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

Blastocystis - 'Monsters Inside Me'

I woke up this morning, grabbed my smartphone and went on to check my Blastocystis Google Alert. There was one entry, and this was the one:

Now, I could probably do a post with hundreds of examples showing how the internet abounds with material that may misguide/misinform people on Blastocystis pathogenicity. As such, this video is a nice example of how you can diligently manipulate people into thinking that severe, debilitating disease can be caused by Blastocystis.

Now, before I move on, I have to say that if this is a documentary, I'm very sorry for the couple in this video who have suffered the pain and consequences of sudden debilitating illness. Precautions have to be taken when you are exposed to sewage to avoid contracting infections.

The symptoms that are described in the video - including the weight loss - could be attributable to many different bacterial and viral pathogens, even parasites such as Cryptosporidium and maybe also Giardia; to this end, the video provides us with no information on other pathogens found in the patient's stool. Even in the event that Blastocystis was the only potential pathogen found, other pathogens may have been overlooked if sensitive diagnostics were not taken into use.

It is possible that Bill Wilson contracted Blastocystis only after signing on to his plumbing contract, but it is also possible that he had it a long time before. Many of us (up to 30% of the healthy Danish population) are colonised, and colonisation is often chronic.

We are informed that the patient receives a course of metronidazole, a drug that is often used to treat Blastocystis, but which in fact has a limited efficacy in vivo when used alone. Bill apparently clears his symptoms after metronidazole treatment, but we do not know whether in fact Bill also clears his Blastocystis infection, which could be determined by post-treatment stool tests. Metronidazole is capable of clearing a large number of anaerobic bacterial and protozoan species, and it is not unlikely that the drug has eradicated one or more pathogens that Bill could have contracted during his work (or elsewhere), and so symptom relief may be due to clearance of a non-Blastocystis pathogen instead.

Finally, it may be so that symptom disappearance coincides with spontaneous pathogen resolution. Cryptosporidiosis, for instance, can cause quite debilitating disease even in immunocompetent individuals, causing the infected individuals to lose a lot of water due to diarrhoea lasting for days or even weeks, but the disease is usually self-limiting.

So, this video tells a story that makes the audience automatically think that Bill Wilson's disease is due to Blastocystis. Apart from the statement 'Complications from a Blastocystis hominis infection can be fatal' and the explanation of how metronidazole works on Blastocystis, there is not really any statements or information in the video that do not make sense; the video is just put together in a way so that the viewer automatically deduces that Blastocystis is the culprit. A diligent act of manipulation!

Please note that this post is about how information on Blastocystis can be conveyed to an audience and not about the particular case as such.

Reference:

Stensvold CR, Smith HV, Nagel R, Olsen KE, & Traub RJ (2010). Eradication of Blastocystis carriage with antimicrobials: reality or delusion? Journal of clinical gastroenterology, 44 (2), 85-90 PMID: 19834337

This Month in Blastocystis Research (MAY 2013)

Now, we have a situation. Last month, I came up with the idea of the post series 'This Month in Blastocystis Research' developed for discussing a couple of papers on Blastocystis appearing recently in pubmed. However, this month only one Blastocystis release has emerged. It is in Turkish with an English abstract and so I'm not in the optimum position to review it. Overall, I'm not entirely clear on why the authors have chosen to publish the work. The paper is apparently about PCR amplification of Blastocystis specific DNA (using the barcoding primers) with subsequent cloning with a view to producing subtype information that could have been obtained simply by direct sequencing. At least when the goal is to subtype a particular positive sample, PCR + sequencing should suffice. Obviously, if you want to explore intra-subtype diversity, cloning is very useful. But it is time consuming for subtyping and also expensive. Therefore, for plain subtyping, I recommend the protocol that I put out on youtube a few weeks ago. The phylogenetic tree produced by the authors looks unfamiliar to me in that the clustering of the subtypes is quite different from the phylogenies inferred by other groups; this should not have anything to do with the SSU rDNA region explored; rather it may boil down to issues with alignment editing or the algorithm chosen for phylogenetic analysis. Well, we should be looking forward to more subtype data from Turkey! Incidentally, I was once involved in a Turkish study where we found ST1, ST2 and ST3 mainly, while ST4 was rare.

