Showing posts with label genetic diversity. Show all posts
Showing posts with label genetic diversity. Show all posts

Wednesday, December 11, 2013

Molecular Epidemiology: Developing a Language

Initiatives towards standardising diagnostic methods and convening on taxonomy and reference data is extremely important in a world where multiple research teams independently carry out research using molecular markers to identify and differentiate species and genotypes of infectious organisms; such activity is crucial to identify patterns of transmission, differences in virulence, and opportunities for control and intervention. Without such standards, efforts to survey and surveil such organisms would be more or less futile, and so they are the backbone of molecular epidemiology.

Having seen that a variety of morphologically similar but genetically diverse Blastocystis organisms found in humans could in fact colonise a range of different hosts, we realised back in 2006 that all these variants could not all be 'Blastocystis hominis', which was then the species name used for Blastocystis found in humans, and together with colleagues we took to revisiting Blastocystis terminology: We recognised that we did not know enough about host specificity and genetic diversity to be able to come up with relevant species names, and so we invented (or maybe not invented, but at least 'formalised') the subtype system, a sort of a barcode system, where genetically similar (typically 98-100%) organisms are assigned to the same subtype, hence ST1, ST2, ST3, etc., which we today now know so well.

Slapeta now suggests a barcoding system for Cryptosporidium. This single-celled parasite takes a major toll on the health of infants and toddlers in developing countries (in some places surpassed only by norovirus), and may also cause debilitating disease in immunocompromised. The nomenclature for Cryptosporidium is very complicated for those of us who are not experts; for instance, I only recently realised that C. parvum may now only refer to the Mouse I genotype and not the 'common' or 'traditional' C. parvum (which now appears to be C. pestis), which is common in both humans and cattle. However, there is a debate going on as to which taxonomy should be followed, and whether this novel leap in 'Cryptosporidium taxonomy revision' can be endorsed by Slapeta's fellow Crypto experts, remains to be seen. Contentiousness aside, barcoding Cryptosporidium does seem relevant due to the fact that the host specificity of Cryptosporidium is relatively loose; for instance humans and cattle are known to share at least 9 species of Cryptosporidium... 

In his paper, Jan Slapeta lists all the known species of Cryptosporidium (in the 'revised' terminology), and even includes GenBank reference strains for common molecular markers such as actin, HSP70 and COWP1 used for genotyping. Interestingly, he does not include the GP60 marker, a molecular marker for which the terminology is also discordant.

Slapeta moreover includes a file with reference SSU rDNA sequences that enable a standardisation of genetic analyses. This year, we did in fact a similar thing for Blastocystis: Along with our 2013 Protist paper surveying Blastocystis subtypes in animals (including the identification of a couple of new subtypes!), we uploaded a reference alignment consisting of some complete SSU rRNA gene sequences present in GenBank; one or more for each of the now known 17 subtypes; more will be added as more subtypes are discovered. The file can be downloaded when accessing the online version of the paper, and we hope that everyone interested in analysing sequences that represent potentially novel subtypes will use this reference alignment (which has been edited to eliminate regions of ambiguous base alignment); it should be quite helpful. Again, I also bring your attention to the pubmlst Blastocystis database, where fast files obtained by Blastocystis barcoding can be queried in batches for quick analysis of large amounts of sequence data. There's a Youtube video here on Blastocystis barcoding and how to use the pubmlst database.

Consensus on methods, terminology and diagnostic algorithms is essential to developing a common language and understanding of how infectious organisms impact our lives; without it,  confusion wreaks havoc with our efforts.


Alfellani MA, Taner-Mulla D, Jacob AS, Imeede CA, Yoshikawa H, Stensvold CR, & Clark CG (2013). Genetic diversity of Blastocystis in livestock and zoo animals. Protist, 164 (4), 497-509 PMID: 23770574

Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, Faruque AS, Zaidi AK, Saha D, Alonso PL, Tamboura B, Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, Oundo JO, Hossain A, Das SK, Ahmed S, Qureshi S, Quadri F, Adegbola RA, Antonio M, Hossain MJ, Akinsola A, Mandomando I, Nhampossa T, Acácio S, Biswas K, O'Reilly CE, Mintz ED, Berkeley LY, Muhsen K, Sommerfelt H, Robins-Browne RM, & Levine MM (2013). Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet, 382 (9888), 209-22 PMID: 23680352

Šlapeta J (2013). Cryptosporidiosis and Cryptosporidium species in animals and humans: a thirty colour rainbow? International Journal for Parasitology, 43 (12-13), 957-70 PMID: 23973380  

