B, Wawrzyniak I, Da Silva C, Diogon M, Viscogliosi E, Brochier-Armanet C, Couloux A, Poulain J, Segurens B, Anthouard V, Texier C, Blot N, Poirier P, Ng GC, Tan KS, Artiguenave F, Jaillon O, Aury JM, Delbac F, Wincker P, Vivarès CP, & El Alaoui H (2011). Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite. Genome Biology, 12 (3) PMID: 21439036

Stensvold CR, Lebbad M, Victory EL, Verweij JJ, Tannich E, Alfellani M, Legarraga P, & Clark CG (2011). Increased sampling reveals novel lineages of Entamoeba: consequences of genetic diversity and host specificity for taxonomy and molecular detection. Protist, 162 (3), 525-41 PMID: 21295520

This Month In Blastocystis Research (JUL 2013)

The open access journal 'Tropical Parasitology' (published by the Indian Academy of Tropical Parasitology) has included a symposium on Blastocystis in their January-June (Vol. 3) issue (available here). The symposium comprises three papers; one is on "taxonomy, biology and virulence", the next is on genetic diversity and molecular methods for diagnosis and epidemiology, and the last one is on treatment controversies. I believe that it may take quite a while before these papers will appear in PubMed.

The first paper written by Drs Parija and Jeremiah sums up a few of the aspects related to (especially historical) taxonomic issues and very little on the actual biology of Blastocystis. Meanwhile, there is quite a substantial section on Blastocystis morphology. Regarding virulence, the authors mention the possibility that differences in virulence may be due to differences in subtypes, but that subtyping alone does not predict pathogenicity which in part may be due to varying levels of intra-subtype genetic variation. The authors also briefly mention some of the morphological and phenotypical observations that have been associated with 'pathogenic Blastocystis', such as the amoeboid stage, large cells, rough surface, slow growth rate, and increased binding to lectins. It is always interesting to speculate on such associations, but it must be kept in mind that results from in-vitro experiments may not necessarily reflect in-vivo situations.

One topic that keeps popping up in the literature - and also in two of the papers here in this symposium - is the possibility of 'amoebic forms' of Blastocystis being associated with symptomatic infection. This hypothesis was introduced in 2006 by Tan and Suresh, I believe; Scanlan (2013) speculated that amoeboid forms might be the nutrient acquiring form potentially selecting for bacterial virulence or certain bacterial communities through grazing; please go here for more thoughts from a previous blog post.

My own experience on Blastocystis morphology mainly stems from looking at cultures, and since we practically only get isolates from patients with gastrointestinal disease, I don't know what Blastocystis cultures from asymptomatic individuals look like. A dear colleague of mine - Marianne Lebbad, a brilliant Swedish parasitologist with many years in business - sent me the picture below (light microscopy of a faecal concentrate) and speculates that Blastocystis might be able to form groups/clusters of cells, maybe even with the ability to form a mono-layer on the surface of the gut mucosa? I've never observed the cluster formation in cultures, but then again, we have no idea of whether the stages seen in in vitro cultures (microaerophilic environment) are identical to the in vivo stages (strictly anaerobic), and exactly how Blastocystis lives and multiplies in the colon... Anyway, the idea of biofilm comes into mind. It would be nice to learn more from colleagues with a similar experience.

Light microscopy of Blastocystis apparently forming a cluster of cells; we wonder whether the cells are in fact 'glued' together and if so, how? Courtesy of Dr Marianne Lebbad.

Moving on to the next paper, this one was written by me and deals mostly with issues and developments within the field of diagnostics, molecular characterisation, and molecular epidemiology. The target audience comprises clinical microbiologists and those involved in Blastocystis epidemiology and genetic diversity research. Included is a table, which is basically a reproduction of the one included in the recent paper by Alfellani et al. (2013) displaying the distribution of subtypes in humans across different geographical regions. I hope that the open access feature of this paper will prompt even more researcher into Blastocystis epidemiology! At least it is currently listed on the site as 'popular'!

The third paper in the string is written by Drs Sekar and Shanthi. These authors put emphasis on the conspicuous lack of data on the metabolic processes of Blastocystis, making it difficult to establish how to best approach antibiotic intervention; we must anticipate that with more genomic and transcriptomic data analyses arriving within a foreseeable future we will soon know much more about this. They also reiterate what has been put forth by many, namely that differences in eradication may boil down to differences in drug susceptibility, which again may be due to a variety of reasons, including genetic diversity, which is extreme in Blastocystis.

According to these authors, 'therapy should be limited to patients with persistent symptoms subsequent to a complete work up for alternative etiologies'; at the present stage this appears sensible, although clinicians would probably appreciate a clearer definition of 'symptoms'!

The review goes through some of the drugs most commonly used for treating Blastocystis, including metronidazole, paromomycin and co-trimoxazole, but also includes a few data on the use of the probiotic Saccharomyces boulardii in attempts to eradicate Blastocystis. There is not very much on the mechanisms of drug action, - it's more like a summary of data coming out from different studies, including the few placebo-controlled ones.
Regarding co-trimoxazole (which is also known as 'Bactrim' or 'Septra') this drug combo is often administered to HIV-patients prophylactically against Pneumocystis. In a study of parasites in Danish HIV patients, only 6/96 patients were given co-trimoxazole (unpublished data); two of these patients had Blastocystis. Hence, one 'alternative' way of finding out about the efficacy of co-trimoxazole on Blatocystis is to test the stools from patients undergoing long-term Pneumocystis prophylaxis comparing these patients to a cohort not receiving Pneumocystis prophylaxis but otherwise similar.