Since there are no other papers to discuss, I will try and compensate by providing a link to 'This Week in Parasitism' hosted by Vincent Racaniello and Dickson Despommier, who are going through a case of Blastocystis possibly contracted abroad during a field trip to Bali. Now, there's a lot of digression in this pod cast (some of which is actually quite enjoyable). Also, I do not agree with all the things said about Blastocystis in this conversation. If you cannot make the link work, you can access the podcast directly here. 

I do think it's a bit strange though that given the clinical focus of the talk, there is not a single word on paromomycin. But I guess the overall take home message is that treating Blastocystis is really difficult, and no single type of therapeutic intervention is consistently efficacious. Unfortunately, the two gentlemen do not touch upon the genetic diversity of Blastocystis, which is probably one of the most interesting things about Blastocystis currently known, and which may also be part of the reason why no single treatment modality seems to work every single time.

I wonder whether Blastocystis will always be stuck in shades of grey... or whether at some point we'll be able to make some clear-cut conclusions that will be useful for clinicians and clinical microbiologists...? I hope! And I believe we are certainly on our way!

Anyway, enjoy a bit of Blastocystis causerie!

Suggested reading:

Sakalar C, Uyar Y, Yürürdurmaz MA, Tokar S, Yeşilkaya H, Gürbüz E, Kuk S, & Yazar S (2013). [Cloning of Blastocystis sp Subtype 3 Small-subunit Ribosomal DNA]. Turkiye Parazitolojii Dergisi / Turkiye Parazitoloji Dernegi = Acta Parasitologica Turcica / Turkish Society for Parasitology, 37 (1), 13-8 PMID: 23619039

Ozyurt M, Kurt O, Mølbak K, Nielsen HV, Haznedaroglu T, & Stensvold CR (2008). Molecular epidemiology of Blastocystis infections in Turkey. Parasitology International, 57 (3), 300-6 PMID: 18337161

Nature Editorial (2013). Shades of grey Nature, 497 (7450), 410-410 DOI: 10.1038/497410a

Blastocystis aux Enfers

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

Source

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

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

Blastocystis Highlights 2012

2012 is coming to an end and it is also time for taking stock of the year Blastocystis-wise. We saw many significant scientific papers, among them a paper by Poirier and colleagues, predicting a potential role for Blastocystis in irritable bowel syndrome (IBS), based on analysis of their recent genome data.They propose that Blastocystis is genetically armed with the equipment necessary to cause intestinal dysbiosis, and potentially IBS, which may be a cause of dysbiosis. Indeed, members of this group found that the Blastocystis genome encodes various proteases and hydrolases that, if secreted, may be involved with perturbations of the gut flora; however, we need transcriptional profiling or similar studies to find out, whether these enzymes are actually expressed. Some species of Entamoeba are also in possession of multiple "virulence genes", but for some species they apparently remain un-expressed, and most Entamoeba species are still considered harmless.

Video Abstract on Blastocystis Paper on Search for Drug Targets

Please watch this video abstract co-authored by one of my colleagues, Mark van der Giezen, about the search for suitable drug targets in Blastocystis.

The whole paper can be found here.

The "Flagyl" Poll

For some reason the "Flagyl" poll in the right side bar of this blog was reset; the number of votes was approaching 100. The question was

"For those who have received metronidazole (Flagyl or Protostat) treatment for Blastocystis, please indicate whether you experienced no, transient or permanent improvement (or none of the above)"

The interesting thing is that there was a tie between "no improvement" and "transient improvement", and although this poll could have been heavily biased in numerous ways, it is still completely in line with our experience: Many patients report transient alleviation of symptoms, while others have no clinical benefit from Flagyl. Flagyl is an antibiotic targeting a wide range of bacteria and single-celled parasites. It is sometimes successful in terms of eradicating Dientamoeba fragilis, one of the most common parasites in the human intestine, and a parasite which may cause symptoms especially in children (we are currently conducting a randomised control clinical trial at Statens Serum Institut to explore clinical and microbiological effect of metronidazole treatment of children with D. fragilis).

