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 

Do IBS Patients Lack Blastocystis and Dientamoeba??

I feel like sharing data from a poster created by one of my colleagues, Dr Laura Rindom Krogsgaard who works at Køge Sygehus, Denmark. She presented the poster last month at the United European Gastrointestinal (UEG) Week in Berlin.

Laura is currently doing a very interesting survey on IBS and IBS-like symptoms in Danish individuals. Her first publication was on the epidemiology of IBS in Denmark (see literature list below). She performed a web-based survey, using YouGov Zapera, and questionnaires were emailed to a web panel (n = 19,567) representative of the general Danish population aged 18-50 years containing info on gender, age, geography and type of intestinal symptoms (if any). IBS and subtypes were estimated by the Rome III criteria. Of 6,112 responders, 979 (16%) fulfilled the Rome III criteria for IBS and had no organic diagnosis likely to explain their symptoms. IBS subtypes detected included  mixed IBS (36%), IBS with diarrhea (33%), IBS with constipation (18%), and unsubtyped IBS (11%).

At the Laboratory of Parasitology, we helped Laura analyse stool samples from survey participants for parasites. Not surprisingly, Blastocystis and Dientamoeba were by far the most common parasites detected; however, it appeared that individuals with IBS symptoms were less likely to be colonised by these parasites than their controls! Which means that we have a situation reminiscent of that seen in IBD patients, only less pronounced. 

Laura was able to survey symptom developement over 1 year and compare this to the incidence of Blastocystis and Dientamoeba, and none of the parasites (indvidually or in co-infection) were linked to symptom development.

Indeed, Laura's data are in line with the general tendency that we see for Blastocystis (see figure below). Blastocystis appears to be rare in individuals with perturbation of the intestinal microbiota (due to antibiotic treatment, inflammation, infection, diet, etc.), while common in healthy individuals, most of whom are probably characterised by high gut microbial diversity and thereby - apparently - the right substrate/growth conditions for Blastocystis.

Literature:

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

The entire poster "Dientamoeba fragilis and Blastocystis: Two parasites the irritable bowel might be missing" presented at the UEGweek can be viewed here via SlideShare.

Dying to know about Dientamoeba?

It's difficult to say 'Blastocystis' without saying 'Dientamoeba fragilis'. Both parasites tend to be extremely common in countries where other intestinal parasites (e.g. Entamoeba, Giardia, Cryptosporidium) are of low endemic occurrence, and they are often seen together in patient samples. It is only due to the recent introduction of DNA-based diagnostic methods (PCR) that we now know that these parasites are much more common than previously anticipated.

So, while I'm trying to encourage guest bloggers, I thought I'd introduce a 'guest star' - Dientamoeba!

Dientamoeba fragilis trophozoites with the characteristic binucleated feature.

The parasite belongs to the trichomonads, which also comprise parasites such as Histomonas meleagridis (the cause of 'blackhead disease' in turkeys) and - more distantly - Trichomonas vaginalis.

At our Parasitology Lab at Statens Serum Institut in Copenhagen we have been using real-time PCR for specific detection of Dientamoeba fragilis in faecal samples from patients with gastrointestinal symptoms for quite a few years now. In the period of 2008-2011 we analysed 22,484 stool samples for D. fragilis. The overall prevalence of the parasite in these samples was 43% but depended mainly on age (Figure 1). D. fragilis prevalence appears to fluctuate dramatically depending on the age group. Highest prevalence was seen among 7-year-olds, and a second 'peak' is seen in the parental age suggesting that infected children pass on infections to their parents. 

Figure 1:  Prevalence of D. fragilis as a function of age. (For more information, see Röser et al., 2013b).

Intestinal protozoa are transmitted faecal-orally and most of them have a cyst stage. However, a few protozoa appear not to have a cyst stage, among them D. fragilis. There is a lot of evidence that Histomonas meleagridis is transmitted by eggs of Heterakis gallinae, a nematode of galliform birds. Conspicuously, we recently demonstrated the presence of D. fragilis DNA in surface-sterilised eggs of Enterobius vermicularis (pinworm). The implications of this finding are unclear but could suggest a similar vector-borne transmission of D. fragilis.

As in so many other situations it is not possible to dish out simple guidelines as to when to test for and treat D. fragilis. It is clear that many carriers experience few or no symptoms at all, but there are several case reports demonstrating symptom relief in patients eradicated of D. fragilis. We published one such case recently in 'Ugeskrift for Læger' - the journal of the Danish Medical Association. Basically, the report describes lasting symptom relief after documented eradication of D. fragilis using high dose metronidazole. However, the patient's symptoms returned after a year, and  real-time PCR revealed D. fragilis positive stools. Eradication was achieved using paromomycin (250 mg x 3 for nine days).

Contrary to Blastocystis, this parasite exhibits remarkably limited genetic diversity. We recently analysed three different genetic loci (18S, actin, elongation factor 1-alpha), and we confirmed that only 2 genotypes exist, one of which is very rare. Genetically, however, the two genotypes are quite different, and it will be interesting to compare the nuclear genomes of the two, once they have become available.

Dientamoeba has been speculated to be a neglected cause/differential diagnosis of irritable bowel syndrome (IBS). We once found a statistical significant association between IBS and Dientamoeba; however, other more recent and more targeted studies (one of which is ongoing) have not confirmed this association. However, multiple factors could interact and analysing only simple associations such as symptoms related to parasite presence/absence may be a limiting approach; for instance, infection load/intensity may play a role, and other factors such as host genetics/susceptibility and microbiota ecology may be significant factors influencing on clinical outcome as well. On that note, we have observed some very low Ct values in our real-time PCR results for some of our D. fragilis positive patients, suggesting massive infections. D. fragilis infections are probably often long lasting (months), and if symptoms appear in the initial phase of infection only, cross-sectional studies of prevalence and clinical presentation will be potentially misleading. Large longitudinal cohort studies of pre-school children with monitoring of incidence of pinworm and D. fragilis infections would be extremely informative.

