This Month in Blastocystis Research (NOV 2014): Blasting Blastocystis Edition

The 'This Month' post is triggered by a paper emerging in the journal Gut Pathogens describing a clinical pilot study on the efficacy of triple antibiotic therapy in Blastocystis positive IBS patients. The article is free for download here. The triple therapy consisted of fourteen days of diloxanide furoate 500 mg thrice daily, trimethoprim/sulfamethoxazole (cotrimoxazole) 160/80 mg twice daily, and secnidazole 400 mg thrice daily. Six of ten patients achieved eradication. Please have a look at the paper for more information.

Sometimes I get contacted by people who have been trying to get rid of Blastocystis. And on the odd occasion, I receive accounts that I'd like to share - completely anonymously of course - hoping that the information will benefit those interested and that I can stimulate interest in the field a bit. But also because I think that sometimes people expose themselves to MASSIVE antibiotic treatment that might cause more harm than good (microbiota perturbation).

Below you'll find three examples dealing with the eradication of Blastocystis. Kindly note that this is not a post on IF or WHEN one should seek to eradicate Blastocystis, and please also note that this should not be interpreted as 'medical advice'.

I obtained permission from the patients in Examples #1 and #2 to share their stories, which have been eidted slightly for clarity.


Example #1:

"Two years ago, I was declared positive for Blastocystis after traveling to India. My symptoms included abdominal pain, weight loss, rectal itching, constipation or diarrhoea (yes, it's supposed to be 'or') -
I could be constipated for 7-10 days and then have a big diarrhoea "in one go". I took:

• January 2012: Fasygin (tinidazole) twice daily for 3 days => still positive after treatment.

• February 2012: Bactrim Forte (co-trimoxazole) three times daily for 10 days => still positive after treatment.

• March 2012: A combination of Bactrim Forte (cotrimoxazole) three times daily for 10 days and Tiberal (ornidazole) twice a day for 5 days. Then, Intetrix (tiliquinol) twice a day for 10 days => still positive after treatment. 

• May 2012: first-line-treatment from Australia = combination of Bactrim Forte (co-trimoxazole) twice a day for 10 days / Secnidazole 3 times a day for 10 days / Diloxanide Furoate 3 times a day for 10 days  => 3 consecutive Blastocystis-negative stools (tests in July 2012).

Then no symptoms anymore till February 2014, when the same symptoms came back, and I was stool-positive for Blastocystis. I took:

• March 2014: Flagyl (metronidazole) 3 times a day for 10 days, then a combination of Paromomycin 6 times a day for 10 days / Doxycyclin 2 times a day for 10 days / Bactrim Forte (co-trimoxazole) 3 times a day for 10 days / Saccharomyces boulardii 4 times a day for 10 days.

• Test in April: Stool-positive for Blastocystis.

• May 2014: Nitazoxanide 2 times a day for 10 days / Furazolidone 3 times a day for 10 days / Secnidazole 3 times a day for 10 days.

I'm now in a period with phases (after pain during 2/3 weeks, no more pain during 2/3 weeks, then pain again, then no more...). All tests were carried out in the same way at the same lab."

The patient's current Blastocystis carrier status remains unknown. However, the present story demonstrates the ferocious concoctions taken into use to clear Blastocystis.

Example #2:

"Metronidazole for 10 days failed, then, a few months later, I tried metronidazole plus paromomycin for 10 days, flanked with 3 doses of nitazoxanide and one dose of albendazole, and I am now convinced that that heavy chemo-treatment worked, since several tests, including my most recent one, have been negative since that multi-drug treatment. Some lingering mild symptoms, possibly related, or not, kept me wondering, but I am now convinced the bugs are gone." 

Example #3:

The last story is my own, and it describes how I inadvertently lost my Blastocystis strain. Please note that I have no financial interests to disclose. Moreover, I don't believe that I ever suffered symptoms from Blastocystis colonisation.