Many people will get diagnosed with Blastocystis without knowing whether they might also be positive for D. fragilis (and vice versa). It is a complex situation, since both parasites are common, they are difficult to detect unless you use PCR or other specialised analyses, and in most labs they are not tested for on a routine basis. And if they happen to be part of the panel of organisms that is tested for, it may be so that insensitive methods are used for their detection, which means that only a fraction of the cases will be detected. So, this is a bit of a conundrum in itself!

So, it's not easy to know what causes the temporary alleviation in some patients. Is it due to parasite recrudescence? Is it due to parasite eradication with subsequent re-infection? And which parasite? Blastocystis? Dientamoeba? Any others? Or, is it due to perturbation of the intestinal flora in a "positive" direction, which is then gradually going back to normal? Placebo effect? There are possibly many more explanations...

However, deep sequencing of faecal samples pre- and post treatment of parasite-positive patients will probably answer many of our questions...

Literature:

Engsbro AL, Stensvold CR, Nielsen HV, & Bytzer P (2012). Treatment of Dientamoeba fragilis in Patients with Irritable Bowel Syndrome. The American journal of tropical medicine and hygiene PMID: 23091195

Engsbro AL, & Stensvold CR (2012). Blastocystis: to treat or not to treat ... But how? Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 55 (10), 1431-2 PMID: 22893582

Additional Comments on Blastocystis Treatment

I want to thank for the many emails I get! Unfortunately, I cannot respond to each one of them, in part due to time limits, in part since some of them are a bit off my topic or very difficult to answer. However, a few words on Blastocystis treatment (again!), which will hopefully satisfy some of the readers: 

Differences in the reported efficacy (microbiological and clinical cure) of certain drugs or drug combinations may be due to one or more of the following:

1) Actual differences in efficacy due to differences in pharmacokinetics, and -dynamics. Some drugs used for treatment of intestinal parasites are absorbed quickly from the intestine, while others are practically not absorbed at all (but stay in the intestinal lumen). For instance: Metronidazole is absorbed almost 100% in the proximal part of the intestine and may very well fail to reach Blastocystis, which resides is in the large intestine.

2) Different methods are used for evaluating treatment efficacy. If insensitive methods are used, the efficacy of any drug will be overestimated. Culture in combination with PCR is clearly advantageous in terms of evaluating microbiological efficacy since it will detect viable cells (see previous blog posts).

3) Drugs used in Blastocystis treatment may have broad spectrum antibiotic activity (e.g. metronidazole) and thus affect the surrounding microbiota, which again may influence the ability of Blastocystis to continue establishment. Hence indirect drug actions may play a role too. 

Could vegetables contribute to Blastocystis transmission?

4) Diet. What types of food do we eat? I notice that some people undergoing treatment for “blastocystosis” are cautious about eating carbs, for instance, and turn to vegetables only or at least non-carb diets, thinking that by cutting out carbs, they will cut off the "power supply" to Blastocystis. I’m not sure that this approach is very effective and it’s also important to acknowledge that the processing and metabolism of the foods that we ingest are complex. I hope to be able to do a blog post once on short-chain fatty acids, for instance. Again, changes in our diets may influence our bacterial flora which again may have an impact on Blastocystis. Importantly, we don’t know much about potential transmission of Blastocystis from raw vegetables and whether this could be a potential source infection (vegetables contaminated with Blastocystis).

5) Which leads to the next issue: The issue of re-infection. With so many people infected by Blastocystis (probably between 1-2 b people) it is likely that many of us are often exposed to the parasite. If we receive treatment but are not cut off from the source of infection, microbiological and clinical cure will be short-lived if at all possible.

6) Compliance - some drugs have serious adverse effects, and so, failure to reach microbiological cure may stem from failure to comply with drug prescriptions.

7) Differences in drug susceptibility. There is evidence from in vitro studies that Blastocystis subtypes exhibit differences in drug susceptibility.