Dr Dennis Röser here at the SSI is currently finishing a randomised controlled treatment trial of D. fragilis in children, testing the clinical efficacy of metronidazole treatment versus placebo. Results are expected next year, so watch out for a 'D. fragilis special' by Dr Röser in 2014! It appears a lot easier to eradicate D. fragilis than Blastocystis - at least on a short term basis with metronidazole having an efficacy of about 70% or so (unconfirmed).

A couple of reviews free for download are available; please see literature list below or go here and here.

Suggested 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, 87 (6), 1046-52 PMID: 23091195   

Johnson EH, Windsor JJ, & Clark CG (2004). Emerging from obscurity: biological, clinical, and diagnostic aspects of Dientamoeba fragilis. Clinical Microbiology Reviews, 17 (3) PMID: 15258093

Ogren J, Dienus O, Löfgren S, Iveroth P, & Matussek A (2013). Dientamoeba fragilis DNA detection in Enterobius vermicularis eggs. Pathogens and Disease PMID: 23893951  

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

Röser D, Simonsen J, Nielsen HV, Stensvold CR, & Mølbak K (2013b). Dientamoeba fragilis in Denmark: epidemiological experience derived from four years of routine real-time PCR. European Journal of Clinical Microbiology & Infectious Diseases : official publication of the European Society of Clinical Microbiology, 32 (10), 1303-10 PMID: 23609513  

Stark DJ, Beebe N, Marriott D, Ellis JT, & Harkness J (2006). Dientamoebiasis: clinical importance and recent advances. Trends in Parasitology, 22 (2), 92-6 PMID: 16380293  

Stark D, Barratt J, Roberts T, Marriott D, Harkness J, & Ellis J (2010). A review of the clinical presentation of dientamoebiasis. The American Journal of Tropical Medicine and Hygiene, 82 (4), 614-9 PMID: 20348509

Stensvold CR, Clark CG, & Röser D (2013). Limited intra-genetic diversity in Dientamoeba fragilis housekeeping genes. Infection, Genetics and Evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 18, 284-6 PMID: 23681023

Stensvold CR, Lewis HC, Hammerum AM, Porsbo LJ, Nielsen SS, Olsen KE, Arendrup MC, Nielsen HV, & Mølbak K (2009). Blastocystis: unravelling potential risk factors and clinical significance of a common but neglected parasite. Epidemiology and infection, 137 (11), 1655-63 PMID: 19393117

The Potential Role of Our Microbiome Ecosystems

For those who like these pop-sci articles on the still somewhat conjecture-like but very inspiring theories about the role of our intestinal microbiome in health and disease, here's a link to an article from The Economist (18 AUG 2012):

The Human Microbiome: Me, myself, us

And let me reiterate: We still don't know much about mikro-eukaryotes in all this... do they play a role as well? And how do they cope with different types of microbiomes?

Anyways, enjoy!

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

Intestinal Symptoms

For over a century, the clinical significance of Blastocystis has puzzled medical doctors scientists. After realising the extensive genetic diversity in Blastocystis, one of the current main hypotheses is that Blastocystis subtypes differ in terms of clinical significance. In other words: Symptoms, such as diarrhoea or other intestinal upset, may be associated only with one or more subtypes, while other subtypes are strict commensals.

Blastocystis is very difficult to eradicate and colonisation is chronic. Do symptoms caused by potentially  pathogenic subtypes persist or do they develop initially only to diminish after host immunological adaptation? Do fluctuations in symptoms reflect fluctuations in parasite load? Such issues ire important when interpreting results generated from cross-sectional surveys of subtypes in various cohorts.
Moreover, intestinal symptoms are difficult to define. Diarrhoea may be defined by 3 stool passages per day or more, while many other symptoms can be very difficult to define, if at all possible. Irritable bowel syndrome (IBS) and - to some extent - food allergy may both be considered differential diagnoses of symptomatic Blastocystis infections.

IBS diagnosis is currently defined by the Rome III criteria, and there are at least three types of IBS, namely IBS with diarrhoea, IBS with constipation and IBS with a mixture of diarrhoea and constipation.

Symptoms may be experienced differently from person to person. While abdominal cramping is perceived mostly as a symptom and something unpleasant, flatulence may by many be seen as a sign of a "healthy tummy" (e.g. due to consumption of a high fibre diet), although "inconvenient". Some individuals may very well tolerate intermittent intestinal symptoms and do not consult their GPs or other health care professionals, while others may be much more sensitive to any changes in for instance stool patterns.

What some people do not realise is that many methods fail to detect Blastocystis. PCR and culture are the most sensitive methods, but are still only rarely used. Moreover, PCR is also suitable for the detection of Dientamoeba fragilis, which is a parasite often seen in co-infection with Blastocystis. These two parasites are probably the most common single-celled eukaryotes in the human intestine.

This means that complete and accurate microbiological make-ups are far from always performed. And so, incomplete microbiological examination coupled with differential diagnostic challenges, potential immunological adaptation and the very subjective components of symptom presentation renders our quest for clear-cut associations extremely challenging. Blastocystis will often be seen as the culprit of symptoms, possibly simply to the reason that it is the only potential microbial pathogen that has been demonstrated in a stool sample. Cohort studies using sensitive diagnostic methods for pathogen surveillance are expensive, but may be one of the few only ways forward with regard to epidemiological studies that can assist us in resolving the clinical significance of Blastocystis.