I spent most of my childhood in the countryside in Denmark. Moving down from Norway, my parents had bought a small farm, although they were not farmers. We did have some animals though, e.g. cats, a dog, chickens, sheep, and at some point even a couple of tortoises. I don't think I ever received antibiotics throughout childhood, except from once when being hospitalised due to surgery back in 1975. In 1990, I sustained a severe bicycle accident and was admitted to hospital; I believe I must have received some antibiotics back then, too. I have travelled extensively, and spent several months in e.g. Laos and Thailand in 2003/2004, three weeks in India in 2007, etc.

I started testing myself for Blastocystis only in 2009, and just like at least 20% of mankind, I was positive. Since then I re-checked myself every now and then, and I was always positive for the same strain (evidenced by DNA analysis), ST1 allele 4. However, in early 2014 I had major dental surgery, and I was prescribed tablets three times daily for six days. These tablets contained amoxicillin (500 mg) + the beta-lactamase inhibitor clavulanic acid (125 mg). A couple of weeks after completing antibiotic treatment, I tested myself a couple of times, and Blastocystis had vanished! Also today there is no sign of it...

I wish that I had been able to map my intestinal bacterial communities both before and after treatment to identify the effect of the drugs on my gut microbiota, thinking that Blastocystis disappeared due to microbiota perturbation rather than a direct effect on the parasite. I don't remember changing anything in my diet around the time of 'conversion'; only thing that I can think of is that - for a reason I no longer remember - I took to ingesting large amounts of freshly chopped ginger and consumed quite a few cups of 'ginger tea' (basically just a ginger infusion) around that time. But since ginger consumption is very common in parts of the world where Blastocystis is common, I don't attribute eradication to ginger consumption. I may be wrong of course.

For now, I just wanted to post the information and let the examples speak for themselves.

Reference: 

Nagel R, Bielefeldt-Ohmann H, & Traub R (2014). Clinical pilot study: efficacy of triple antibiotic therapy in Blastocystis positive irritable bowel syndrome patients. Gut pathogens, 6 PMID: 25349629

This Month in Blastocystis Research (OCT 2014) - Trick or Treat Edition

Over the past 30 days I've hardly had any time to focus on Blastocystis. I've been busy preparing for and attending UEGWeek 2014, preparing abstracts for next year's ECCMID conference in Copenhagen, and I've also put a lot of effort into preparing proposals for this round of grant calls from the Danish Council for Independent Research. 

Among other things, we are applying for money to develop DNA-probe based diagnostics, including a unique software, for use in the clinical microbiology lab that entirely circumvents the use of PCR (and thereby amplification bias) and that screens sequence data in real-time. An issue with current state-of-the-art in the area is that no software is available to relevantly and reliably handle the tons of sequence data that next/third/fourth generation sequencing devices are capable of producing. The proposed software will have a vast application range, applicable not only to clinical microbiology but also other areas of microbiology, such as food control, water sanitation, and monitoring of microbes in oil, soil, etc.

We are applying for about 270.000 Euros, and although this doesn't sound like an awful lot of money, competition is extremely fierce for this type of grant (although I'm not sure that the competition has to do exclusively with the scientific and innovative quality of the proposal...). So, let's see if it's going to be trick or treat!

Earlier this month, I was honoured to give a talk in Padova at the XXX National Congress of the Italian Society of Protistology on Blastocystis and its role in health and disease. I also got the chance to listen to some of the remarkable talks delivered by passionate colleagues of the society. There was quite a lot on endosymbionts of protists. The development of mitochondria in eukaryotic cells is a classical example of endosymbiosis; however, there are numerous examples of e.g. bacteria infecting protists, including the parasitic ones. Legionella, for instance, may be found in Acanthamoeba,  known to host a variety of bacterial endosymbionts. Along the same lines, I wish that studies could be made to look up potential endosymbionts in Blastocystis; endosymbionts which may confer disease, and the varying/unstable presence of which might explain the irregularity in symptoms reported by Blastocystis carriers? The question about endosymbionts in Blastocystis is interesting not only from a metabolic and horizontal gene transfer point-of-view, but also in the perspective of Blastocystis potentially serving as a vector, a vehicle for transmission of bacteria and maybe viruses... A nice paper on endosymbiotic associations within protists is available for a free download here. Rickettsia, for instance, are obligate intracellular bacteria found as endosymbionts in different types of eukaryotes, including amoebae, but also in endothelial cells (which are not phagocytic by nature, similar - presumably - to the case of Blastocystis), and some of these rickettsia are  known as causes of spotted fever and typhus. I think that Zierdt is the only one until now who has studied endosymbiosis in Blastocystis...

