Showing posts with label epidemiology. Show all posts
Showing posts with label epidemiology. Show all posts

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

Thursday, May 9, 2013

YouTube Video on Blastocystis Subtyping

For those who want to venture into Blastocystis subtyping - the easy way - I've recorded and uploaded a video on YouTube fyi.




For even more information, please visit a selection of relevant blog posts here.

Monday, April 29, 2013

'Invasive Blastocystis' in ECCMID 2013

ECCMID - the annual European Congress of Clinical Microbiology and Infectious Diseases (hosted by ESCMID) is currently taking place in Berlin. This year, I'm not attending, but I've been scanning the abstract book for 'Blastocystis', and it appears that an oral presentation was scheduled for yesterday in the "Emerging Infectious Diseases" section:

First of all: it's great to see fellow researchers screening larger (i.e. hundreds) of faecal DNAs by PCR for Blastocystis. I wish more people would do that to produce reliable data on prevalence and subtypes.

Now, as I've already mentioned, there are currently mainly two methods in use for subtyping, barcoding and STS PCR, and recently I evaluated these. To cut a long story short, barcoding is recommended for subtyping, since the STS method, which was used in the study by Tarasova et al. (abstract), appears to miss the majority of ST4 strains (the major genotype), and moreover, no STS primers exist for ST8 and ST9 (or any of the other 8 subtypes identified to date, but which have only been found in animals). So, the subtype data found in this study should be interpreted with this in mind.

Importantly however, I'm not sure whether the authors used the original Yoshikawa STS terminology or the terminology acknowledged in our 2007 consensus.

First, let us assume that consensus terminology is used. Then it's surprising to find ST5 in human samples in the first place, and finding a ST5 prevalence of 45% in a cohort of humans included in a larger study like this is very unlikely based on current evidence of more than 3,000 observations from all over the world, where the overall prevalence of ST5 in humans is <1%. Also, finding so much ST6 is also really striking. Also, if the consensus terminology is used, then I'm a bit puzzled why the authors put emphasis on ST7 not being found, since ST7 is relatively rare in humans.

And so let us assume that consensus terminology was not used, and the original Yoshikawa terminology was used instead. This would translate into STs 4, 6, and 7 not being detected in the CVH group. Which makes sense, since ST6 is extremely rare (at least in Europe), ST7 is only seen on occasion, and, as I said, the majority of ST4 infections are likely to go undetected by the STS method. However, ST4 appears quite common in Europe, and I suspect that it should be quite common in St Petersburg as well. But then there is one thing that comes to my mind: If ST4 infections are common, then there should be a relatively large number of samples detected by PCR which were untypable by PCR...and there is no information on untypable positive samples in the abstract...
But what is more:  STS subtype 5 translates into ST2 in consensus terminology, and similarly STS subtype 6 equals ST5 (yes, it may seem confusing, but we have provided a table in the 2007 consensus paper to make this easy). This means that no matter which of the two terminologies were used, ST5 is seen in abundance in patients with CVH in St Petersburg! Which is a very remarkable observation, and maybe more interesting than the rest of the data, which  I, by the way, find a bit difficult to follow (I expected to learn something about Blastocystis invasion, when I read the title of the abstract, but there is no data or information on invasiveness... and I'm very curious as to how the authors managed to obtain such a high number of samples from 'healthy people'! To evaluate the prevalence of Blastocystis in the control group, demographic data are needed, and a prevalence as low as 5.3% among healthy individuals makes me suspect that this control group consisted of newborns/toddlers who generally have a low prevalence of Blastocystis). Also, since when was ST1 'zoonotic'?

Anyway, often conference abstract are previews of upcoming articles, and so I expect that there will be a paper out soon from this group, and hopefully these issues will be clarified. The occasional confusion in Blastocystis epidemiology could be reduced to a minimum if everyone got into using barcoding and the Blastocystis 18S subtyping site (and go here for a video introduction to Blastocystis subtyping).

Are some citizens of St Petersburg infected by Blastocystis sp. ST5, a subtype seen primarily in livestock and African apes? Source

References:
Tarasova E, Suvorova M, Sigidaev A, Suvorov A. Blastocystis invasion in patients with chronic viral hepatitis in Saint Petersburg. ECCMID 2013 abstract O338.

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

Stensvold CR (2013). Comparison of sequencing (barcode region) and sequence-tagged-site PCR for Blastocystis subtyping. Journal of Clinical Microbiology, 51 (1), 190-4 PMID: 23115257

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

Friday, April 26, 2013

This Month in Blastocystis Research (APR 2013)

I've been extremely bored all day writing up my evaluation of a (not so interesting) PhD thesis, and I thought I'd spice up my day by introducing a new series of posts on this blog inspired by so many other blogs, namely: This Month in Blastocystis Research! A place for me to go through some of the most recent papers on Blastocystis.

