This Month In Blastocystis Research (JUL 2014)

For Spanish-speaking Blastocystis geeks, this summer must have been a real treat: Londoño-Franco and colleagues published a paper in Biomédica on Blastocystis in children and Colombia. But not only did they look for Blastocystis in faecal samples, they also sampled from finger nails, house floors, toys, tap water,  vegetables, other food items, etc... It is extremely rare to see studies aiming to identify sources of potential transmission, and I thought that this study would merit a blog post (unfortunately, I will have to rely on the Google translated version with all its potential limitations; I excuse for any misunderstandings).

Of course one of the big questions still remaining in Blastocystis research is: From where do we get this parasite? With more than one billion people colonised on the globe, the transmission pressure must be massive, and it's tempting to expect infectious cysts (or other stages) being more or less ubiquitous. There is some evidence accumulating that the parasite can be water-borne, and we also know that zoonotic transmission can occur (although relatively rarely, supposedly). However, this study takes things way further:

The authors carried out their study in Calarcá where they identified a prevalence of Blastocystis (based on microscopy of stool concentrates) of 57.5% in 275 children less than 5 years old; children aged 48 months or more were more prone to be positive than those who were younger. This is something we see a lot, and it either suggests a cumulative effect of colonisation (once established, colonisation is chronic), or that the behaviour (~exposure) or intestinal microbiota of older children favours colonisation.

Agua de panela (source).

Blastocystis was also found in dogs (63.3%), cats (56.3%), and poultry (35.7%). Moreover, it was found in tap water (38.5%), on toys (29.9%), baby bottles (18.5%), and under the nails of infected children (42.2%), their siblings (44.8%), and their mothers (34.2%). Among the vegetables that are typically consumed raw, it was found most frequently in lettuce (66.7%), and, in descending order, in tomato (44.4%), carrots (37.5%), cabbage (28.6%) and onion (25%). A high occurrence was seen in containers used to store 'aqua de panela', which is allegedly some kind of sugar water (haven't had the opportunity to sample it myself), with 47.7% of the samples positive. I believe that this drink is used as a sweetener and possibly also as a refreshment/energy drink, and maybe served with for instance cheese (image). Taken into account that Blastocystis is not exactly fussy about growth medium requirements, it may not be surprising at all to learn that this type of drink serves as a perfect stronghold for Blastocystis. 

The authors also explored a number of other things, among them i) the relative occurrence of cysts and vacuolar stages in the different types of samples and ii) whether any symptoms experienced over the past month could be attributed to Blastocystis, and iii) risk factors for colonisation. However, Google translate plays tricks on me on some of these bits, so I won't try to go more into detail with these findings. Suffice to say that the approach of distinguishing between different stages should help researchers find out more about which stage(s) that is/are responsible for transmission. Also, if for instance vacuolar stages are found in agua de panela and not cysts, then this might indicate that Blastocystis is actually growing in the drink? Which again is interesting because this would mean that Blastocystis capable of infecting humans can grow at temperatures lower than 37 degrees C.

Now, I could only have great confidence in the diagnostic work carried out by this team; however, I would have absolutely loved molecular confirmation of all of these findings. Also, maybe it would have been an idea to try and culture some of the Blastocystis found on fomites and in food/water to test for viability, or, as mentioned by the authors themselves, to test for viability using trypan blue. However, the authors should be praised for their perseverance and ingenuity, and I hope that this study will inspire other colleagues to pursue and expand on these initiatives and ideas.

This month saw a number of different Blastocystis-related papers, among them a paper from Klimes et al. on issues with Blastocystis genome annotation and polyadenylation-mediated termination codon creation in nuclear mRNA transcripts. Moreover, there's a paper on population structure analysis of seven eukaryotic microbial lineages, including Blastocystis, that apparently makes it possible to infer variable impacts of genetic exchange in populations of predominantly clonal micro-pathogens  (in fact the authors used our MLST data for ST3 in their analyses!). Finally, our colleagues in České Budějovice have produced an interesting review on self-infections with parasites; in the paper they point to the traditional focus on sussing out the pathogenic potential of parasites instead of trying to identify the potentially positive effects of parasite colonisation. Definitely worth a read!

Reference:

Londoño-Franco AL, Loaiza-Herrera J, Lora-Suárez FM, & Gómez-Marín JE (2014). [Blastocystis sp. frequency and sources among children from 0 to 5 years of age attending public day care centers in Calarcá, Colombia]. Biomedica : Revista del Instituto Nacional de Salud, 34 (2), 218-27 PMID: 24967927 

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 PMID: 25015079 

Lukeš J, Kuchta R, Scholz T, & Pomajbíková K (2014). (Self-) infections with parasites: re-interpretations for the present. Trends in Parasitology PMID: 25033775

Tomasini N, Lauthier JJ, Ayala FJ, Tibayrenc M, & Diosque P (2014). How often do they have sex? A comparative analysis of the population structure of seven eukaryotic microbial pathogens. PLoS One, 9 (7) PMID: 25054834 

A Penny For Your Thoughts

So, what should we do about Blastocystis? What do we want to know?

I believe the imminent answer to the latter question is easy: We want to know whether it’s pathogenic, whether we should treat it and how. But I also think that there are many other interesting aspects of Blastocystis which are also of broad interest to the general public, namely: How about the many cases of asymptomatic Blastocystis carriage? What does Blastocystis do in our guts? Could it have any potentially beneficial impact on our health?

Given the fact that Blastocystis has not been implicated in any outbreaks (admittedly: I guess that no one actually ever looked for Blastocystis in outbreak investigations... except for me!), I reckon that the chance of it being involved in acute diarrhoea is small. So, in that respect it's very different from the other intestinal protists such as Giardia, Cryptosporidium, Cyclospora, microsporidia, even Entamoeba histolytica. It's actually more reminiscent of helminth infections, which are are often chronic, and when light hardly give rise to symptoms (depending on species that is!).So I'm more thinking along the lines of co-evolution, adaptation, etc.

Maybe future research will call for a shift in paradigm, but until then I think that we should do what we already can, just at a larger scale and see where it takes us, namely:

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

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.