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

Striepen B (2013). Parasitic infections: Time to tackle cryptosporidiosis. Nature, 503 (7475), 189-91 PMID: 24236315

Xiao L, Ryan UM, Fayer R, Bowman DD, & Zhang L (2012). Cryptosporidium tyzzeri and Cryptosporidium pestis: which name is valid? Experimental Parasitology, 130 (3), 308-9 PMID: 22230707 

Wednesday, October 16, 2013

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

Wednesday, July 10, 2013

This Month In Blastocystis Research (JUL 2013)

The open access journal 'Tropical Parasitology' (published by the Indian Academy of Tropical Parasitology) has included a symposium on Blastocystis in their January-June (Vol. 3) issue (available here). The symposium comprises three papers; one is on "taxonomy, biology and virulence", the next is on genetic diversity and molecular methods for diagnosis and epidemiology, and the last one is on treatment controversies. I believe that it may take quite a while before these papers will appear in PubMed.

The first paper written by Drs Parija and Jeremiah sums up a few of the aspects related to (especially historical) taxonomic issues and very little on the actual biology of Blastocystis. Meanwhile, there is quite a substantial section on Blastocystis morphology. Regarding virulence, the authors mention the possibility that differences in virulence may be due to differences in subtypes, but that subtyping alone does not predict pathogenicity which in part may be due to varying levels of intra-subtype genetic variation. The authors also briefly mention some of the morphological and phenotypical observations that have been associated with 'pathogenic Blastocystis', such as the amoeboid stage, large cells, rough surface, slow growth rate, and increased binding to lectins. It is always interesting to speculate on such associations, but it must be kept in mind that results from in-vitro experiments may not necessarily reflect in-vivo situations.

One topic that keeps popping up in the literature - and also in two of the papers here in this symposium - is the possibility of 'amoebic forms' of Blastocystis being associated with symptomatic infection. This hypothesis was introduced in 2006 by Tan and Suresh, I believe; Scanlan (2013) speculated that amoeboid forms might be the nutrient acquiring form potentially selecting for bacterial virulence or certain bacterial communities through grazing; please go here for more thoughts from a previous blog post.

My own experience on Blastocystis morphology mainly stems from looking at cultures, and since we practically only get isolates from patients with gastrointestinal disease, I don't know what Blastocystis cultures from asymptomatic individuals look like. A dear colleague of mine - Marianne Lebbad, a brilliant Swedish parasitologist with many years in business - sent me the picture below (light microscopy of a faecal concentrate) and speculates that Blastocystis might be able to form groups/clusters of cells, maybe even with the ability to form a mono-layer on the surface of the gut mucosa? I've never observed the cluster formation in cultures, but then again, we have no idea of whether the stages seen in in vitro cultures (microaerophilic environment) are identical to the in vivo stages (strictly anaerobic), and exactly how Blastocystis lives and multiplies in the colon... Anyway, the idea of biofilm comes into mind. It would be nice to learn more from colleagues with a similar experience.

Light microscopy of Blastocystis apparently forming a cluster of cells; we wonder whether the cells are in fact 'glued' together and if so, how? Courtesy of Dr Marianne Lebbad.

Moving on to the next paper, this one was written by me and deals mostly with issues and developments within the field of diagnostics, molecular characterisation, and molecular epidemiology. The target audience comprises clinical microbiologists and those involved in Blastocystis epidemiology and genetic diversity research. Included is a table, which is basically a reproduction of the one included in the recent paper by Alfellani et al. (2013) displaying the distribution of subtypes in humans across different geographical regions. I hope that the open access feature of this paper will prompt even more researcher into Blastocystis epidemiology! At least it is currently listed on the site as 'popular'!

The third paper in the string is written by Drs Sekar and Shanthi. These authors put emphasis on the conspicuous lack of data on the metabolic processes of Blastocystis, making it difficult to establish how to best approach antibiotic intervention; we must anticipate that with more genomic and transcriptomic data analyses arriving within a foreseeable future we will soon know much more about this. They also reiterate what has been put forth by many, namely that differences in eradication may boil down to differences in drug susceptibility, which again may be due to a variety of reasons, including genetic diversity, which is extreme in Blastocystis.

According to these authors, 'therapy should be limited to patients with persistent symptoms subsequent to a complete work up for alternative etiologies'; at the present stage this appears sensible, although clinicians would probably appreciate a clearer definition of 'symptoms'!

