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.

Although subtype data are now available from almost 30 countries, there are still major regions where extremely little sampling has been done, e.g. sub-Saharan Africa and the Americas. Despite this, we can already now see a picture building up of variable geographic distribution of Blastocystis subtypes. The most striking thing is that ST4 is common in Europe, but remains uncommon or virtually absent in most other parts of the world.

The distribution of subtypes across different geographical regions is seen above and based on 3,171 observations. Clear differences can be seen, and further sampling will reveal whether these differences are as pronounced as seen here. It should also be noted that this data summary does not take any differences in clinical phenotypes into account, nor data on demographics such as age, gender, and exposures simply due to the reason that such information is not consistently available.

Until now, only ST1, ST2 and ST3 have been found in humans in the entire American continent. This is different, from many European countries, where especially ST4 but also to a certain extent ST7 are quite common. Moreover, ST1 appears to be the most common ST in America, while ST3 is the most common ST in most other regions. ST2 has a far greater prevalence in America than in other regions. However, only three American countries have been sampled (USA, Colombia and Brazil), and the American data only make up 3% of the total data.

Also in sub-Saharan Africa, where three countries have been sampled so far (Liberia, Nigeria and Tanzania), ST1 and ST3 predominate, while ST2 and ST4 are much less common. Again, there are no observations on other subtypes in humans in these regions, and the data make up less than 2% of the entire data set.

There is one major cave-at in all of this: Many studies have used subtype-specific primers, the so-called STS primers, for subtyping. In my recent comparison of the STS method with the barcoding method, I pointed out several things, among these:

1) STS primers are insensitive in terms of picking up ST4. This means that ST4 may go undetected in studies using the STS method. So, it remains to be seen whether the absence of ST4 in some regions is indeed factual or due to inappropriate methodology. Interestingly, ST4 appears to be more common among patients with intestinal symptoms than asymptomatic individuals.

2) There are no STS primers for subtypes other than ST1-ST7, so any ST8s or ST9s will not be found in studies using the STS method.

Therefore: Use barcoding!
Barcode data can be submitted to subtype calling using our 18S database - it's quick, simple, completely standardised and obviates the need for phylogenetic reconstructions. Moreover, submitting your barcode sequences to the 18S database gives you the 18S allele and thereby adds further resolution to your data. However, in the event that you have a new subtype or allele, no match will be found in the database, and therefore 1) phylogenetic analysis should be performed, and 2) the sequence should be submitted to the 18S data base for reference (please contact me, and I'll help you do it).

Insight from subtype analysis of various cohorts of people and animals across different geographical regions is extremely useful in our quest to identify transmission dynamics and whether clinical outcome of Blastocystis carriage is dependent on subtype. As I mentioned in my previous post further data from Blastocystis genomics and transcriptomics will be available in the next couple of years and this will probably enable us to identify whether differences in potential virulence between subtypes can be identified.

Our paper appearing in Acta Tropica moreover addresses some other interesting points, for instance subtypes detected in IBS patients and control groups in various countries; I plan to deal with this topic in a separate blog post, but if you are curious then have a look at the paper (see reference section for details).

So, where are we on Blastocystis subtypes? Well, as mentioned here there is a strong indication of geographical variation in subtype distribution. Moreover, there are indications of differences in clinical significance. The extent of animal-to-human transmission is not known, but may be strongly overestimated, at least for common subtypes; only studies using highly discriminatory molecular markers can assist us in reaching conclusions.

Incidentally, if you missed out on the 2012 Blastocystis highlights, - look up my previous post!


Alfellani, M., Stensvold, C., Vidal-Lapiedra, A., Onuoha, E., Fagbenro-Beyioku, A., & Clark, C. (2013). Variable geographic distribution of Blastocystis subtypes and its potential implications Acta Tropica DOI: 10.1016/j.actatropica.2012.12.011

Domínguez-Márquez, M., Guna, R., Muñoz, C., Gómez-Muñoz, M., & Borrás, R. (2009). High prevalence of subtype 4 among isolates of Blastocystis hominis from symptomatic patients of a health district of Valencia (Spain) Parasitology Research, 105 (4), 949-955 DOI: 10.1007/s00436-009-1485-y

Stensvold, C. (2012). Comparison of Sequencing (Barcode Region) and Sequence-Tagged-Site PCR for Blastocystis Subtyping Journal of Clinical Microbiology, 51 (1), 190-194 DOI: 10.1128/JCM.02541-12

Stensvold, C., Alfellani, M., & Clark, C. (2012). Levels of genetic diversity vary dramatically between Blastocystis subtypes Infection, Genetics and Evolution, 12 (2), 263-273 DOI: 10.1016/j.meegid.2011.11.002

Stensvold, C., Christiansen, D., Olsen, K., & Nielsen, H. (2011). Blastocystis sp. Subtype 4 is Common in Danish Blastocystis-Positive Patients Presenting with Acute Diarrhea American Journal of Tropical Medicine and Hygiene, 84 (6), 883-885 DOI: 10.4269/ajtmh.2011.11-0005

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