This Month in Blastocystis Research (DEC 2016)

I would like to end the year by briefly highlight three of the most important/interesting papers in Blastocystis research published in 2016 (and not co-authored by me).

The first article that comes to my mind is one by Pauline Scanlan and colleagues, who took to investigating the prevalence of Blastocystis in US households (family units). The reason why I'm mentioning this article is not so much due to its approach; it's much more related to the fact that even when molecular methods are used (i.e., highly sensitive methods), the prevalence in this population was only 7%, and the vast majority of Blastocystis carriers were adults. The prevalence is much lower in this population (Colorado) than in a country such as Denmark. I'm interested in knowing the reason for this difference. Are people in Colorado less exposed or are they less susceptible than people in Denmark? I'm also interested in knowing why there was only one child among the carriers... we see similar trends elsewhere: Blastocystis is a parasite that emerges only in adolescence and adulthood. Meanwhile, we see a lot of Dientamoeba in toddlers and smaller children, with more or less all children being infected at some point - at least in Denmark; here, geographical differences may exist as well. Mixed infection with Blastocystis and Dientamoeba in adults is not uncommon, so it's not that they outcompete each other.

Next up, is the article by Audebert and colleagues who published in the Nature-affiliated Scientific Reports on gut microbiota profiling of Blastocystis-positive and -negative individuals. I already made a small summary of the article in this post.

While we gain valuable insight into gut microbiota structure, we also need to know what these microbes are able to do. We need to know about the interaction with the host and how they influence our metabolism. I hope to see more studies emerging on the metabolic repertoire of Blastocystis and how the parasite may be capable of influencing the diversity and abundance of bacterial, fungal and protist species in the gut. What would also be useful is a drug that selectively targets Blastocystis so that we can be able to selectively eradicate the parasite from its niche in order to see what happens to the surrounding microbiota and - if in vivo - to the host.

The last article is authored by my Turkish colleagues Özgür Kurt, Funda Dogruman-Al, and Mehmet Tanyüksel, who pose the rhetorical question: "Blastocystis eradication - really necessary for all?" in the special issue on Blastocystis in Parasitology International. For some time I have been thinking of developing a reply to the authors as a Letter to the Editor with the title "Blastocystis eradication - really necessary at all?" Nevermind, quite similar to what we did back in 2010, the authors review the effect of various drugs that have been used to try eradicate Blastocystis. Moreover, they acknowledge the fact that Blastocystis is often seen in healthy individuals, and that its role in the development of gut microbiota and host immune responses should be subject to further scrutiny. They even suggest that the role of Blastocystis as a probiotic should be investigated. It's great to see clinicians think along these lines, since this is an important step towards expanding the revolution lately seen in Blastocystis research, exemplified by studies such as that by Audebert et al. mentioned above.

So, wishing you all a Happy New Year and a great 2017, I'd like to finish by encouraging you to stay tuned; soon, I will be posting some very... interesting... neeeeeeewwwws...

References:

Audebert C, Even G, Cian A, Blastocystis Investigation Group., Loywick A, Merlin S, Viscogliosi E, & Chabé M (2016). Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota. Scientific Reports, 6 PMID: 27147260 

Kurt Ö, Doğruman Al F, & Tanyüksel M (2016). Eradication of Blastocystis in humans: Really necessary for all? Parasitology International, 65 (6 Pt B), 797-801 PMID: 26780545

Scanlan PD, Knight R, Song SJ, Ackermann G, & Cotter PD (2016). Prevalence and genetic diversity of Blastocystis in family units living in the United States. Infection, Genetics and Evolution, 45, 95-97 PMID: 27545648

Happy World Digestive Health Day!

Today is World Digestive Health Day!

The theme is 'Gut Microbes -  Importance in Health and Disease'.

United European Gastroenterology (UEG), a professional non-profit organisation combining all the leading European societies concerned with digestive diseases, has launched a short video to raise awareness of such diseases:

In 2000, 600 million patients suffered from a gastrointestinal disease. By 2025, this is predicted to double to 1.2 billion (source).

For those interested (and with access!), there is a special issue on 'The Gut Microbiome in Health and Disease' in the journal Gastroenterology, one of the most renown and established journals in the field.

