This Month in Blastocystis Research (JAN 2016)

Three publications have caught my attention over the past month.

The first one is by my Turkish colleagues Kurt, Dogruman-Al, and Tanyüksel. They just published the paper "Eradication of Blastocystis in humans: Really necessary for all?" This title implies that treatment of Blastocystis is recommendable in some cases. The authors appear to acknowledge the view that treatment should be given to symptomatic carriers when all other causes of gastrointestinal symptoms have been rule out, - the popular 'last-resort' approach.

What I think is really useful and admirable is that the authors leave so many questions open/unanswered, despite the fact that they have been "in business" for so many years, representing some of the most avid Blastocystis researchers. It becomes clear from reading the paper that even in 2016, we still do not know how to eradicate Blastocystis from the intestine in those cases where we'd really like to try and do so. Importantly, the authors give examples of data supporting the fact that treatment failure may be due to failure of the drug to reach the parasite as well as treatment resistance. They also highlight the possibility that eradication of Blastocystis by antibiotic/anti-protozoal agents may be due to microbiota perturbation rather than a direct action on Blastocystis. I also very much appreciate the fact that the authors are embracing the necessity of studying Blastocystis in a parasite-microbiota-host context in order to be able to draw useful conclusions on its role in human health and disease.

Das and colleagues just published data on Blastocystis and subtypes of Blastocystis in IBS patients and controls in New Delhi, India. Using multiple traditional and DNA-based methods, they found that in their study material, the prevalence of Blastocystis was higher among patients with IBS than among healthy controls. It is not exactly clear how the controls were picked and what type of study population they represented. What I found really useful is the fact that they not only carried out subtyping of Blastocystis, but also identified subtype alleles. The subtypes and alleles found in the study were very similar to those found recently by Pandey et al. (2015) in Maharashtra, India.  Interestingly, it appears that only two subtypes are found in humans in India, namely ST1 and ST3. However, only two studies from India are available on subtypes in humans, to my knowledge, and so we need much more data to draw conclusions.

The last paper that I'm going to address is one by Zanzani and colleagues. When I read the abstract I almost dislocated my lower jaw from stupefaction: Studying the gastrointestinal parasitic fauna of captive non-human primates (Macaca fascicularis), they found a variety of protozoa and helminths, which is not surprising at all. Neither is it surprising that most macaques were positive for Blastocystis. Now, what really made my jaw drop was the fact their data on the subtypes found in the macaques challenged the host specificity of Blastocystis identified so far: They reported finding ST1, ST2, ST3, ST5, and ST7. And so, I had a closer look at the methods used to obtain data on subtypes. I take the liberty of questioning the data, since the authors report using a set of primers for amplification of Blastocystis DNA targeting the SSU rRNA gene, while using the STS primers developed by Yoshikawa et al. as sequencing primers! I guess that it is possible that the description of the methods was flawed (should have been picked up by the reviewer though), in which case I hope that an erratum will be developed and published.

References:

Das R, Khalil S, Mirdha BR, Makharia GK, Dattagupta S, & Chaudhry R (2016). Molecular Characterization and Subtyping of Blastocystis Species in Irritable Bowel Syndrome Patients from North India. PloS One, 11 (1) PMID: 26784888  

Kurt Ö, Doğruman Al F, & Tanyüksel M (2016). Eradication of Blastocystis in humans: Really necessary for all? Parasitology International PMID: 26780545

Pandey PK, Verma P, Marathe N, Shetty S, Bavdekar A, Patole MS, Stensvold CR, & Shouche YS (2015). Prevalence and subtype analysis of Blastocystis in healthy Indian individuals. Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, 31, 296-9 PMID: 25701123  

Zanzani SA, Gazzonis AL, Epis S, & Manfredi MT (2016). Study of the gastrointestinal parasitic fauna of captive non-human primates (Macaca fascicularis). Parasitology Research, 115 (1), 307-12 PMID: 26374536  

Yoshikawa H, Wu Z, Kimata I, Iseki M, Ali IK, Hossain MB, Zaman V, Haque R, & Takahashi Y (2004). Polymerase chain reaction-based genotype classification among human Blastocystis hominis populations isolated from different countries. Parasitology Research, 92 (1), 22-9 PMID: 14598169

This Month in Blastocystis Research (APR 2015)

#ECCMID2015 took place in Copenhagen. It was a great venue with a lot of interesting sessions. My favourite presentation was by Dr Paul D Cotter. We have had the pleasure of doing some work together with Dr Pauline D Scanlan as the main driving force. In his talk, Dr Cotter highlighted the emergence of research exploring whether certain organisms are pathobionts or probionts; among these, Blastocystis. Among many things, Dr Cotter reviewed the two recent Blastocystis-specific publications by Scanlan et al. focusing on the commonness and stability of Blastocystis colonisation [1] and on the need to use subtype-specific PCRs to detect and identify mixed subtype colonisation/infection [2].