There is paper out by Gould and Boorom who look at the stability of Blastocystis surface antigen over time. They show that detection of Blastocystis by an immunofluorescense assay (IFA) is not hampered after1 year of storage of faecal material in formalin compared to results immediately after the sampling point. Detection of Blastocystis by IFA is something that is not often used (that's my impression, anyway), but makes sense in cases where laboratory analyses can be performed only weeks-months after sample collection (e.g. during field work), in which case samples need to be preserved. We usually, however, recommend storing faecal material in (70%) ethanol (in the relationship 1 part faecal sample to 4 parts of ethanol), where the sample is mixed with the ethanol initially by vortexing the tube (typically a 2 mL Eppendorf tube) for 5-10 min, and subsequently keeping the tubes away from light until further processing. Importantly, in contrast to formalin-fixed stool, ethanol-fixed stool can be made highly suitable for PCR by just washing the samples x3 in PBS prior to DNA extraction. An example of this methodology can be seen in our study of Blastocystis in members of the Tapirapé tribe in Mato Grosso, Brazil (go here for a free download).

I'd wish that Gould and Boorom had validated their findings by running a PCR on the samples too (specificity and sensitivity testing). The IFA assay was also used in a publication from 2010 by Dogruman-Al et al., who found a diagnostic sensitivity of the IFA assay of 86.7% compared to culture; also here, adding PCR would have been relevant to better determine the diagnostic qualities of the IFA assay.

I was lucky to be involved in field work in the Lao PDR in 2003 conducted by regional WHO authorities; preserving and analysing faecal samples for parasites by microscopy (Kato Katz) and - later - PCR was what we did!

Adding to the endless row of cross-sectional prevalence papers, there is an article out just now by Abdulsalam et al. (2013) on Prevalence, predictors and clinical significance of Blastocystis sp. in Sebha, Libya (free for download here). The study used culture (Jones' medium) as diagnostic modality and confirmed the existence of frequent asymptomatic carriage. The authors used questionnaire info and multivariate statistical analysis to identify risk factors for Blastocystis carriage among 380 individuals aged 1-75, and what I find really interesting is that they found that participants aged > 18 years were much more prone to having Blastocystis than participants < 18 years (P < 0.001). This is something that we see in Denmark too, and I'm currently trying to collect "sufficient proof"! Whether this is an age accumulation effect due to the chronicity of colonisation remains to be investigated. The authors also found that carriers were more likely to experience symptoms than those who were not carriers (P < 0.001), mainly abdominal pain (P < 0.001), but notably not diarrhoea (P = 0.117).
It's a pity that molecular data was not included the study from Libya. Incidentally, our group recently published subtype data from Sebha, Libya, and it appears that Blastocystis found in humans in Libya mainly belongs to ST1, whereas ST3 is often the most common subtype in most other countries, and what is more: ST4 appears virtually absent in Libya and the rest of Africa... But let's see: The investigators might have more data up their sleeve waiting to be published...

May I also again draw your attention to our recent paper on Blastocystis in non-human primates, in which we find that despite the fact that there is a great overlap of subtypes in human and non-human primates, it appears that ST1 and ST3 strains found in non-human primates differ genetically from those found in humans, indicating cryptic host specificity. We have additional data supporting the theory that Blastocystis in humans is a result of human-to-human transmission (anthroponotic) rather than animal-to-human (zoonotic) transmission. Which is really interesting, since the theory of zoonotic transmission of Blastocystis has been heavily (I dare not say purported, so I'll say) propagated. Having said that, I think we still need to look much deeper into barcoding of Blastocystis from pets and other synanthropic animals before we can make more poignant conclusions.

And, finally, yet another add for our recent review on Recent Development in Blastocystis Research!

Please note that I'm happy to take suggestions for future posts, and I'd also like to encourage guest blogging!

