This Month In Blastocystis Research (MAR 2014)

If there's one paper that really made my eye balls pop over the past 30 days, it's the paper appearing a couple of days ago in BMC Infectious Diseases by Safadi et al. on Blastocystis in a cohort of Senegalese children. The paper is open access and can be downloaded here. But I'll be jumping right at it:

A 100% prevalence of Blastocystis in a cohort of 93 Senegalese children! 

The children represented a mixed group of children with and without symptoms. And yes, they were all colonised!

Are Senegalese children obligate carriers of Blastocystis? Image courtesy of whl.travel.

I will not at all try and discuss the potential clinical implications of this. I don't think we currently have the appropriate tools to ascertain to which extent a 100% Blastocystis prevalence is a public health problem. 

However, technically and scientifically, I'm extremely pleased to see a study like this one. My group and some of my colleagues have somewhat similar data in the pipeline, and it's great to see this next generation of survey data emerging from different regions of the world, based on the use of highly sensitive molecular tools to screen for Blastocystis. I cannot emphasise the importance of this too much.

The authors hoovered faecal samples from the children for Blastocystis-specific DNA using both PCR + sequencing (barcode region) and real-time PCR. Importantly, quite a few samples negative by barcoding were positive by real-time PCR, and so if the authors had included only PCR + sequencing, the prevalence would have been only 75% or so. It may be not very surprising that barcoding PCR did not pick up all cases of Blastocystis, but then again, it has always been known that the barcoding PCR is not diagnostic - one of the primers, RD5, is a general eukaryotic primer, while the other one, BhRDr is Blastocystis-specific. Also, the PCR product is about 600 bp; diagnostic PCRs should preferably be designed to produced much smaller amplicons (100 bp or so) for a variety of reasons.

The research team subtyped all samples, and found ST3 to be the most prevalent subtype - colonising about 50% of the children. ST1 and ST2 were also common, while ST4 was found in only 2 children and only in mixed infections. Mixed subtype infections was seen in 8 cases. Note the small fraction of ST4. This subtype is very common in Europe but seems to be rare in most other regions.

There is no doubt that we with molecular tools are now starting to obtain data that represent a more precise snapshot of reality than before when tools of low sensitivity and unable to give strain information were used. And while qPCR can take us a long way in terms of precisely distinguishing positive from negative samples, we still have an amplification step that may interfere with the DNA information that we obtain. The French group involved in this study has over multiple studies done  an admirable job in terms of pursuing the extent of mixed subtype infections. Whether the data are based on sequencing of PCR products amplified by genus-specific primers, or whether real-time PCR  using genus-specific primers is used, it can still be argued that these methods have limitations due to application of genus-specific primers in both cases. It is going to be interesting to compare the evidence that we have collected from subtyping over the past few years with analysis of metagenomics data, which are independent of PCR amplification, and thus not subject to potential bias. 

A 100% prevalence means that transmission pressure is massive. Three subtypes are common. Still, mixed infections are present in less than 10%. If this is indeed a realistic picture, this may imply that once established, a Blastocystis strain is capable of keeping other strains at bay? In keeping with waht I said above, it is also possible that the extent of mixed infections is higher, and that the PCR methods only detect the more predominant strain, making the prevalence of mixed ST infection seem low.

It's tempting to believe that such a high prevalence of Blastocystis compared to Europe is due to exposure to contaminated water, but how does this explain a whopping 30% Blastocystis prevalence in the background population in Denmark, a country characterised by supreme hygienic standards and 'perfect plumbing' with all potable water being pumped up from the ground (ie. hardly no surface water)? Have all individuals positive for Blastocystis in Denmark been out traveling to more exotic countries with less well controlled water infrastructures? Or is Blastocystis just highly transmissible through e.g. direct contact? And will all who are exposed develop colonisation? What are the determinants? It's probably not fair to dismiss the idea of Blastocystis being waterborne (as one of the modes of transmission) due to the fact that Blastocystis has not been cause of waterborne outbreaks. If Blastocystis is non-pathogenic, it can easily be transmitted by water. In fact, if Blastocystis is waterborne and never gives rise to outbreaks, what does this tell us about it's pathogenic potential? Well, acute disease such as that seen for some bacteria, viruses, and Cryptosporidium, Giardia and microsporidia is probably not something that is associated with the organism.

I could have wished for allele analysis of the subtypes detected. It should be possible in all cases where barcode sequences were available, - simply and easy using this online tool. But the data is available in GenBank so everyone interested can have a look. 

There is plenty of interesting things to address, but for now I'll leave it here, and on behalf of all of us interested in Blastocystis research just thank the people behind the paper for publishing this important study!