The review goes through some of the drugs most commonly used for treating Blastocystis, including metronidazole, paromomycin and co-trimoxazole, but also includes a few data on the use of the probiotic Saccharomyces boulardii in attempts to eradicate Blastocystis. There is not very much on the mechanisms of drug action, - it's more like a summary of data coming out from different studies, including the few placebo-controlled ones.
Regarding co-trimoxazole (which is also known as 'Bactrim' or 'Septra') this drug combo is often administered to HIV-patients prophylactically against Pneumocystis. In a study of parasites in Danish HIV patients, only 6/96 patients were given co-trimoxazole (unpublished data); two of these patients had Blastocystis. Hence, one 'alternative' way of finding out about the efficacy of co-trimoxazole on Blatocystis is to test the stools from patients undergoing long-term Pneumocystis prophylaxis comparing these patients to a cohort not receiving Pneumocystis prophylaxis but otherwise similar.

I find it a bit peculiar though to go through a review on treatment data that does not at one single point mention the need for sensitive diagnostics when evaluating courses of treatment and the identification of carriers and non-carriers. Also, there are some passages which are quite difficult for me to follow, for instance p. 36, second column, bottom section.

I hope that this symposium will inspire some of our colleagues and contribute to an increased understanding of Blastocystis.



Parija SC & Jeremiah SS (2013). Blastocystis: Taxonomy, biology and virulence Tropical Parasitology DOI: 10.4103/2229-5070.113894
Stensvold CR (2013). Blastocystis: Genetic diversity and molecular methods for diagnosis and epidemiology Tropical Parasitology DOI: 10.4103/2229-5070.113896  

Sekar U & Shanthi M (2013). Blastocystis: Consensus of treatment and controversies Tropical Parasitology DOI: 10.4103/2229-5070.113901


Scanlan PD (2012). Blastocystis: past pitfalls and future perspectives. Trends in parasitology, 28 (8), 327-34 PMID: 22738855

Stensvold CR, Nielsen SD, Badsberg JH, Engberg J, Friis-Møller N, Nielsen SS, Nielsen HV, & Friis-Møller A (2011). The prevalence and clinical significance of intestinal parasites in HIV-infected patients in Denmark. Scandinavian Journal of Infectious Diseases, 43 (2), 129-35 PMID: 20936912  

Tan TC & Suresh KG (2006). Predominance of amoeboid forms of Blastocystis hominis in isolates from symptomatic patients. Parasitology Research, 98 (3), 189-93 PMID: 16323025

Friday, June 21, 2013

This Month In Blastocystis Research (JUN 2013)

Another paper in the string of publications coming out from the PhD study by Dr Alfellani (London School of Hygiene and Tropical Medicine) has just appeared in PubMed.

Dr Alfellani and his colleagues have done a great job in analysing a multitude of samples from humans, non-human primates and animals; I have previously blogged about their observations from studies of human and non-human primates. Moreover, they have surveyed available data in order to better discuss their own findings, and the work has contributed significantly to what today is known about the host specificity, genetic diversity, phylogeography and general molecular epidemiology of Blastocystis.

Alfellani's most recent paper is published in the journal Protist, and it deals with the 'Genetic Diversity of Blastocystis in Livestock and Zoo Animals'.

It is quite a large paper which includes a lot of new information and a comprehensive (and hopefully exhaustive) table summarising Blastocystis subtype data in all relevant hosts (humans, non-human primates, other mammals and birds).

I will highlight a couple of things from the paper:

1. Apart from reporting on virtually complete SSU rDNA sequences from a couple of subtypes for which entire SSU rDNA sequences have yet not been available, we also report on three novel subtypes. Until recently, we only knew about 14 subtypes (ST1-ST14), of which ST1-ST9 can be found in humans. Now, three additional subtypes have been identified; ST15 in artiodactyls (camel and sheep) and non-human primates (chimpanzee and gibbon), ST16 in kangaroos, and ST17 in gundis.

The Gundi (Ctenodactylus gundi) is a rodent living mainly in the deserts of Northern Africa. (Source)

2. Novel data arising from analysis of faecal samples from humans and animals in Sebha, Libya, strongly indicate that humans and animals in this area are infected by different subtypes: Humans appear to carry ST1, ST2, and ST3, while synanthropic animals (artiodactyls in this case) mostly have ST5 and ST10 infections, suggesting that livestock is not a major contributor to human Blastocystis infection.