After the congress in Padova, I got a chance to pay my first visit ever to Venice, which was nothing short of brilliant.

Venice, October 2014.

I did have an hour here and there, however, to look up newest 'releases' on Blastocystis, and I'm just going to highlight a few of them.

Unfortunately in Russian and not available for download on any of the servers that I can access, there's a paper describing the finding of dividing (i.e. alive) forms of Blastocystis in a liver abscess in an immunocompromised woman. The question here is of course, did the parasite end up here by chance (fistula and/or secondary to bacterial invasion?) or by independent invasion? Hope to receive a copy of the paper at some point... and a translation!

There is a paper in a journal called 'Case Reports in Medicine' on what is called a co-infection of Schistosoma and Blastocystis in a 37-year-old male with chronic kidney disease, in whom Blastocystis was speculated to be the cause of chronic IBS-like symptoms. However, there is a number of issues that I would like readers of the paper to focus on: Apparently, the patient had Schistosoma mansoni detected in the urine suggesting schistosomiasis of the bladder. But how was Schistosoma detected? It doesn't say. Was it by microscopy? The patient was ab-positive, but still intestinal schistosomiasis was not ruled out (by e.g. PCR on faecal DNA, microscopy for ova and parasites, rectal biopsy, etc.). The patient responded well to praziquantel treatment and got rid of symptoms, including the intestinal symptoms ascribed to Blastocystis, for which the patient was prescribed metronidazole. We know that Blastocystis is only rarely eradicated by metronidazole alone, and indeed, the article does not provide data on post-treatment stool examination to see whether Blastocystis was still there. I think there is a chance that Blastocystis was an incidental finding and that intestinal symptoms in this case were due to Schistosoma. Given our recent data and improved diagnostic techniques, Blastocystis will more often now than ever become an incidental finding on routine analysis of faecal samples.

There is a paper by Fletcher and colleagues coming out in Journal of Public Health Research studying the prevalence and geographical distribution of enteric protozoan infections in Sydney, Australia, which I haven't had a chance to study in detail. I just want to emphasize that this study found Blastocystis prevalence to be increasing by age, a finding adding support to accumulating data suggesting that Blastocystis is more common in adults than in children, which is interesting from a clinical, epidemiological, and ecological point of view.

Hope to be able to address an interesting and brand new paper on Blastocystis treatment in Faculty of 1000 very soon. 

Happy Halloween!

References:

Fletcher S, Caprarelli G, Merif J, Andresen D, Hal SV, Stark D, & Ellis J (2014). Epidemiology and geographical distribution of enteric protozoan infections in Sydney, Australia. Journal of Public Health Research, 3 (2) PMID: 25343139

Nagel R, Bielefeldt-Ohmann H, & Traub R (2014). Clinical pilot study: efficacy of triple antibiotic therapy in Blastocystis positive irritable bowel syndrome patients. Gut Pathogens, 6 PMID: 25349629

Nowack EC, & Melkonian M (2010). Endosymbiotic associations within protists. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 365 (1541), 699-712 PMID: 20124339

Prodeus TV, Zelia OP, Khlebnikova TA, & Pikul' DA (2014). [Extraenteric infection caused by Blastocystis spp. in a female patient with liver abscess]. Meditsinskaia Parazitologiia i Parazitarnye Bolezni (2), 6-9 PMID: 25296418

Young CR & Yeo FE (In Press). Blastocystis and Schistosomiasis coinfection in a patient with chronic kidney disease. Case Reports in Medicine http://www.hindawi.com/journals/crim/2014/676395/ 

UEG Week 2014

The United European Gastroenterology (UEG) Week 2014 in Vienna is just around the corner. The conference kicks off on Saturday the 18th of October and judging from the program it will be a very interesting and inspiring event with so much to choose from that you will hardly be able to make up your mind about what to attend.

The general UEG website has now changed to the UEG Week Live site, which can be accessed here; however, you can still access the regular site here.