Suggested reading:

Abdulsalam AM, Ithoi I, Al-Mekhlafi HM, Khan AH, Ahmed A, Surin J, & Mak JW (2013). Prevalence, predictors and clinical significance of Blastocystis sp. in Sebha, Libya. Parasites & Vectors, 6 PMID: 23566585

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, 1-6 PMID: 23561720

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

Clark CG, van der Giezen M, Alfellani MA, & Stensvold CR (2013). Recent developments in Blastocystis research. Advances in Parasitology, 82, 1-32 PMID: 23548084

Dogruman-Al F, Simsek Z, Boorom K, Ekici E, Sahin M, Tuncer C, Kustimur S, & Altinbas A (2010). Comparison of methods for detection of Blastocystis infection in routinely submitted stool samples, and also in IBS/IBD Patients in Ankara, Turkey. PloS One, 5 (11) PMID: 21124983 

Gould R, & Boorom K (2013). Blastocystis surface antigen is stable in chemically preserved stool samples for at least 1 year. Parasitology research PMID: 23609598

Malheiros AF, Stensvold CR, Clark CG, Braga GB, & Shaw JJ (2011). Short report: Molecular characterization of Blastocystis obtained from members of the indigenous Tapirapé ethnic group from the Brazilian Amazon region, Brazil. The American Journal of Tropical Medicine and Hygiene, 85 (6), 1050-3 PMID: 22144442

Tuesday, April 9, 2013

Blastocystis in Non-Human Primates

If my recent blog post "Blastocystis aux Enfers" could be described as "Blastocystis meets Dante Alighieri", then this post might come across as "Blastocystis meets Sir David Attenborough" (with all due respect to both of these gentlemen!).

Non-human primates (NHPs) include apes (hominoids), Old World monkeys (cercopithecoids), New World Monkeys (ceboids) and prosimians such as lemurs. I have been so fortunate to be involved in a study of Blastocystis in NHPs; a study which was led by Dr Alfellani with several co-investigators, and which has just appeared online in the journal Parasitology (click here to be diverted to the the website - first view article section).

The study is the first of its kind aiming to provide a substantial insight into the host specificity of Blastocystis in NHPs and included subtype observations for 441 captive and free-living animals representing no less than 30 genera; most of the data were generated during the study, while sporadic observations from similar studies were also included.

It was a huge study with a lot of interesting information, and I will try and summarise some of the points here.

Apes such as bonobos, chimps, gorillas and orangutans were colonised by some of the most common subtypes in humans, namely ST1, ST2, and ST3, accounting for about 77% of the cases. Contrary to humans though, ST5 also appeared rather common, accounting for about almost 14% of the cases, and some of the gibbons studied had ST8. Interestingly, a chimp and a gibbon were found to be hosts of a new subtype, ST15.

Old World monkeys were studied to an even larger extent, and again, ST1, ST3 and ST2 predominated, accounting for about 95% of all cases of single subtype infection. Here ST5 was also seen (2%) but only in langurs/lutungs and vervet monkeys. Interestingly, ST8 was seen only in 1/226 cases. ST13 was found by colleagues in Tanzanian colobus monkeys (Petrasova et al., 2011), and 8% of the 226 cases represented mixed/unknown subtype infections.

Woolly monkey (Lagothrix lagotricha) (Source)

New World monkeys included in the study were mainly represented by woolly monkeys, and these were colonised first and foremost by ST8 (49%), but ST3, ST2, ST1 were also found. So was a single case of ST4, which in general appears to be surprisingly rare among NHPs.

A few observations on lemurs were included, and such animals appear to host a vast variety of subtypes with no particular predilection, hence ST1, ST2, ST4, ST8, ST10 and ST15.

Ring-tailed lemur (Lemur catta) (Source).

The most striking differences between humans and NHPs in terms of colonisation by Blastocystis subtypes is that humans are very rarely colonised by ST5, while this subtype appears common in apes and Old World monkeys. ST8 was seen only in arboreal apes and in woolly and howler monkeys, which are also tree-dwellers, and it is tempting to think that ST8 is found mainly in tree-dwelling NHPs; to my knowledge, ST8 has not been found in non-primate hosts, except for once in a bird. Human colonisation by ST8 has been demonstrated only very rarely, for instance in a Danish woman returning from holiday in Indonesia and in animal keepers. Conversely, ST4 is seen extremely rarely in NHPs, while very common in humans in some parts of the world, apparently especially in Europe. These clear discrepancies in subtype distribution in humans and NHPs may boil down to host specificity and/or apparent geographically restricted range of some subtypes.

Another striking observation was that cryptic host specificity exists in ST1 and ST3, meaning that ST1 and ST3 strains found in NHPs overall differ genetically from strains found in humans belonging to the same subtypes, adding support to our previous findings.This suggests that humans are generally colonised by other strains than those found in NHPs. It will be interesting to see, whether other types of hosts sharing these subtypes carry distinct, host-specific strains. While MLST is probably the best way of testing for this, a lot of information can be obtained simply by barcoding. Pets, for instance, may share subtypes seen in humans, and so barcoding of "pet blasto" may be one of the very interesting pathways to knowledge.