I would also like to bring your attention to the 5th ASM Conference on Beneficial Microbes, September 27-30 in Washington DC. Deadline for submission of abstracts is July 14.

Wishing everyone a nice World Digestive Health Day!

Parasite-Microbiota-Host Interactions

One of the current mantras in microbiology is that 'bacterial cells in the human body outnumber human cells 10 to one'. This has been known for a long time, but I guess that the main reason why this is being hyped nowadays is due to the fact that current technologies now enable us to look at entire microbial communities in a given ecological niche at any time point and how for instance they relate to health and disease.

Casadevall and Pirofski already made this point clear back in 2000 in their great minireview in the journal Infection and Immunity on 'Host-Pathogen Interactions: Basic Concepts of Microbial Commensalism, Colonization, Infection, and Disease'. This is a great paper that helps us understand and distinguish between the many ways microbes impact on our body. First and foremost, it allows us to understand that microbes can be commensals in some hosts but cause disease in other hosts, and that very few microbes are obligate pathogens.

Anatomical drawing of abdomen, ca. 1900 (Elisa Schorn). Source.

Commensalism is defined by these authors as 'a state of infection that results in either no damage or clinically inapparent damage to the host, though it can elicit an immune response'. And a commensal is a 'microbe that induces either no damage or clinically inapparent damage after primary infection; a state that is thought to be established early in life'. In terms of the antibody response, 'it is not known whether these immune responses reflect the occurrence of an unidentified form of damage to the host'. Importantly, 'commensals also synthesise metabolites that are essential nutrients for the host'.

According to Casadevall and Pirofski, colonisation is 'a state of infection that results in a continuum of damage from none to great, with the latter leading to the induction of host responses that could eliminate or retain the microbe, or progress to chronicity or disease; for organisms that induce no damage during infection this state is indistinguishable from commensalism'.

I guess that to this end comes the concept of tolerance...Some single-celled parasites are common in young mammals such as calves and lambs (for instance Cryptosporidium and - it appears - microsporidia (unpublished data)), but appear to be cleared by host immune response mechanisms, while other parasites are establishing chronic colonisation, - parasites such as Entamoeba and Blastocystis. The latter parasites may also colonise humans for years on end... So why do we not eliminate these parasites? Blaser writes back in 1997: 'Failure to eliminate the parasite implies that the cost to the host is greater than the benefit. This may be due to high costs (e.g., loss of vital (...) functions), or significant benefits (e.g., protection against lethal diseases), or that both cost and benefit are relatively low'.

In Blastocystis research most scientists appear to be preoccupied by identifying a role for Blastocystis in disease, driven by the black and white concept that either it's pathogenic or not... or at least that if it can cause disease, it's by definition a pathogen! I think the paper by Casadevall and Pirofski shows with great clarity why we should try and take a much more differentiated view on Blastocystis and it's role in health and disease.

As mentioned in a previous blog post, Blastocystis is practically an obligate finding in some societies, while more rare in others. In some communities it may be common to contract it in very early childhood (infants/toddlers), while in other communities you may not be infected or infected only in adulthood. In Denmark, the prevalence in the healthy adult population is about 30%, and there may be countries where the prevalence is even lower - typically in regions, where the general population has been 'intestinally defaunated' (presumably due to excessive hygiene combined with a Westernised diet). And so, in some places this parasite is getting so uncommon that it may at some point become a cause of disease, a so-called 'emerging pathogen'... Conjectural maybe, but still not far fetched.

Simultaneously, evidence is emerging that intestinal microbial eukaryotes (Blastocystis and Dientamoeba fragilis) are significantly more common in healthy individuals than in patients with gastrointestinal disease such as IBS and - especially - IBD, suggesting that these parasites are protective of functional and organic bowel disease. Do they prime our immune system in a beneficial way? Do they select for beneficial bacteria? Do they keep potential harmful microbial intruders at bay? Could they be synthesising metabolites beneficial to the host just like ciliates involved in fermentation processes in the large intestine of various herbivorous mammals?

This is why the exploration of the structure and function of intestinal pro- and eukaryotic communities is so important. For instance, can we link Blastocystis to any intestinal microbial patterns? At our lab, we think we can, and it's something that we will try and explore further (if funding can be obtained). Our null hypotheses include the following:

1) The distribution of pro- and eukaryotes is random (for instance: Blastocystis is not statistically associated with the presence of particular bacteria or other eukaryotes (fungi, parasites)).