To this end, there is growing evidence of quite a substantial degree of host specificity of Blastocystis.  Even when subtypes overlap between humans and animals, we have accumulating evidence that the strains found in humans and animals are different. This means that the hypothesis that animals constitute an important reservoir of human Blastocystis infections currently has very limited support. It is my clear impression that when a strain of ST6 or ST8 is detected in humans, this strain has most probably been transmitted from an animal source. But we very rarely see these subtypes in humans, at least in Europeans.

It will be extremely interesting to see how the universe of Blastocystis subtypes unfolds... by genetically characterising strains in humans and non-human hosts, we are building up a clearer picture of transmission patterns and evolutionary biology, including our adaptation to Blastocystis, and the parasite's adaptation to us and other hosts.

It is noteworthy that we are starting to see different subtypes in rodents. We have previously thought that generally, rodents were infected by ST4. But now we know that many rodents are not infected, and we also know that rodents may harbour subtypes other than ST4.

So,17 subtypes of Blastocystis are now known. We have probably only seen the top of the iceberg, since many host species have not yet been sampled from, and it is likely that we will see quite a few STs being identified in the nearest future. To this end it is necessary to have a consensus regarding the identification of novel subtypes. Along with the Protist paper we have uploaded a supplementary file (Appendix A, TXT format) with aligned reference sequences that can be used for phylogenetic analysis,  hoping that it will be useful to our colleagues. In a future blog post I will try to address the issues of identifying new subtypes more specifically.

ST15 is one of the more interesting subtypes since it appears to have quite a low host specificity - infecting both non-human primates and artiodactyls. Yet, we have come across it only now. ST15 and ST17 are remarkable in the way that they appear to be closer related to herptile and arthropod lineages, respectively, than to lineages from mammals.

Please note that virtually complete sequences of ST10, ST13, ST14, ST15, and ST17 analysed in the study have been released in GenBank just now.

Further reading:

Alfellani MA, Taner-Mulla D, Jacob AS, Imeede CA, Yoshikawa H, Stensvold CR, & Clark CG (2013). Genetic Diversity of Blastocystis in Livestock and Zoo Animals. Protist, 164 (4), 497-509 PMID: 23770574

Alfellani MA, Stensvold CR, Vidal-Lapiedra A, Onuoha ES, Fagbenro-Beyioku AF, & Clark CG (2013). Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Tropica, 126 (1), 11-8 PMID: 23290980

Alfellani MA, Jacob AS, Perea NO, Krecek RC, Taner-Mulla D, Verweij JJ, Levecke B, Tannich E, Clark CG, & Stensvold CR (2013). Diversity and distribution of Blastocystis sp. subtypes in non-human primates. Parasitology, 140 (8), 966-71 PMID: 23561720

Sunday, September 2, 2012

Bugs Galore!

After spending more than 8 years in clinical microbiology with special reference to parasitology, I’ve come to realise that it truly is a bug’s life! Use of nucleic acid-based methods such as PCR in routine clinical microbiology diagnostic labs have revealed that single-celled parasites are colonising the intestine of up to 50% of the Danish population! And so what? Well, this finding has several implications.

A couple of months ago I revisited Why it is a bugs life by Jörg Blech (The Guardian (2002)). Speaking of numbers, - I wonder which one is the most successful eukaryote in terms of numbers? Blastocystis? Dientamoeba? Or any other “Parasite sp.”? After realising that microscopy methods allow us to see only the very tip of the iceberg and after adding PCR to our routine diagnostics, we have found a few examples of “novel” parasitic species and many more may be in store for us. Morphologically identical organisms, such as those belonging to Iodamoeba bütschlii, may be found in both human and non-human hosts and may differ genetically across the nucelar small subunit rRNA gene by up to more than 30%! This is quite astonishing given the fact that the difference between human and murine small subunit rDNA is about 1%! Since these data have been established only recently, obviously no one knows the respective clinical significance of these morphologically similar but genetically very different lineages, and further studies may reveal differences in pathogenicity as seen in other amoebic genera. Blastocystis and Entamoeba coli are somewhat similar examples.

Our results reveal that faecal-oral transmission is much more common in Denmark - a highly industrialised country where drinking water comes from waterworks (i.e. no surface water supplies), where outbreaks even due to bacteria are scarce, and where authorities spend 1.2 billion DKK on food safety and control. Today, 90% of dwellings in Denmark (5.6m citizens) are connected to efficient sewage systems, and Denmark has more than 1,400 treatment plants to purify wastewater from households, businesses and institutions. But somewhere the chain pops off… Even in Denmark it is “bugs galore”, which means that faecal exposure is much more common that we would probably like to think. Intestinal protists (primarily Blastocystis and Dientamoeba) are telltales of exposure to faecal contamination and faecal-oral transmission.