Based at TEAGASC - Ireland, the Cotter/Scanlan group is one of the teams interested in looking into the ecology of Blastocystis (and other microbial eukaryotes of the gut), including its influence of the parasite on gut microbiota/microbiome (structure and function of our all gut organisms) and vice versa, and I'm sure that there will be a lot of interesting data coming out from their lab in the near future.

Cotter mentioned that Blastocystis has been subject to bad science. This may be due to a number of reasons. When developing hypotheses, we have a tendency of opting for dichotomous outcomes - either it is this or that, - maybe that's the very nature of hypotheses. If the clinical significance of Blastocystis is dependent on a number of different things such as co-colonising microbes (cross-talk), differences in host immunity response, Blastocystis subtype, and host diet for instance, then the true tapestry of physiological/biological/clinical mechanisms is likely to be extremely difficult to uncover. Moreover, despite the fact that so many people are curious about the public health significance of Blastocystis, apparently very little funding for targeted Blastocystis research exists. This means that mostly minor and not so significant studies ("cheap studies") on Blastocystis are available. Relatively little seminal research has been done in the clinical field (including the field of gastroenterology), and most studies on Blastocystis are cross-sectional and descriptive and usually not very well designed/carried out (use of diagnostic tools with limited sensitivity, for instance).

Maybe things will change when more and more people realise that we might be able to use Blastocystis as a biomarker/surrogate marker of intestinal homeostasis...

In my opinion the following topics would make for good research projects:

1) Large studies of both diseased cohorts and healthy individuals, including Blastocystis subtype data and data on accompanying protists, bacteria and fungi (16S/18S/ITS profiling).
2) Manipulaton studies where Blastocytsis (cysts) are introduced in ecosystems (in vitro or in vivo) to monitor potential changes.
3) Animal models using cyst challenge (to look at microbiota profile changing upon challenge, and, if in vivo colitis models are used, impact on host immunity)
4) Longitudinal microbiome studies of patients with and without Blastocystis.
5) Investigation of Blastocystis as a biomarker/surrogate marker of microbiota profiles and gut microbiome homeostasis... similar to my recent blog post: 'Show me your gut bacteria, and I'll tell you if you have Blastocystis!'
6) Comparative genomics (virulence gene identification for instance).
7) Identification of Blastocystis-specific signatures in metagenomics data sets.
8) Identification of drugs that have anti-Blastocystis properties, since currently, there is no drug regimen that consistently enables eradication of Blastocystis.

Speaking of which: We just published data in Journal of Ethnopharmacology on the anti-Blastocystis activity of 24 plant parts from 21 medicinal plants from Ghana [3]. We performed in vitro challenge of 48 h Blastocystis cultured cells (subtype 4) using ethanolic, warm and cold water plant extracts. Screening of these 24 different plant parts showed significant anti-Blastocystis activity of six of the ethanolic extracts: Mallotus oppositifolius, IC₅₀, 24h 27.8 µg/mL; Vemonia colorata, IC₅₀, 24h 117.9 µg/mL; Zanthoxylum zanthoxyloides, cortex IC₅₀, 24h 255.6 µg/mL; Clausena anisata, IC₅₀, 24h 314.0 µg/mL; Z. zanthoxyloides, radix IC₅₀, 24h 335.7 µg/mL and Eythrina senegalensis, IC₅₀, 24h 527.6 µg/mL. The reference anti-protozoal agent metronidazole (MTZ) had an IC₅₀, 24h of 7.6 µg/mL. Since cultures were xenic, antimicrobial activity was tested against two Gram-positive and two Gram-negative bacteria for all 24 plant parts at a final concentration of 1 mg/mL. Only C. anisata showed antimicrobial activity at a concentration of 800 µg/mL.

Hence, M. oppositifolius showed nearly as good activity as the reference anti-protozoal drug MTZ. Historically, the active plants found in this study have been used against dysentery, diarrhoea or other stomach disorders. Nowadays they are not used specifically for dysentery, but they are being used as medicinal plants against various stomach disorders.

Our book 'Biology of Foodborne Parasites' is out and available for ordering.

Incidentally, Blastocystis earned a designated chapter in the book 'Biology of Foodborne Parasites' which is now out and available for ordering here. It was fun writing it up, and hope that the chapter will be of interest to health care professionals and students around the world. The book also contains  introductions to the public health importance of foodborne parasites, molecular biological techniques in studies of foodborne parasites, and detection of parasites in foods.

References:

[1] 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, 90 (1), 326-30 PMID: 25077936   

[2] Scanlan PD, Stensvold CR, & Cotter PD (2015). Development and application of Blastocystis subtype-specific PCR reveals that mixed subtype infections are common in a healthy human population. Applied and Environmental Microbiology PMID: 25841010

[3] Bremer Christensen C, Soelberg J, Stensvold CR, & Jäger AK (2015). Activity of medicinal plants from Ghana against the parasitic gut protist Blastocystis. Journal of Ethnopharmacology PMID: 25773490

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