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 (SEP 2015)

The month of September saw the publication of the first data on Blastocystis subtypes going out from Qatar. Abu-Madi and colleagues--who have already been quite prolific in terms of surveying intestinal parasitic infections in Qatar--studied the positive rate of Blastocystis in 608 apparently healthy subjects arriving in Qatar for the first time, identifying a prevalence of 71% as identified by PCR. Strikingly, the positive rate by microscopy of the corresponding samples was only 7%. Three subtypes were idenfied, with ST3 being the most common subtype, followed in prevalence by ST1 and ST2. The study is important for at least two reasons: It confirms the drawback of basing Blastocystis epidemiological research on data generated using microscopy alone, and it confirms the virtual absence of ST4 outside of Europe.

Increased sensitivity of PCR relative to microscopy was also confirmed in a study carried out in Malaysia (I presume) by Ragavan and colleagues. This group surveyed the Blastocystis positivity rate among IBS and non-IBS patients analyzing colonic aspirates, including a total of 109 individuals. Given the data available on Blastocystis prevalence, I was quite surprised to learn that this group failed to detect Blastocystis in any of the samples by microscopy and culture. Using PCR (the subtype-specific [STS] primers were used as diagnostic primers), the group identified Blastocystis in 6 IBS patients and 4 non-IBS patients. Also these figures appear quite low. However, there is very little information available on the non-IBS patients, and since all study individuals were subject to colonscopy, this group of individuals might be suffering chronic and potentially severe intestinal disease, including for instance colorectal cancer, inflammatory bowel disease, etc., which would explain the low prevalence of Blastocystis observed among these individuals. Indeed, evidence is accumulating that the more "gut healthy" you are, the larger the probability of being Blastocystis-positive. I noticed that the colonic aspirates were spun down using 3,000 rpm prior to culture and microscopy; this process might have had an impact on cell viability and morphology; still, DNA should be detectable following this process. Meanwhile, we recently showed (Scanlan et al., 2015) that the sensitivity of the STS primers is relatively low, which is why the use of real-time PCR is recommendable for PCR-based screening. To see an example of how the STS primers perform relative to barcoding primers, go here (Suppl Table 2).
Moreover, care should be taken when reading this paper, since I'm fairly convinced that the subtype terminology used in the study is different from the consensus terminology (Stensvold et al., 2007). It says that the subtypes detected included ST2, ST3, ST4, and ST5; if this reflects the terminology that went along with the original description of the STS primers, these subtypes correspond to ST7, ST3, ST6, and ST2, which to me would be a more likely subtype distribution, taking this particular region into consideration, and given the fact that ST5 appears to be extremely rare in humans. 

It's always interesting to expand on the natural host spectrum of Blastocystis. The parasite has been found in a perplexing array of hosts, but some host specificity has been observed. When it comes to animals held by humans as livestock or pets, we know that pigs and cattle are commonly, if not consistently, colonised by Blastocystis with some quite specific subtypes. With regard to pets, dogs and cats have been found positive, but there seems to be increasing evidence that these animals are not natural hosts (see also Wang et al., 2013). Osman and colleagues, recently published a survey on Cryptosporidium and Blastocystis in dogs using sensitive molecular methods, demonstrating a prevalence of Blastocystis of only about 3%. Moreover, the subtypes 2 and 10 were found, and ST10 is found mostly in cattle, and never before in dogs, as far as I know, which could suggest accidental colonisation - and possibly not a very long-lasting one. Similarly, when humans are found to be colonised with subtypes rarely found in humans, such as ST6, ST7, and ST8, it would be interesting to know for how long these subtypes are capable of "staying put" in the human intestine.

References

Abu-Madi M, Aly M, Behnke JM, Clark CG, & Balkhy H (2015). The distribution of Blastocystis subtypes in isolates from Qatar. Parasites & Vectors, 8 PMID: 26384209

Osman M, Bories J, El Safadi D, Poirel MT, Gantois N, Benamrouz-Vanneste S, Delhaes L, Hugonnard M, Certad G, Zenner L, & Viscogliosi E (2015). Prevalence and genetic diversity of the intestinal parasites Blastocystis sp. and Cryptosporidium spp. in household dogs in France and evaluation of zoonotic transmission risk. Veterinary Parasitology PMID: 26395822   

Ragavan, N., Kumar, S., Chye, T., Mahadeva, S., & Shiaw-Hooi, H. (2015). Blastocystis sp. in Irritable Bowel Syndrome (IBS) - Detection in Stool Aspirates during Colonoscopy PLOS ONE, 10 (9) DOI: 10.1371/journal.pone.0121173  

Scanlan PD, Stensvold CR, & Cotter PD (2015). Development and Application of a Blastocystis Subtype-Specific PCR Assay Reveals that Mixed-Subtype Infections Are Common in a Healthy Human Population. Applied and Environmental Microbiology, 81 (12), 4071-6 PMID: 25841010   

Stensvold CR, Suresh GK, Tan KS, Thompson RC, Traub RJ, Viscogliosi E, Yoshikawa H, & Clark CG (2007). Terminology for Blastocystis subtypes--a consensus. Trends in Parasitology, 23 (3), 93-6 PMID: 17241816

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

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