I find it a bit peculiar though to go through a review on treatment data that does not at one single point mention the need for sensitive diagnostics when evaluating courses of treatment and the identification of carriers and non-carriers. Also, there are some passages which are quite difficult for me to follow, for instance p. 36, second column, bottom section.

I hope that this symposium will inspire some of our colleagues and contribute to an increased understanding of Blastocystis.

References:

SYMPOSIUM

Parija SC & Jeremiah SS (2013). Blastocystis: Taxonomy, biology and virulence Tropical Parasitology DOI: 10.4103/2229-5070.113894
 
Stensvold CR (2013). Blastocystis: Genetic diversity and molecular methods for diagnosis and epidemiology Tropical Parasitology DOI: 10.4103/2229-5070.113896  

Sekar U & Shanthi M (2013). Blastocystis: Consensus of treatment and controversies Tropical Parasitology DOI: 10.4103/2229-5070.113901

OTHER:

Scanlan PD (2012). Blastocystis: past pitfalls and future perspectives. Trends in parasitology, 28 (8), 327-34 PMID: 22738855

Stensvold CR, Nielsen SD, Badsberg JH, Engberg J, Friis-Møller N, Nielsen SS, Nielsen HV, & Friis-Møller A (2011). The prevalence and clinical significance of intestinal parasites in HIV-infected patients in Denmark. Scandinavian Journal of Infectious Diseases, 43 (2), 129-35 PMID: 20936912  

Tan TC & Suresh KG (2006). Predominance of amoeboid forms of Blastocystis hominis in isolates from symptomatic patients. Parasitology Research, 98 (3), 189-93 PMID: 16323025

This Month In Blastocystis Research (JUN 2013)

Another paper in the string of publications coming out from the PhD study by Dr Alfellani (London School of Hygiene and Tropical Medicine) has just appeared in PubMed.

Dr Alfellani and his colleagues have done a great job in analysing a multitude of samples from humans, non-human primates and animals; I have previously blogged about their observations from studies of human and non-human primates. Moreover, they have surveyed available data in order to better discuss their own findings, and the work has contributed significantly to what today is known about the host specificity, genetic diversity, phylogeography and general molecular epidemiology of Blastocystis.

Alfellani's most recent paper is published in the journal Protist, and it deals with the 'Genetic Diversity of Blastocystis in Livestock and Zoo Animals'.

It is quite a large paper which includes a lot of new information and a comprehensive (and hopefully exhaustive) table summarising Blastocystis subtype data in all relevant hosts (humans, non-human primates, other mammals and birds).

I will highlight a couple of things from the paper:

1. Apart from reporting on virtually complete SSU rDNA sequences from a couple of subtypes for which entire SSU rDNA sequences have yet not been available, we also report on three novel subtypes. Until recently, we only knew about 14 subtypes (ST1-ST14), of which ST1-ST9 can be found in humans. Now, three additional subtypes have been identified; ST15 in artiodactyls (camel and sheep) and non-human primates (chimpanzee and gibbon), ST16 in kangaroos, and ST17 in gundis.

The Gundi (Ctenodactylus gundi) is a rodent living mainly in the deserts of Northern Africa. (Source)

2. Novel data arising from analysis of faecal samples from humans and animals in Sebha, Libya, strongly indicate that humans and animals in this area are infected by different subtypes: Humans appear to carry ST1, ST2, and ST3, while synanthropic animals (artiodactyls in this case) mostly have ST5 and ST10 infections, suggesting that livestock is not a major contributor to human Blastocystis infection.

To this end, there is growing evidence of quite a substantial degree of host specificity of Blastocystis.  Even when subtypes overlap between humans and animals, we have accumulating evidence that the strains found in humans and animals are different. This means that the hypothesis that animals constitute an important reservoir of human Blastocystis infections currently has very limited support. It is my clear impression that when a strain of ST6 or ST8 is detected in humans, this strain has most probably been transmitted from an animal source. But we very rarely see these subtypes in humans, at least in Europeans.

It will be extremely interesting to see how the universe of Blastocystis subtypes unfolds... by genetically characterising strains in humans and non-human hosts, we are building up a clearer picture of transmission patterns and evolutionary biology, including our adaptation to Blastocystis, and the parasite's adaptation to us and other hosts.

It is noteworthy that we are starting to see different subtypes in rodents. We have previously thought that generally, rodents were infected by ST4. But now we know that many rodents are not infected, and we also know that rodents may harbour subtypes other than ST4.

So,17 subtypes of Blastocystis are now known. We have probably only seen the top of the iceberg, since many host species have not yet been sampled from, and it is likely that we will see quite a few STs being identified in the nearest future. To this end it is necessary to have a consensus regarding the identification of novel subtypes. Along with the Protist paper we have uploaded a supplementary file (Appendix A, TXT format) with aligned reference sequences that can be used for phylogenetic analysis,  hoping that it will be useful to our colleagues. In a future blog post I will try to address the issues of identifying new subtypes more specifically.

ST15 is one of the more interesting subtypes since it appears to have quite a low host specificity - infecting both non-human primates and artiodactyls. Yet, we have come across it only now. ST15 and ST17 are remarkable in the way that they appear to be closer related to herptile and arthropod lineages, respectively, than to lineages from mammals.

Please note that virtually complete sequences of ST10, ST13, ST14, ST15, and ST17 analysed in the study have been released in GenBank just now.

Further reading:

Alfellani MA, Taner-Mulla D, Jacob AS, Imeede CA, Yoshikawa H, Stensvold CR, & Clark CG (2013). Genetic Diversity of Blastocystis in Livestock and Zoo Animals. Protist, 164 (4), 497-509 PMID: 23770574

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

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, 140 (8), 966-71 PMID: 23561720