Thursday, July 12, 2012

10,000 views and a Blastocystis Salute!

I've been writing this blog since late March, and today this blog was visited by viewer #10,000. I think that calls for a salute!

And what could be more appropriate than a piece of music attributed to Blastocystis? Please click here and read and listen for yourselves! And by the way: This is not the only orchestral work that has been written with Blastocystis in mind...

(Please note that this blog can now be accessed simply via http://blastocystis[dot]net - thanks for visiting!)

Tuesday, July 10, 2012

Blastocystis Culture in Jones' Medium

Upon request I have now posted the protocol on one of the simplest media used for Blastocystis culture, Jones' Medium, - please go to the tab (page) "Lab Stuff".

You can read about Blastocystis culture in some of my other blog posts, use the search box or the labels feature.

Please be aware that this is for xenic culture only - i.e. culture in the presence of bacteria. It's quick, inexpensive, very reliable (at least for human samples) and isolates can be kept this way for months/years - all you need is an incubator.

Extracting DNA from cultures and using it for subtyping usually yields excellent results.

I have never tried to cryopreserve (freeze down) Blastocystis using Jones' Medium, but it is possible (at least when Robinson's Medium is used).

More reading:

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

And, if you are interested in culture of intestinal protists in general, why not look up

Clark CG, & Diamond LS (2002). Methods for cultivation of luminal parasitic protists of clinical importance. Clinical microbiology reviews, 15 (3), 329-41 PMID: 12097242

Saturday, July 7, 2012

Blastocystis Nutrition

A reader of this blog asked me about the nutritional requirements of Blastocystis and whether I thought the parasite can be eradicated by fasting.

Given my background (I'm not a dietitian for starters), I guess my best way of approaching this is by drawing on my experience from the lab. When we diagnose Blastocystis, we have multiple methods to choose from, some of which are better than others (please look up previous posts here for more information). Short term (i.e. 24-48 h) in-vitro culture at 37 °C in Jones' medium is almost as sensitive as PCR (molecular detection). This means that if viable Blastocystis is present in a faecal sample, then it will most probably "come up" in culture, which means that in a day or two, we will be able to detect those "characteristically non-characteristic" soap bubble structures (the vacuolar stage) by light microscopy of a small portion of the culture - they will be all over the place!

So, what's Jones' medium? Well, Blastocystis can be cultured in a variety of different media, some of which are very primitive. Jones' medium is probably one of the simplest media, and consists mainly of electrolytes, yeast extract (contains nucleic acids) and horse serum (containing lipids). Importantly, we don't even have to add starch to the medium, when we culture Blastocystis xenically (i.e. under non-sterile conditions and this is what we always do when using culture diagnostically). Blastocystis has also been grown in a saline-serum medium, again in the presence of bacteria.

Apart from providing the anaerobic environment required for Blastocystis to thrive, bacteria most probably constitute a significant source of nutrients for the parasite. We can consistently keep strains of Blastocystis in xenic culture for weeks, months, years, observing vigorous growth, and it is clear that the bacteria and the simple medium supply nutrients in abundance. I have never managed to axenise (i.e. eliminate bacteria from) a culture, but others have been successful at times. One of the pioneers in Blastocystis research, Charles H. Zierdt, noted that the axenisation of Blastocystis usually takes weeks/months with a continuous reduction of bacterial numbers and species, until one species, usually a Bacteroides sp., remains; elmination of the last bacterial species may or may not result in axenisation, simply depending on the need for bacterial support. One of our future goals is to characterise the bacterial flora in individuals with and without Blastocystis.

I believe that even during fasting, Blastocystis will have plenty of access to essential nutritional components. It is possible that fasting may impact the intestinal bacterial flora, and if Blastocystis is dependent on a certain bacterial flora, it may be so that the parasite can be "manipulated" by manipulating the intestinal flora.

Useful reading:

Clark CG, & Diamond LS (2002). Methods for cultivation of luminal parasitic protists of clinical importance. Clinical microbiology reviews, 15 (3), 329-41 PMID: 12097242
 
Zierdt CH (1991). Blastocystis hominis--past and future. Clinical microbiology reviews, 4 (1), 61-79 PMID: 2004348

Wednesday, July 4, 2012

Share Your Experience

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


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

Thanks.

Sunday, July 1, 2012

Do I Get Diagnosed Correctly?

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


Further reading:

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

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

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

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

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

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

Friday, June 29, 2012

On Blastocystis and Animal Models

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

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

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

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

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

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

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

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

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

Further reading:

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

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

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

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

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

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

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

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

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

Wednesday, June 27, 2012

Dressed Up For The Party But No Invitation...

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

The Latest News from the Human Microbiome Project



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

Monday, June 25, 2012

Microbiome Blog Posts

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

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

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

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

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

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

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

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

More from NY Times, this time by Carl Zimmer:

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

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

And finally - to top it off:

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

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