In Denmark, 90% of dwellings are connected to efficient sewage systems, and the country has more than 1,400 treatment plants.

However, we might also learn to see these parasites as other types of indicators. In our experience Danish patients with inflammatory bowel disease (IBD) represent a cohort of people whose gut flora is remarkably different from that of other cohorts (patients with irritable bowel syndrome (IBS) and patients with non-IBD/non-IBS diarrhoea): Apparently IBD patients don’t harbour parasites. This can in part be explained by the fact that some IBD patients have had bowel resection, but even IBD patients with in intact bowel system are generally negative for parasites.

We know that in highly developed countries the prevalence of helminth infections has gone down over the past few decades due to improved hygiene measures, but maybe also due to other reasons, which have not been clarified, but as we have seen, many of us are still positive for one or more intestinal parasites. However, most IBD patients do not have any parasites at all. This correlates well with the hygiene hypothesis, and it may be so that not only helminths, but also amoebae, which are able to colonise our guts for months and even years, may be co-responsible for 1) preventing us from developing inflammatory bowel disease and other autoimmune diseases by immunomodulatory mechanisms, and 2) maintaining a sound intestinal flora and ecology. Or is it so that these protists are dependent on a certain gut ecology or gut flora in order to colonise our intestines for a longer period, and in this way, they can be seen as indicators of a certain gut microbiota? Do they have any modulatory functions or do they happen to "lead their own life"?

As a parasitologist and worshipper of most things eukaryotic, I was both pleased and disconcerted after leaving the MetaHIT conference in Paris in March. Pleased, since the stratification of people into enterotypes and correlation of enterotypes to disease phenotypes suited my naïve, B/W perception of the world, but disconcerted since all presentations and posters addressed only bacteria (and virus to a minor extent, - maybe one on archaea even?). But, how about intestinal yeasts and parasites? Where in the gene catalogues and pools of metagenomic data could I find information on eukaryotes? Nowhere. Which hopefully boils down to methodological limitations rather than absence of interest.

The concept of paving an avenue of new knowledge with metagenomics data is holistic in its approach, but it currently fails to encompass a common part of the intestinal microbiota, possibly due to methodological limitations. However, we are probably facing the imminent inclusion of eukaryotic data in metagenomic studies, and this will enable us to investigate the potential role of intestinal protists and maybe yeasts as biomarkers of certain enterotypes and maybe even disease or health phenotypes.

Further reading:

Stensvold CR, Lebbad M, & Clark CG (2012). Last of the human protists: the phylogeny and genetic diversity of Iodamoeba. Molecular biology and evolution, 29 (1), 39-42 PMID: 21940643

Stensvold CR (2012). Thinking Blastocystis out of the box. Trends in parasitology, 28 (8) PMID: 22704911

Thursday, July 19, 2012

Micro-Eukaryotic Diversity in The Human Intestine

While we’re currently being flooded by papers on the intestinal microbiome, we still have very few dealing with the intestinal “micro-eukaryome” (forgive me my "badomics", I should have known better after reading this piece by Dr Eisen).

Hamad et al., just published their work on “Molecular Detection of Eukaryotes in a Single Human Stool Sample from Senegal” in PLoS One. They used a panel of 22 broad-specificity eukaryotic primers on genomic DNA extracted directly from faeces, cloned PCR products and did a blast search of the resulting sequences. They found about 18 micro-eukaryotic species in this particular faecal sample, most of which were fungi, and only two of which were “parasites”, namely Blastocystis sp. (subtype not given) and Entamoeba hartmanni, a so-called non-pathogenic amoebic species.They used both culture and culture-independent methods (PCR directly on genomic DNA from faeces) for the detection of intestinal fungi.

The study is interesting for a number of reasons:

1) It is one of the few papers out there on micro-eukaryotic diversity in faecal samples (other ones are listed in the reading list below), and we still know very little about micro-eukaryotes' potential interaction with the host and their ecological niche.