In the absence of sound data that take all of the above factors into account, it is not possible for me (or anyone) to predict exactly which drug (combo) that will work and which will not. I think that it is important that GPs or specialists who take an interest in treating Blastocystis collaborate with diagnostic labs that are experts on Blastocystis diagnostics. If any drug or drug combo enabling microbiological cure can be identified, such pilot data can be used to design randomised controlled treatment studies that again will assist us in identifying whether Blastocystis eradication leads to clinical improvement.

I will try and provide some thougths on other future directions for Blastocystis research soon. Stay tuned!

To Treat or Not To Treat... But How?

In the "To Treat or Not To Treat" series (please look up previous post here), we have come to the "...But How?" episode.

Blastocystis may be susceptible to a number of drugs - in vitro. In vitro is not the opposite of in vivo. In vitro just  means that the test has been done on an organism that has been isolated from its usual habitat and tested e.g. in a flask, test tube, etc. In the lab, strains can be challenged and manipulated in multiple ways, but there is no guarantee that the outcome of an in vitro susceptibility test is reproducible in vivo, i.e. when the organism is challenged in its natural habitat and under "natural" conditions. Hence, if you test Blastocystis against metronidazole or any other compound (such as iodine) in vitro, and you observe an effect, you cannot rely on being able to reproduce the effect in vivo. This is due to a variety of reasons including pharmaco-kinetics and pharmaco-dynamics, including the ability of the drug to reach the parasite in its ecological niche, impact of the drug on other micro-organisms, drug interactions, strain-dependent differences in susceptibility (including inherent or acquired resistance), etc.

We recently described a case in which a woman with irritable bowel syndrome (according to the Rome III criteria) had both Blastocystis subtype 9 (ST9) and Dientamoeba fragilis. In order to try and eradicate the parasites and to see whether any eradication would impact on her clinical situation, she received multiple courses of antibiotic treatment:

1. Metronidazole (750 mg x 3/d for 10 days)
2. Tetracycline (500 mg x 4/d for 10 days)
3. Trimethoprim + Sulfamethoxazole (TMP 800 mg + SXT 160 mg x 2/d for 7 days)
4. Mebendazole + Metronidazole (100 mg x 2 separated by 2 weeks; subsequently metronidazole as in 1.)
5. Paromomycin + Metronidazole (PM 500 mg + MZ 170 mg x 3/d for 10 days)

Mebendazole was given to the entire household due to suspicion of pinworm infection running in the family that could be a potential reservoir of D. fragilis (re-)infection.

No clinical alleviation was seen throughout this period.

PCR-based detection of Blastocystis and D. fragilis was used to evaluate  faecal samples 5-10 days post-treatment: Microbiological effect was seen only on D. fragilis which was cleared only after treatment with PM + MZ (5).

So, Blastocystis "survived" this series of antimicrobial treatment. In Denmark, no further relevant treatment options are available for general use (actually, even the use of Humatin (PM) needs a special license).

None of the patient's family members or pets were found to be colonised by the same strain, probably indicating that there was no "local" reservoir for ST9, and that the repeated finding of ST9 was not due to re-infection.

It may be so that Blastocystis requires a certain intestinal bacterial flora to establish. However, we expect that substantial perturbations in the intestinal flora must have taken place during the patient's various treatments, and therefore Blastocystis must be able to quickly overcome and adapt to such perturbations. It may add to the conundrum that in this case the woman harboured ST9, which is only very rarely seen in humans, and we might therefore deduce that its presence would be more volatile. No animal/environmental reservoir has yet been identified for ST9.

There is no doubt that microbiomic profiling of the intestinal flora would be of great benefit in a case like this. If data could be achieved on the impact of these drugs on the relative bacterial structure and function by metagenomic approaches, then this would allow us to explore the changes in the general flora that Blastocystis is capable of withstanding. Certainly, none of these drugs had a measurable in-vivo protistocidal effect on Blastocystis when administered as shown.

I re-emphasise that it is far from certain that Blastocystis is capable of inducing disease, directly or indirectly, and hence, we do not know if, and in which situations, we should aim at eradicating it. Suffice it to say, that in our hands and with the compounds that are available for general use in Denmark, it is apparently extremely challenging to eradicate Blastocystis, if at all possible.