The programme for the entire venue can be downloaded here. There is also a very useful conference app (look for 'UEG Week 2014' in iTunes or go here), and a pathways tool, which you can download here.

However, I would like to invite you on a guided tour of some of the #UEGWeek topics for your inspiration. So, if you're interested in the clinical management and research developments in upper GI diseases, particularly in Helicobacter pylori, you may want to visit this blog post.

There's a panoply of interesting presentations and discussions on the menu for those who, like me, take an interest in the role of gut microbiota in health and disease, and to guide you through the jungle of treats, we developed this post. I doubt that there will be a lot on common intestinal microbial eukaryotic communities, but I'll try and see if I can stimulate the debate here and there...!

This Month in Blastocystis Research (SEP 2014)

Before leaving for Venice and Padova to introduce Blastocystis to the XXX National Congress of The Italian Society of Protistology (ONLUS), allow me to kick in just a few words for the September issue of 'This Month in Blastocystis Research'.

I will highlight two papers.

The first is a study from the US (Yes, - US data! How rare is that?). The team investigated the prevalence and subtype distribution of Blastocystis among client-owned and shelter-resident cats and dogs. Studies of Blastocystis in companion animals are actually quite rare. The authors used nested PCR for detection, followed by sequencing of PCR products. Interestingly, Blastocystis was not detected in any of the >100 fecal samples from client-owned animals. By comparison, Blastocystis was detected in 10/103 (9.7%) shelter-resident canines, and 12/103 (11.65%) shelter-resident felines. There was no significant difference in Blastocystis spp. carriage rates between the shelter-resident dogs and cats. It is likely that differences in diet and other types of exposure account for Blastocystis being found in shelter-resident animals and not in domestic animals. As for cats and dogs, we don't really know much about what to expect subtype-wise. These animals harboured ST10 mostly, a subtype that has only been found in artiodactyls, NHPs, and lemurs, so far, and - taking these new data into account - with little apparent host preference.

Viktor - an avid fox hunter (in 2007).

Other subtypes included ST1 and one case of ST3, and one case of what was most likely a new subtype - maybe! But then, few animals were positive, and given the different data on subtypes in cats and dogs, it's much too early to speculate on host specific subtypes... for now, it appears that there are none, and that maybe cats and dogs are not really natural hosts? A study by Wang and colleagues identified a plethora of subtypes in dogs: Among 22 positive dogs, most of which were from India, ST1, ST2, ST4, ST5, and ST6 were found. Again, nested PCR was used, and I might have a slight concern that this type of PCR approach is so sensitive that it will pick up the smallest quantity of Blastocystis, maybe even dead Blastocystis or other stages of Blastocystis not necessarily colonising the host (contamination, etc.). But I don't know. The authors of the US study noted that Blastocystis was unlikely to be associated with disease of the animals and were unable to establish a reservoir for human colonisation/infection in these animals.

I never got around to checking Viktor (our cat, pictured above) for Blastocystis. Now it's too late.

I would like to move on to another study. This time the data is from a paper that has just appeared in press in Clinical Gastroenterology and Hepatology. We  analysed faecal DNAs from patients diagnosed with irritable bowel syndrome and healthy individuals. The reason for doing this was due to the fact that intestinal parasite have been speculated to play a role in the development of IBS, a disease affecting about 16% of the adult Danish population. And so we thought that the prevalence of common parasites such as Blastocystis and Dientamoeba fragilis might be higher in IBS patients than in healthy individuals. The study was led by Dr Laura R Krogsgaard, who took a quite unusual approach to collecting questionnaires and faecal samples, namely by collaborating with the company YouGov Zapera.  
We obtained faecal samples from 483 individuals, of whom 186 were cases – ie. patients with IBS – and 297 were healthy controls. DNA was extracted directly from the stool using the easyMag protocol, and the faecal DNAs were submitted to real-time PCR based screening for Blastocystis, Dientamoeba, Entamoeba histolytica and E. dispar, Cryptosporidium, and Giardia.