2) Many fungal species were detected by cloning of PCR products obtained by various primer pairs. It is possible that many of these are fungi stemming from the environment and diet, and not actually fungi colonizing the intestinal tract of this person; indeed the primers were able to pick up eukaryotic DNA such as that from tomatoes and common hop, stemming from the person’s diet. This is also one of the draw-backs of studies of fungi in stool samples: Even for mycologists it may prove difficult to determine which fungi are likely to be colonisers rather than fungi in transit due to environmental exposure, including diet. Analysis of consecutive samples from the same individual(s) (similar to the approach by Scanlan and Marchesi (2008)) will assist in identifying which fungi are stable and probable colonisiers. Similar to other studies, the investigators highlight the disparate findings resulting from the use of culture-dependent and culture-independent analyses; culture may be a way of identifying which ones of the many fungi detected by PCR that are actual colonisers.

3) We still don’t know much about what to expect when we take an approach like this. In the present study, multiple primer pairs were put into use, and 11 primer pairs yielded PCR products. The primer pairs amplified products of different lengths (some of them covering the complete SSU rDNA (18S)), and large products can sometimes be difficult to amplify and/or sequence for a variety of reasons; also preferential amplification may be a limiting factor. What would sometimes be useful is an in-silico analysis of the spectrum of organisms covered – at least theoretically - by each set of primers. In the papers I’ve seen so far aiming to display the eukaryotic diversity in human stool, Blastocystis has been a consistent finding, while Dientamoeba fragilis, which, at least in Denmark is almost as prevalent as Blastocystis (in some cohorts even more prevalent) and can be seen in co-infection, has not been reported so far. When you are presented with a list like the one presented by Hamad et al., you are inclined to believe that this list is exhaustive, but I think in-silico analysis data on such broad-specificity primers used for the detection of eukaryotic DNA would help us validate the use of these primers. Another approach to test the applicability of this methodology is to construct samples of DNA from known organisms in different ratios... and then test how the primers and cloning perform. What is also important is the very method of DNA extraction... obviously, our ability to detect DNA from any organism relies on our ability to extract DNA from it.

4) The study of micro-eukaryotes and their roles in health and disease includes first and foremost knowledge about which species and lineages that can be found and which ones that are the most common. Molecular methods are needed to identify the organisms in our intestine, since for instance parasites that look the same (morphological identity) can be genetically diverse with differing abilities to cause disease. We know from studies of micro-eukaryotes in ruminants that for instance some ciliates can be directly beneficial to the host, while others - such as cryptosporidia - are virtually obligate pathogens causing watery diarrhoea. Moreover, some organisms, including micro-eukaryotes, may be extremely difficult to culture even short-term, and also microscopy has limitations.

While we are still searching for virulence genes and other effector proteins in common micro-eukaryotes such as Blastocystis and Dientamoeba fragilis which could potentially cause disease directly, we also need to look for more indirect effects. Although much lower in numbers than our bacteria, (some) micro-eukaryotes may predate on beneficial bacteria to an extent where dysbiosis is reached. "Defaunation" of the intestine is speculated to be associated not only with impaired absorption of nutrients, but also with the development of severe disesases such as colon cancer and if micro-eukaryotes are able to skew our flora, this may have indirect impact on our health; many of our commensal bacteria are essential to some of our vital body functions, - indeed our intestinal flora can be viewed as a separate organ (see previous blog posts).

In the era of "omics" and "ngs" tools, it is interessesting to see a paper on global microbiotic diversity using a "conventional" cloning and sequencing approach in 2012. It may be one of the last papers of its kind?

To sum up: it is clear that a healthy intestine may be populated by a variety of micro-eukaryotes and future studies of the structure and function of the intestinal microbiome including micro-eukaryotes will help us understand their role in health and disease.

Let me end this post by uploading an image depicting "A Tree of Eukaryotes" (including Blastocystis) from an excellent protist blog by a colleague - my rendition here is practically useless, but I hope it might tease you to go and look at it in detail on "Welcome to the Ocelloid" by Psi Wavefunction.

Further reading:

Hamad I, Sokhna C, Raoult D, & Bittar F (2012). Molecular detection of eukaryotes in a single human stool sample from senegal. PloS one, 7 (7) PMID: 22808282

Pandey PK, Siddharth J, Verma P, Bavdekar A, Patole MS, & Shouche YS (2012). Molecular typing of fecal eukaryotic microbiota of human infants and their respective mothers. Journal of biosciences, 37 (2), 221-6 PMID: 22581327

Scanlan PD, & Marchesi JR (2008). Micro-eukaryotic diversity of the human distal gut microbiota: qualitative assessment using culture-dependent and -independent analysis of faeces. The ISME journal, 2 (12), 1183-93 PMID: 18670396