Microbe Resilience (Source)

Further reading:

Coyle CM, Varughese J, Weiss LM, & Tanowitz HB (2012). Blastocystis: to treat or not to treat... Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 54 (1), 105-10 PMID: 22075794

Engsbro AL, & Stensvold CR (2012). Blastocystis: To Treat Or Not To Treat...But How? Clinical infectious diseases : an official publication of the Infectious Diseases Society of America PMID: 22893582

Stensvold CR, Smith HV, Nagel R, Olsen KE, & Traub RJ (2010). Eradication of Blastocystis carriage with antimicrobials: reality or delusion? Journal of clinical gastroenterology, 44 (2), 85-90 PMID: 19834337

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

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.

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

Brave New World

Using Blastocystis as an example, we have only recently realised the fact that conventional diagnostic methods in many cases fail to detect Blastocystis in faecal samples, which is why we have started using molecular diagnostics for Blastocystis. I was also surprised to realise that apparently no single drug can be used to treat Blastocystis, and that in fact we do not know which combo of drugs will actually consistently eradicate Blastocystis (Stensvold et al., 2010).

There will come a time - and it will be soon - where it will be common to use data from genome sequencing of pathogenic micro-organisms to identify unique signatures suitable for molecular diagnostic assays and to predict suitable targets (proteins) for chemotherapeutic intervention; in fact this is already happening (Hung et al., in press). However, despite already harvesting the fruits of recent technological advances, we will have to bear in mind that the genetic diversity seen within groups of micro-organisms infecting humans may be quite extensive. This of course will hugely impact our ablility to detect these organisms by nucleic acid-based techniques. For many of the micro-eukaryotic organisms which are common parasites of our guts, we still have only very little data available. For Blastocystis, data is building up in GenBank and at the Blastocystis Sequence Typing Databases, but for other parasites such as e.g. some Entamoeba species, Endolimax and Iodamoeba, we have very little data available. We only recently managed to sequence the small subunit ribosomal RNA gene of Iodamoeba, and we demonstrated tremendous genetic variation within the genus; it is now clear that Iodamoeba in humans comprises a species complex rather than "just" Iodamoeba bütschlii (Stensvold et al, 2012).

Cysts of Iodamoeba

Ribosomal RNA is present in all living cells and is the RNA component of the ribosome. We often use this gene for infering phylogenetic relationships, i.e. explaining how closely or distantly related one organism is to another. This again assists us in hypothesising on transmission patterns, pathogenicity, evolution, drug susceptibility and other things. Since ribosomal RNA gene data are available for most known parasites, we often base our molecular diagnostics on such data. However, the specificity and sensitivity of our molecular diagnostic assays such as real-time PCRs are of course always limited by the data available at a given point in time (Stensvold et al., 2011). Therefore substantial sampling from many parts of the world is warranted in order to increase the amount of data available for analysis. In terms of intestinal micro-eukaryotes, we have only seen the beginning. It's great to know data are currently builiding up for Blastocystis from many parts of the world, - recently also from South America (Malheiros et al., 2012) - but the genetic diversity and host specificity of many micro-eukaryotes are still to be explored. It may be somewhat tricky to obtain information, since conventional PCR and sequencing offer significant challenges in terms of obtaining sequence data; such challenges can potentially be solved by metagnomic approaches - today's high throughput take on cloning; however, although the current next generation sequencing technology hype makes us feel that we are almost there, it seems we still have a long way to go - extensive sampling is key!

Cited literature:

Hung, G., Nagamine, K., Li, B., & Lo, S. (2012). Identification of DNA Signatures Suitable for Developing into Real-Time PCR assays by Whole Genome Sequence Approaches: Using Streptococcus pyogenes as a pilot study Journal of Clinical Microbiology DOI: 10.1128/JCM.01155-12

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

Stensvold, C., Lebbad, M., & Clark, C. (2011). Last of the Human Protists: The Phylogeny and Genetic Diversity of Iodamoeba Molecular Biology and Evolution, 29 (1), 39-42 DOI: 10.1093/molbev/msr238  