And nope, this is no April Fool!

Literature:

El Safadi D, Gaayeb L, Meloni D, Cian A, Poirier P, Wawrzyniak I, Delbac F, Dabboussi F, Delhaes L, Seck M, Hamze M, Riveau G, & Viscogliosi E (2014). Children of Senegal River Basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infectious Diseases, 14 (1) PMID: 24666632

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

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

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

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.

Blog Feedback

I'm very thankful for all the positive feedback I get from readers across the globe, mostly by email. Due to time limits I can only respond to 5-10% of the mail, and I'm sorry for not getting back to the rest of you.

Meanwhile, this blog currently holds more than 60 posts, and you will also find a lot of key words in the right side bar, so take your time and browse a few posts or look up a few relevant key words, -  you might find an answer to one or more  of your questions.

Having said that, I try to read all my email, and I am listening! The feedback and questions that I get are vital for our work and help us identify the avenues that we need to take to unveil the many mysteries of Blastocystis.

And let me just say this for now: A proper microbiological work-up (by state-of-the-art methods, including PCR for intestinal parasites), is something that is offered on a routine basis in only very few laboratories, and also the number of clinically orientated Blastocystis research centres can be counted on one hand, I believe. Subtyping of Blastocystis is currently done mostly in epidemiological surveys (as part of research projects), and I suspect that our lab is one of the very few labs in the world doing subtyping on a routine basis.

Oh, and I've been asked by some readers about how to get blog updates. It's easy: You can follow this blog by email, - just scroll down and find "follow by email" in the right side bar and enter your email address. You can also subscribe to posts via atom (go to the very bottom of the page).

And then here's a little something about stomach acidity and intestinal microbiota from Scientific American, - but make sure to read the comments underneath the post too!
 

A Penny For Your Thoughts

So, what should we do about Blastocystis? What do we want to know?

I believe the imminent answer to the latter question is easy: We want to know whether it’s pathogenic, whether we should treat it and how. But I also think that there are many other interesting aspects of Blastocystis which are also of broad interest to the general public, namely: How about the many cases of asymptomatic Blastocystis carriage? What does Blastocystis do in our guts? Could it have any potentially beneficial impact on our health?

Given the fact that Blastocystis has not been implicated in any outbreaks (admittedly: I guess that no one actually ever looked for Blastocystis in outbreak investigations... except for me!), I reckon that the chance of it being involved in acute diarrhoea is small. So, in that respect it's very different from the other intestinal protists such as Giardia, Cryptosporidium, Cyclospora, microsporidia, even Entamoeba histolytica. It's actually more reminiscent of helminth infections, which are are often chronic, and when light hardly give rise to symptoms (depending on species that is!).So I'm more thinking along the lines of co-evolution, adaptation, etc.

Maybe future research will call for a shift in paradigm, but until then I think that we should do what we already can, just at a larger scale and see where it takes us, namely:

Where Are We On Blastocystis Subtypes?

As mentioned, Blastocystis exhibits remarkable intrageneric diversity, which is continuously being explored by us and our colleagues. We are convinced that the genus of Blastocystis comprises multiple species, but for now we call them "ribosomal lineages" or "subtypes" and allocate numbers to each subtype, hence ST1, ST2, etc. While the number of subtypes that can be found in humans remains stable, we and our colleagues are still expanding the subtype universe in non-human hosts (I will be blogging on this shortly).

Barcoding currently represents state-of-the-art in Blastocystis subtyping, and luckily this method appears to gain a foothold in labs across the world.

Nine subtypes have been found in humans, but some of them only on rare occasions. A recent study going out from London School of Hygiene and Tropical Medicine and led by Dr Alfellani and published just now in Acta Tropica looked at 356 Blastocystis sequences from samples from the UK and Libya, but also from sub-Saharan Africa, namely Liberia and Nigeria.

Is This A New Subtype?

To quote one of my colleagues attending the recent IWOP 2012 meeting in Tarrytown, NY, Blastocystis subtyping in humans and animals is becoming 'trendy', and so we keep trying to advocate for a standardisation of the metholodology of Blastocystis subtyping.

We recently changed the title of our page at www.pubmlst.org/blastocystis so that now it is called Blastocystis Subtype (18S) and Sequence Typing (MLST) Databases, and we added some text to front page:

In terms of genetic markers, the barcode region (Scicluna et al., 2006) is by far the best represented in publicly available sequence databases, and the correct subtype can be identified by BLAST analysis in the sequence database at the present site. Blasting against this database has the added advantages, compared to using GenBank, of automatically assigning allele types to the SSU-rDNA as well as using the consensus subtype nomenclature (unlike GenBank where the subtype is included only if one was part of the accession submission and no attempt to impose a standard nomenclature is made). In case the sequence does not match any of the ones in the database despite full coverage of the region, this indicates that the sequence represents a new allele or maybe even a new subtype depending on the amount of variation. If a new subtype is suspected, we suggest doing PCR and sequencing of the complete SSU rRNA gene with subsequent phylogenetic analysis using reference sequences.