Above you see the results of the various analyses. Blue columns represent healthy individuals, and orange columns represent IBS patients. Fifty percent of the healthy controls were positive for one or more parasites, while this proportion was significantly lower in IBS patients, 36%. Also for each individual parasite, the number of positive cases was higher among controls than among patients with IBS. Dientamoeba was the most common parasite among healthy controls and IBS patients. In terms of Blastocystis subtypes, the distribution of subtypes between the two groups was non-significant (data not shown).We ended up by concluding that our findings indicated that these parasites are not likely to play a direct role in the pathogenesis of IBS. Longitudinal studies are required to understand their role in gastrointestinal health. 

Still, the role of Blastocystis in human health and disease remains ambiguous, although lots of interesting data is emerging. In order to try and understand the theories behind Blastocystis' potential able to generate disease, I would like to point the readers' attention to a new review, developed by Ivan Wawrzyniak and his prolific colleauges.

Ciao!

References

Krogsgaard LR, Engsbro AL, Stensvold CR, Vedel Nielsen H, & Bytzer P (2014). The Prevalence of Intestinal Parasites is not Greater Among Individuals with IBS: a Population-Based Case-Control Study. Clinical Gastroenterology and Hepatology : the official clinical practice journal of the American Gastroenterological Association PMID: 25229421

Krogsgaard LR, Engsbro AL, & Bytzer P (2013). The epidemiology of irritable bowel syndrome in Denmark. A population-based survey in adults ≤50 years of age. Scandinavian Journal of Gastroenterology, 48 (5), 523-9 PMID: 23506174

Ruaux CG, & Stang BV (2014). Prevalence of Blastocystis in Shelter-Resident and Client-Owned Companion Animals in the US Pacific Northwest. PloS One, 9 (9) PMID: 25226285  

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

Wawrzyniak I, Poirier P, Viscogliosi E, Dionigia M, Texier C, Delbac F, & Alaoui HE (2013). Blastocystis, an unrecognized parasite: an overview of pathogenesis and diagnosis. Therapeutic Advances in Infectious Disease, 1 (5), 167-78 PMID: 25165551 

This Month in Blastocystis Research (AUG 2014)

Some August highlights in Blastocystis research:

1) The PRE-IOPCA Molecular Parasitology Workshop took place from the 7-10 August at CINVESTAV, Mexico City. Top-motivated students from some 10-15 countries worked hard from 7 am to 7 pm in dry+wet lab sessions, and we all had a really great time, thanks to both participants and fantastic organisers. There was a 4 h session on Blastocystis molecular epidemiology, and I was pleased to learn that some of the participants currently work with (or plan to work with) Blastocystis. I look forward to doing something similar in Ankara, Turkey on the 27th of May next year (www.blastomeeting2015.com - did you bookmark it?).

Most of the task force of the Molecular Parasitology Workshop (ICOPA 2014).

2) At the actual ICOPA conference, I chaired a session on Blastocystis in the context of IBS, with talks delivered by Ken Boorom, Pablo Maravilla, Pauline Scanlan and myself. In the audience I was honoured to see and meet Dr Hisao Yoshikawa, who has been a main contributor to Blastocystis research over the past 25 years or so (you can look up the publications by Dr Yoshikawa here). Considering the focus of this post, I guess that Pauline's talk was of particular interest, since she presented the data that we just published in FEMS Microbiology and Ecology:

3) The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota. That's the title of the paper appearing in FEMS Microbiology and Ecology. The study, led by Pauline, showed that Blastocystis was present in 56% of 105 healthy adults, which is much higher than previously reported from an industrialised county (Ireland). Moreover, a diversity of different subtypes (species) were detected and Blastocystis was present in a subset of individuals sampled over a period of time between six and ten years, indicating that it is capable of long-term host colonisation. These observations show that Blastocystis is a common and diverse member of the healthy gut microbiota, thereby extending our knowledge of the microbial ecology of the healthy human intestine. And one of the interesting things here is: Why do we see this great divide? Why does half of the population appear colonised while the other half not? What are the factors driving successful Blastocystis colonisation? Would some people be refractory to colonisation or does it really boil down to some sort of enterotype-driven phenomenon as previously indicated?

