This Month in Blastocystis Research (JAN 2016)

Three publications have caught my attention over the past month.

The first one is by my Turkish colleagues Kurt, Dogruman-Al, and Tanyüksel. They just published the paper "Eradication of Blastocystis in humans: Really necessary for all?" This title implies that treatment of Blastocystis is recommendable in some cases. The authors appear to acknowledge the view that treatment should be given to symptomatic carriers when all other causes of gastrointestinal symptoms have been rule out, - the popular 'last-resort' approach.

What I think is really useful and admirable is that the authors leave so many questions open/unanswered, despite the fact that they have been "in business" for so many years, representing some of the most avid Blastocystis researchers. It becomes clear from reading the paper that even in 2016, we still do not know how to eradicate Blastocystis from the intestine in those cases where we'd really like to try and do so. Importantly, the authors give examples of data supporting the fact that treatment failure may be due to failure of the drug to reach the parasite as well as treatment resistance. They also highlight the possibility that eradication of Blastocystis by antibiotic/anti-protozoal agents may be due to microbiota perturbation rather than a direct action on Blastocystis. I also very much appreciate the fact that the authors are embracing the necessity of studying Blastocystis in a parasite-microbiota-host context in order to be able to draw useful conclusions on its role in human health and disease.

Das and colleagues just published data on Blastocystis and subtypes of Blastocystis in IBS patients and controls in New Delhi, India. Using multiple traditional and DNA-based methods, they found that in their study material, the prevalence of Blastocystis was higher among patients with IBS than among healthy controls. It is not exactly clear how the controls were picked and what type of study population they represented. What I found really useful is the fact that they not only carried out subtyping of Blastocystis, but also identified subtype alleles. The subtypes and alleles found in the study were very similar to those found recently by Pandey et al. (2015) in Maharashtra, India.  Interestingly, it appears that only two subtypes are found in humans in India, namely ST1 and ST3. However, only two studies from India are available on subtypes in humans, to my knowledge, and so we need much more data to draw conclusions.

The last paper that I'm going to address is one by Zanzani and colleagues. When I read the abstract I almost dislocated my lower jaw from stupefaction: Studying the gastrointestinal parasitic fauna of captive non-human primates (Macaca fascicularis), they found a variety of protozoa and helminths, which is not surprising at all. Neither is it surprising that most macaques were positive for Blastocystis. Now, what really made my jaw drop was the fact their data on the subtypes found in the macaques challenged the host specificity of Blastocystis identified so far: They reported finding ST1, ST2, ST3, ST5, and ST7. And so, I had a closer look at the methods used to obtain data on subtypes. I take the liberty of questioning the data, since the authors report using a set of primers for amplification of Blastocystis DNA targeting the SSU rRNA gene, while using the STS primers developed by Yoshikawa et al. as sequencing primers! I guess that it is possible that the description of the methods was flawed (should have been picked up by the reviewer though), in which case I hope that an erratum will be developed and published.

References:

Das R, Khalil S, Mirdha BR, Makharia GK, Dattagupta S, & Chaudhry R (2016). Molecular Characterization and Subtyping of Blastocystis Species in Irritable Bowel Syndrome Patients from North India. PloS One, 11 (1) PMID: 26784888  

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

Pandey PK, Verma P, Marathe N, Shetty S, Bavdekar A, Patole MS, Stensvold CR, & Shouche YS (2015). Prevalence and subtype analysis of Blastocystis in healthy Indian individuals. Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases, 31, 296-9 PMID: 25701123  

Zanzani SA, Gazzonis AL, Epis S, & Manfredi MT (2016). Study of the gastrointestinal parasitic fauna of captive non-human primates (Macaca fascicularis). Parasitology Research, 115 (1), 307-12 PMID: 26374536  

Yoshikawa H, Wu Z, Kimata I, Iseki M, Ali IK, Hossain MB, Zaman V, Haque R, & Takahashi Y (2004). Polymerase chain reaction-based genotype classification among human Blastocystis hominis populations isolated from different countries. Parasitology Research, 92 (1), 22-9 PMID: 14598169

This Month In Blastocystis Research (JUL 2014)

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

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

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

Agua de panela (source).

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

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

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

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

Reference:

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

Klimeš V, Gentekaki E, Roger AJ, & Eliáš M (2014). A large number of nuclear genes in the human parasite Blastocystis require mRNA polyadenylation to create functional termination codons. Genome Biology and Evolution PMID: 25015079 

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

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

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