Now, the last bit is extremely important. We have seen examples of researchers (including ourselves!) assigning sequences to a new a subtype in the absence of complete SSU rDNA data (in fact complete sequences for ST10-ST14 are not yet publicly available!). Doing so has a least two major limitations/drawbacks: Far from all SSU rDNA regions have been validated as being representative of the whole SSU rRNA gene in terms of phylogenetic analysis, and therefore phylogenetic inferences based on non-validated regions may have little or at least less support than anticipated. Moreover, if someone analyses e.g. position 600-1600, and phylogenetic analysis based on this region reveals a potentially new subtype, this makes it impossible for his/her colleague who has data covering positions 1-600 from a Blastocystis isolate that may also represent a new subtype to ascertain whether it might be same subtype (see example below)!

Obtaining complete SSU rDNA sequences directly from faecal DNA may be a cumbersome task but is sometimes possible by combining sequence-specific primers with low-specificity primers such as the RD5 and the RD3 primers (Clark, 1997). If a cultured isolate is available, obviously this makes complete SSU rDNA sequencing much easier.

While it appears that the number of subtypes occurring in humans stays around 9, our gut feeling is that we are yet to uncover quite a few subtypes colonising non-human mammals, and it's great to see an increasing number of teams exploring the genetic diversity of Blastocystis. For instance, Dr Ronald Fayer and his group recently published exciting data on a new Blastocystis subtype in cattle, which they named ST14 (Fayer et al., 2012).

Importantly, caution should be taken to avoid creating confusion in subtype terminology. Confusion can arise when independent researchers assign the same new subtype name (e.g. ST14, ST15, etc.) to novel sequences which in fact belong to different ribosomal lineages, or when incomplete SSU rDNA sequence data are used; this situation was seen recently, when Petrasova et al. (2011), assigned a Colobus sequence to ST5, although it was in fact a ST13 sequence (Clark et al., in press); the situation arose, since Petrasova et al. (2011) did not have data covering the region currently available for ST13 (Parkar et al., 2010), and therefore believed that their sequence was a unique ST5 variant. As for ST14, less than 500 bp are currently available, and these 500 bp are not in the barcode region, making it difficult for all teams using barcoding to compare their data. And so we would like to advocate for making complete SSU rDNA sequences publicly available (Genbank) for potentially new subtypes, for at least two reasons:

1. Phylogenetic inferences based on the complete SSU rDNA will be more robust than those obtained from analysing shorter sequence streches.

2. Complete seqeunces are needed for reference since subtype screening typically includes a single round PCR such as barcoding (Scicluna et al., 2006) amplifying about 550 bp; in the situation where complete SSU rDNAs are available for all known subtypes, it will be quick to analyse, whether a sequence may represent a new subtype, since this will be independent on the SSU rDNA region studied.We therefore hope that complete SSU rDNA sequences will soon be made publicly available for ST10-ST14.

So, when does a complete SSU rDNA sequence represent a new subtype? Well, we have a review paper in press in Advances in Parasitology on recent developments in Blastocystis research, which will be published in less than six months probably, and which also touches on this topic; once the paper is published, I will try and make a summary our thoughts on this...

Further reading:

Clark CG (1997). Extensive genetic diversity in Blastocystis hominis. Molecular and biochemical parasitology, 87 (1), 79-83 PMID: 9233675

Fayer R, Santin M, & Macarisin D (2012). Detection of concurrent infection of dairy cattle with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and microscopic methods. Parasitology research PMID: 22710524

Parkar U, Traub RJ, Vitali S, Elliot A, Levecke B, Robertson I, Geurden T, Steele J, Drake B, & Thompson RC (2010). Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers. Veterinary parasitology, 169 (1-2), 8-17 PMID: 20089360

Petrášová J, Uzlíková M, Kostka M, Petrželková KJ, Huffman MA, & Modrý D (2011). Diversity and host specificity of Blastocystis in syntopic primates on Rubondo Island, Tanzania. International journal for parasitology, 41 (11), 1113-20 PMID: 21854778
 
Scicluna SM, Tawari B, & Clark CG (2006). DNA barcoding of blastocystis. Protist, 157 (1), 77-85 PMID: 16431158

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