Stensvold, C., Lebbad, M., & Verweij, J. (2011). The impact of genetic diversity in protozoa on molecular diagnostics Trends in Parasitology, 27 (2), 53-58 DOI: 10.1016/j.pt.2010.11.005

Stensvold, C., Smith, H., Nagel, R., Olsen, K., & Traub, R. (2010). Eradication of Blastocystis Carriage With Antimicrobials: Reality or Delusion? Journal of Clinical Gastroenterology, 44 (2), 85-90 DOI: 10.1097/MCG.0b013e3181bb86ba

Blastocystis network on Facebook

This blog includes everything from updates on Blastocystis research, paper evaluations, polls, links, lab SOPs, to network opportunities and social interaction suggestions for all of us interested in Blastocystis. This time I want to guide your attention towards the Blastocystis network on Facebook. This is a good place to discuss personal experience with e.g. Blastocystis diagnosis and treatment and symptoms. The group is called "Blastocystis sp. (Blastocystis hominis and sp.)". If you have any experience and comments on Flagyl/Protostat (metronidazole), CDD regimens, including Secnidazole, Nitazoxanide, Furazolidone, Septrim (or Bactrim), Diloxanide Furoate, or other agents, please look up the group and share... We need your experience and views.

Blastocystis: To Treat or Not to Treat...

This year, Coyle et al. published a Clinical Practice paper in Clinical Infectious Diseases, a journal with a 5-year impact factor of almost 8. It is still difficult to get papers on Blastocystis published in clinical, peer-reviewed journals of major impact, probably due to the fact that evidence of Blastocystis' pathogenicity is so far only indicative, so it is great to see that the authors have managed to get their manuscript past those iron doors!

A few issues have come to my attention. When reading the abstract the reader will get the impression that subtypes are synonymous with genotypes, which is not the case. In the case of Blastocystis, a subtype is equivalent to a species; one of the reasons why we haven't allocated species names to Blastocystis from humans, other mammals and birds yet, is that we do not have sufficient data on genetic diversity and host specificity to come up with relevant names.

It says in the first page (pdf) that Blastocystis subtype (ST) 3 is found only in humans, which is not true. This subtype is common in non-human primates and can be seen in other, larger animals, including dogs, and also birds, if I remember correctly. However, so far, we only have multilocus sequence typing data from human and non-human primates, and these data indicate that ST3 found in non-human primates is often different from ST3 found in humans.

The authors recommend that asymptomatic individuals with few cysts should not be treated. Then what about asymptomatic individuals with many cysts? Also, with the diagnostic short-comings of microscopy of faecal concentrates, the suggested cut-off at 5 organisms per visual field appears arbitrary and, in best case, fortuitous.

In the abstract, the authors state that metronidazole is the drug of choice, although they appear to be quite aware that this drug has limited effect in terms of eradicating Blastocystis. So, why is metronidazole the drug of choice? Blastocystis is a parasite lodged primarily in the large intestine, and therefore we must anticipate that metronidazole often fails to reach the the parasite in sufficient concentrations due to absorption proximally in the gut. Luminal agents, such as paromomycin, are probably more likely to work, maybe in combination with metronidazole, although we have had a case, where even this combination was not effective.

When reviewing studies of treatment, it is important to acknowledge that insensitive methods have been used to evaluate drug efficacy. Culture combined with PCR is in my opinion the best method available in this respect. I prefer adding culture to the test, since culture detects viable Blastocystis (as opposed to PCR which will detect both viable and non-viable cells). Future randomised controlled treatment studies should therefore use culture and PCR to identify carriers both pre- and post-treatment. Whether Blastocystis-positive stool post-treatment is due to recrudescence, resistance or reinfection is not easily evaluated, but some useful information can be achieved by multi-locus sequence typing of isolates pre- and post-treatment.

Literature cited:

Coyle CM, Varughese J, Weiss LM, & Tanowitz HB (2012). Blastocystis: to treat or not to treat... Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 54 (1), 105-10 PMID: 22075794  

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, Smith HV, Nagel R, Olsen KE, & Traub RJ (2010). Eradication of Blastocystis carriage with antimicrobials: reality or delusion? Journal of clinical gastroenterology, 44 (2), 85-90 PMID: 19834337