2) The introduction of a Blastocystis strain into an intestinal microbial niche does not cause alteration of the composition of the pro- and eukaryotic flora. This can be studied using an animal model, and it is tempting to try and study host immunological parameters during and after challenge with Blastocystis. Also gene expression in both host and microbial communities could be studied.

Take home message is that we should be cautious with regard to deeming a parasite as being either 'pathogenic' or 'non-pathogenic'... parasites may have a multitude of functions and may impact their hosts in a variety of ways that together with all other types of impact from and interactions with other microorganisms (microbiota) results in a health/disease matrix in every single individual.

Finally: Here's to pageview # 200,000! See you in Boston on Sunday morning at the #ASM2014 conference: Passion for Parasites !

Literature:

Blaser, M. (1997). Ecology of Helicobacter pylori in the human stomach. Journal of Clinical Investigation, 100 (4), 759-762 DOI: 10.1172/JCI119588  

Casadevall, A., & Pirofski, L. (2000). Host-Pathogen Interactions: Basic Concepts of Microbial Commensalism, Colonization, Infection, and Disease. Infection and Immunity, 68 (12), 6511-6518 DOI: 10.1128/IAI.68.12.6511-6518.2000

This Month In Blastocystis Research (JAN 2014)

Investigations into the 'biodiversity' existing in a given host is interesting for a number of reasons. One of these reasons has to do with how microorganisms present in our bodies may impact each other or the host. Numerous fields of research are developing at the moment at all levels from studies of gut microbes influencing host microRNA response to bacterial infections to epidemiological/public health studies examining the increased morbidity or risk associated with contracting infection/developing disease given that some infection/disease is present already. Sometimes the reverse is seen, namely that co-infection by multiple parasites is associated with a morbidity lower than the morbidity seen when only one of the parasites is present. For instance, a 2012 study saw that children infected by both Plasmodium falciparum (causing malaria) and hookworm had lower odds of anemia and iron deficiency than their counterparts infected with P. falciparum alone. Other authors have recently reviewed what they see as compelling evidence of chronic viral and helminth coinfections capable of modulating deleterious malaria-specific immune responses. Obviously such types of information are critical to the development of control measures against malaria and helminth infections. A Nigerian colleague of mine is currently exploring co-infections in quite a large Nigerian population, and investigating associations between malaria and soil-transmitted helminths; I'm thankful to be involved.

Along these lines, a new paper sees an inverse association between Blastocystis carriage and tuberculosis/Mycobacterium tuberculosis infections (TB). Dr Franke and colleagues carried out a case–control study to examine associations between parasite infection and tuberculosis (TB) in children in Lima, Peru. Logistic regression analyses revealed that Blastocystis infection was strongly associated with a lower risk of TB (P = 0.002). Hence, the study seems to suggest that Blastocystis carriage may protect against TB. The authors hypothesise that a pro-inflammatory/Th1 response potentially elicited by Blastocystis may protect against other infections, such as TB. While we know very little about Blastocystis induced immunity, little seems to suggest that Blastocystis is related to a chronic pro-inflammatory immunological alert. At least the colonic mucosa of patients with Blastocytis appears to be normal by endoscopy. Given the prevalence of Blastocystis that we have found in the aforementioned Nigerian study, I'd suspect very little TB to be present in this cohort...

The authors highlight a major limitation of their finding, namely the one related to the directionality of the association: It might as well be TB 'protecting' against Blastocystis. Maybe the behaviour (and thereby the exposure to Blastocystis) of patients with TB is different from the behaviour of those who do not have TB; I don't think so though.
I could have wished that a similar analysis had been performed using another common micro-eukaryote in children that the authors did not test for, namely Dientamoeba fragilis. It would be useful to know whether the same association could be identified, or whether the association was specific to Blastocystis. In fact, a general analysis of the microbiota (16S/18S) would have been in place here to learn about other factors potentially responsible for the observations.

Btw, there are some ASM conferences coming up this year relevant to the topic:

1. 5th ASM Conference on Beneficial Microbes, September 27-30, 2014, Washington DC.
2. 1st ASM Conference on Polymicrobial Infections, November 13-16, 2014, Washington DC.