4) I would like to reiterate the paper by Klimes et al. published a study in Genome Biology and Evolution (GBE) on a striking finding in the Blastocystis nuclear genome. Stop codons created by mRNA polyadenylation have been seen so far in mitochondrial genomes only and not in nuclear genomes; however, the authors observered this feature in Blastocystis's nuclear genome. Partly due to limitations of currently available annotation software, this finding ostensibly calls for reannotation of the genome currently available in GenBank (ST7). The paper was highlighted in a separate article in GBE that can be accessed from here.

5) Speaking of Blastocystis genomes: The genome of Blastocystis ST4 (WR1 strain) is now available on-line and can be accessed here.

6) Wang and colleagues studied the location and pathogenic potential of Blastocystis in the porcine intestine. They studied a total of 28 pigs from a commercial piggery, a small animal farm, and a research facility, and all pigs were positive (for ST5, and mixed subtypes were also seen in some). Post-mortem analyses showed that all pigs were consistently found to harbour Blastocystis in the colon, and approximately 90% of the caeca and rectums examined were positive. Some of the pigs were immunosuppresed (Dexamethasone), and interestingly, Blastocystis was occasionally detected in the small intestine, notably in immunosuppressed pigs, suggesting that immunosuprression may alter host-agent relations and predispose to small intestinal colonisation. Histopathological analysis saw the presence of vacuolar and granular forms of Blastocystis, but there was no evidence of attachment or invasion of the intestinal epithelium. The lack of pathology, including inflammation, epithelial damage, mucosal sloughing, and lamina propria oedema, confirmed the trend from a previous study carried out by Ron Fayer's group (see reference below). The study adds to the string of papers finding no evidence in support for Blastocystis causing primary intestinal damage.

6) Lastly, I want to extend a cordial thank you to Shashiraja Padukone and Subhash Chandra Parija, Department of Microbiology, Jipmer, Puducherry, India, for writing up a review of my 'Thoughts on Blastocystis' available on Amazon for the price of only one US$ or so. The review was published recently in Tropical Parasitology and can be accessed here.

And, for those who are worried about researchers 'overselling' microbiome research, there is a small comment in Nature calling for sound scepticism to be applied to research dealing with the relationship between the microbiome and different types of diseases. There is much to be agreed upon, and what I find particularly important, is to take the reductionist approach where possible - in terms of Blastocystis there are lots of ways to study the impact of Blastocystis on bacteria in vitro, and also host microbiota, physiology and immunology in vivo; ways that can be controlled quite diligently. Also, I think that simple validation of methods applied to map e.g. intestinal microbiota is key. This is for some reason something that is generally being utterly and completely ignored. Why?

References 

Fayer R, Elsasser T, Gould R, Solano G, Urban J Jr, & Santin M (2014). Blastocystis tropism in the pig intestine. Parasitology Research, 113 (4), 1465-72 PMID: 24535732

Hanage, W. (2014). Microbiology: Microbiome science needs a healthy dose of scepticism Nature, 512 (7514), 247-248 DOI: 10.1038/512247a

Klimeš V, Gentekaki E, Roger AJ, & Eliáš M (2014). A large number of nuclear genes in the human parasite blastocystis require mRNA polyadenylation to create functional termination codons. Genome Biology and Evolution, 6 (8), 1956-61 PMID: 25015079

Scanlan PD, Stensvold CR, Rajilić-Stojanović M, Heilig HG, De Vos WM, O'Toole PW, & Cotter PD (2014). The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota. FEMS Microbiology Ecology PMID: 25077936 

Venton, D. (2014). Highlight: Not Like a Textbook--Nuclear Genes in Blastocystis Use mRNA Polyadenylation for Stop Codons Genome Biology and Evolution, 6 (8), 1962-1963 DOI: 10.1093/gbe/evu167

Wang W, Bielefeldt-Ohmann H, Traub RJ, Cuttell L, & Owen H (2014). Location and pathogenic potential of Blastocystis in the porcine intestine. PloS One, 9 (8) PMID: 25093578 

Launch of Official Website for 1st International Blastocystis Symposium.

We are now able to introduce the official website for The 1st International Blastocystis Symposium in May 2015, in Ankara, Turkey. Please check it out!

www.blastomeeting2015.com