We found no evidence of those subtypes that we have nicknamed "avian subtypes", namely ST6 and ST7. In some parts of the world, these two subtypes do not appear uncommon in humans; in Denmark and Sweden, for instance, ST7 is seen on quite a few occasions. But, interestingly, both STs are apparently absent in NHPs.

Langurs - the front cover of one of my favourite books showcasing works by the magnificent Walton Ford.

Incidentally, there is a sequence in GenBank from a gorilla (JX159284) which possibly represents a novel subtype, which is related to reptilian Blastocystis, and so it appears that the host spectrum and diversity of Blastocystis in NHPs continues to unfold.

A recent study saw that faecal microbiomes of wild non-human primates co-vary with host species, hence reflecting host phylogeny. This was evidenced by higher intra-species similarity among wild primate species, which may reflect species specificity of the microbiome in addition to dietary influences. This may in part explain the differences in Blastocystis subtypes seen in different NHP host species, but it is also possible that differences in subtypes reflect differences in habitat (and thereby possibly exposure) or geographical differences in subtype distribution. Indeed Homo sapiens is host to a variety of subtypes, and while ST4 is common in Europe, it appears virtually absent in many other parts of the world. Likewise, the differences in the prevalence of ST8 may reflect differences in geographical distribution, habitat and diet (arboreal vs. ground) as well differences in host specificity.

The overall interesting thing here is the schism of exposure vs. host specificity.


Suggested reading:

Alfellani, M., Jacob, A., Perea, N., Krecek, R., Taner-Mulla, D., Verweij, J., Levecke, B., Tannich, E., Clark, C., & Stensvold, C. (2013). Diversity and distribution of Blastocystis sp. subtypes in non-human primates Parasitology, 1-6 DOI: 10.1017/S0031182013000255

Yildirim S, Yeoman CJ, Sipos M, Torralba M, Wilson BA, Goldberg TL, Stumpf RM, Leigh SR, White BA, & Nelson KE (2010). Characterization of the fecal microbiome from non-human wild primates reveals species specific microbial communities. PloS one, 5 (11) PMID: 21103066

Stensvold CR, Arendrup MC, Nielsen HV, Bada A, & Thorsen S (2008). Symptomatic infection with Blastocystis sp. subtype 8 successfully treated with trimethoprim-sulfamethoxazole. Annals of tropical medicine and parasitology, 102 (3), 271-4 PMID: 18348782

Stensvold CR, Alfellani M, & Clark CG (2012). Levels of genetic diversity vary dramatically between Blastocystis subtypes. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 12 (2), 263-73 PMID: 22116021

Monday, January 14, 2013

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:

Saturday, January 5, 2013

Where Are We On Blastocystis Subtypes?

As mentioned, Blastocystis exhibits remarkable intrageneric diversity, which is continuously being explored by us and our colleagues. We are convinced that the genus of Blastocystis comprises multiple species, but for now we call them "ribosomal lineages" or "subtypes" and allocate numbers to each subtype, hence ST1, ST2, etc. While the number of subtypes that can be found in humans remains stable, we and our colleagues are still expanding the subtype universe in non-human hosts (I will be blogging on this shortly).

Barcoding currently represents state-of-the-art in Blastocystis subtyping, and luckily this method appears to gain a foothold in labs across the world.

Nine subtypes have been found in humans, but some of them only on rare occasions. A recent study going out from London School of Hygiene and Tropical Medicine and led by Dr Alfellani and published just now in Acta Tropica looked at 356 Blastocystis sequences from samples from the UK and Libya, but also from sub-Saharan Africa, namely Liberia and Nigeria.


Tuesday, December 18, 2012

Blastocystis Highlights 2012

2012 is coming to an end and it is also time for taking stock of the year Blastocystis-wise. We saw many significant scientific papers, among them a paper by Poirier and colleagues, predicting a potential role for Blastocystis in irritable bowel syndrome (IBS), based on analysis of their recent genome data.They propose that Blastocystis is genetically armed with the equipment necessary to cause intestinal dysbiosis, and potentially IBS, which may be a cause of dysbiosis. Indeed, members of this group found that the Blastocystis genome encodes various proteases and hydrolases that, if secreted, may be involved with perturbations of the gut flora; however, we need transcriptional profiling or similar studies to find out, whether these enzymes are actually expressed. Some species of Entamoeba are also in possession of multiple "virulence genes", but for some species they apparently remain un-expressed, and most Entamoeba species are still considered harmless.


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