On a different note, there is a paper out by Dr Poirier and his colleauges who have been publishing extensively on Blastocystis, and who were the first ever to sequence and annotate a nuclear genome of Blastocystis. In this new article the authors present a new set of primers applicable to all subtypes of Blastocystis found in humans and targeting the DNA of the mitochondrion-like organelle (MLO). This is reminiscent of the primers used for barcoding (18S analysis), and indeed the primers were validated using strains for which 18S data were available.

The paper highlights a variety of interesting topics and discusses the overall applicability of the two methods  (18S barcoding vs. subtyping using the single-copy MLO rDNA). In both of these scenarios the authors cloned the resulting PCR products to compare intra-isolate genetic variability - something which in itself is very interesting. However, cloning of PCR products is something that is not regularly done in most labs due to time and money constraints. Direct sequencing of barcode products reveals the predominating strain in a mixture, given that no other causes of preferential template amplification exist (e.g. selective primers) and hence shows the consensus sequence; sequence traces may partially or completely fail to reflect cases of mixed infection. However, the issue of not detecting mixed ST infections or mixtures of the same subtype is generally recognised.

The authors present a phylogenetic analysis of the MLO rDNA sequences included and come up with a tree topology different to the one usually seen. Usually, ST1, ST2 and ST5 cluster together, ST3, ST4, and ST8 go together, and ST6, ST7, and ST9 go together. Here, ST1 goes with ST2 and ST3. When I studied the MLOs of Blastocystis on genome level, one of my aims was to see if I could identify evolutionary patterns that had so far not been appreciated and that could be exploited in new hypotheses on the epidemiology and clinical significance of the parasite. Also, as the authors suggest here, mitochondrial DNA is haploid and therefore extremely useful when unambiguous base calling is important as it is in typing schemes. However, so far, when doing phylogenetic analysis of MLO nt sequences and even concatenated MLO nad proteins, we have obtained tree topologies identical to that seen when the 18S gene is used. Maybe the different topology seen here may be due to the way the alignment was constructed/edited? In any case, bootstraps are very low, but if confirmed the finding is very interesting.

If you have half an hour, treat yourself to "A resurgence in Field Research is Essential to Better Understand the Diversity, Ecology, and Evolution of Microbial Eukaryotes".

Literature:

Archambaud C, Sismeiro O, Toedling J, Soubigou G, Bécavin C, Lechat P, Lebreton A, Ciaudo C, & Cossart P (2013). The intestinal microbiota interferes with the microRNA response upon oral Listeria infection. mBio, 4 (6) PMID: 24327339

Franke MF, Del Castillo H, Pereda Y, Lecca L, Fuertes J, Cárdenas L, Becerra MC, Bayona J, & Murray M (2013). Parasite Infection and Tuberculosis Disease among Children: A Case-Control Study. The American Journal of Tropical Medicine and Hygiene PMID: 24379242 

Frosch AE, & John CC (2012). Immunomodulation in Plasmodium falciparum malaria: experiments in nature and their conflicting implications for potential therapeutic agents. Expert Review of Anti-Infective Therapy, 10 (11), 1343-56 PMID: 23241191

Heger TJ, Edgcomb VP, Kim E, Lukeš J, Leander BS, & Yubuki N (2013). A Resurgence in Field Research is Essential to Better Understand the Diversity, Ecology, and Evolution of Microbial Eukaryotes. The Journal of Eukaryotic Microbiology PMID: 24325268

Poirier P, Meloni D, Nourrisson C, Wawrzyniak I, Viscogliosi E, Livrelli V, & Delbac F (2014). Molecular subtyping of Blastocystis spp. using a new rDNA marker from the mitochondria-like organelle genome. Parasitology, 1-12 PMID: 24467909 

Righetti AA, Glinz D, Adiossan LG, Koua AY, Niamké S, Hurrell RF, Wegmüller R, N'Goran EK, & Utzinger J (2012). Interactions and potential implications of Plasmodium falciparum-hookworm coinfection in different age groups in south-central Côte d'Ivoire. PLoS Neglected Tropical Diseases, 6 (11) PMID: 23133691