Showing posts with label subtypes. Show all posts
Showing posts with label subtypes. Show all posts

Thursday, December 6, 2018

Is this a new Blastocystis subtype? Maybe not! Here's Why!

The genetic diversity of Blastocystis is becoming comparable to the universe! Seventeen subtypes (which are likely separate species or even genera) have been acknowledged so far, but quite a few more have been mentioned.

However, before assigning new Blastocystis subtype numbers to your SSU rDNA sequences, you'd need to do some QC work on your data. Sometimes we notice sequences deposited in the NCBI Database or included in articles that may look like new Blastocystis subtypes.... but they're most likely not!

I asked Prof Graham Clark from London School of Hygiene and Tropical Medicine, who has more than 20 years' experience in the Blasto business, to give a couple of examples, explaining where issues may arise. He says:


'One of the tasks I do when I have a few minutes to spare is to look at new Blastocystis sequences that have been deposited into GenBank. I am always hoping to stumble across some exciting new subtypes or new hosts that will expand our understanding of diversity in Blastocystis. Only rarely does this happen, however. I do, occasionally, come across sequences that are problematic and it is these that I want to focus on.

Chimaeras: This problem occurs during PCR amplification when one primer binds to a Blastocystis subtype DNA and the other primer binds to a different source of DNA. In the first case I came across the other source was a different Blastocystis subtype, meaning that the sequence at one end of the PCR product matched one subtype and the sequence at the other end matched a different subtype. This observation is mentioned in the paper describing barcoding of Blastocystis (Scicluna et al, 2006). Since then I have seen other chimaeric sequences: one recently was a mixture of Blastocystis plus a plant while another was Blastocystis plus a free-living protist.
Chimaeras are produced when there is incomplete replication of a DNA strand during a cycle. After denaturation in the next cycle, the single stranded partial product can bind to another single stranded product from a different source and synthesis results in a product combining sequences from two sources. The conservation of ribosomal RNA genes means there can be sufficient similarity to allow binding between sequences from distantly related organisms.
Chimaeras are generally only found when the sequences are from cloned ribosomal RNA gene sequences obtained by PCR, although they also occur in some forms of Next Generation  Sequencing. When mixed PCR products are sequenced directly the sequence obtained is the average of all the products in that reaction, and so chimaera sequences will usually be ‘diluted out’ by the major product of the reaction. Only when a single sequence from that mixture is isolated and studied will chimaeras be detected.
If the ‘alien’ region makes up a significant percentage of the sequence then the result of BLAST analysis will show a percentage divergence from known subtypes that indicates it may represent a new subtype. A quick way to evaluate this is to compare the BLAST results using the first and last thirds of the sequence. If it is a new subtype the results should be similar. In a recently detected chimaera, the first third was a 100% match to a known Blastocystis subtype while the last third was a 95% match to asparagus. This approach is an easy way to check whether there is something to get excited about.
A chimaera sequence can sometimes be detected because of its impact on phylogenetic trees. The sequence will be on its own branch, often at the base of a clade containing the subtype found at the Blastocystis-matching end.

Non-Blastocystis Blastocystis sequences: Like chimaeras these are often PCR artefacts, most commonly encountered when amplifying from stool DNA, especially if the stool is non-human. There is an expectation that Blastocystis-specific primers will only amplify Blastocystis DNA but, sadly, that is not always the case. I have personally seen this many times - if Blastocystis DNA is a minority of the eukaryotic DNA in the sample then the likelihood of artefacts increases greatly. These are generally identified easily if the sequence is compared using BLAST against the full nr/nt nucleotide collection in GenBank. However, there is a temptation to limit the search to the genus Blastocystis to speed up the identification process, because that is what you expect it to be. Again because of the conservation of ribosomal RNA genes, if ribosomal RNA genes are amplified there will be a match to Blastocystis, and the divergence will likely suggest, again, a new subtype.  Comparing against the full nucleotide collection will always show whether the sequence is of Blastocystis origin.

Both chimaeras and non-Blastocystis products are easily identified if the correct steps are taken. In conclusion, be suspicious of anything that is significantly divergent to known Blastocystis – it could be an indication of an artefact.'
Fig. 1. A 'Blastaragus' (a chimaera of a Blastocystis and an asparagus)

Fig. 2. An example of a chimaeric DNA sequence (the 'Blastaragus' from Fig. 1). Notice how the consensus sequence starts out as Blastocystis ST14, shifts to asparagus, and then shifts back again to Blastocystis ST14.



I thank Graham, and I really hope that this information will be picked up by many of our colleageus. And please share! Research into Blastocystis is rapdily expanding, and we should all take on the responsibility of QCing our data.

Thanks for listening!

By the way... if you're interested in tutorials on Blastocystis subtyping from our recent workshop in Colombia, please look up Workshop Session 4 in the manual available at this link. 

Hope to be back before Christmas!

Thursday, November 10, 2016

This Month in Blatstocystis Research (OCT 2016)

A few things to highlight:

I'm very pleased to announce the Special Issue on Blastocystis recently appearing in Parasitology International - go here for the list of contents. The papers included in this issue represent the breadth of the contributions made to the 1st International Blastocystis Symposium, which took place last year in Ankara. A couple of review and opinion articles written by members of the Scientific Committee are accompanied by several articles outlining original research findings that were presented at the symposium. This special issue is particularly useful for younger researchers who wish to familiarise themselves with some of the methods that are currently in use in surveys of Blastocystis.
Readers should not expect to find articles on Blastocystis in a microbiota context; nor should they expect to see data from seminal studies that challenge the view that Blastocystis is a possible pathogen. Nevertheless, there is an interesting opinion paper with the title "Eradication of Blastocystis in humans--really necessary for all?"

Led by Dr Alison Jacob and Dr Graham Clark, London School of Hygiene and Tropical Medicine, our group just published an article on a comparative study of Blastocystis mitochondrial genomes. In general, mitochondrial genomes differ vastly in length, structure, and gene content across organisms, and by studying these genomes it has been possible to develop hypotheses on how these organisms have evolved including the adaptive/non-adaptive processes involved in shaping organismal and genomic complexity. Unlike most anaerobic eukaryotes, Blastocystis does not have true mitochondria but has mitochondrion-related organelles (MROs; also referred to as mitochondrion-like organelles [MLO]) that contain a genome. In the study in question, we sequenced and compared mitochondrial genomes from subtypes 1, 2, 3, 4, 6, 7, 8, and 9. All of them have the same genes in the same order, but two curiosities were noted. One gene, called orf160, as stop codons near the beginning of the coding region in most subtypes. A second gene, coding for ribosomal protein S4, lacks a start codon in some subtypes.
In both cases, these characteristics would normally prevent a gene from being expressed, but because these genes are otherwise conserved and most of the gene is 'intact', it seems likely that the genes are functional. Ribosomal protein S4 is considered an essential component of the ribosome needed for protein synthesis in the organelle. How the genes are expressed to produce functional proteins remains a mystery, - just one more peculiarity of Blastocystis!

In the growing pool of articles exploring relationships between intestinal parasites and gut microbiota, I was pleased to discover an article by Iebba et al. (2016) on "Gut microbiota related to Giardia duodeanlis, Entamoeba spp. and Blastocystis hominis infections in humans from Côte d'Ivoire". In this observational study, the authors used qPCR to detect groups of bacteria that are indicative of dysbiosis vs eubiosis, dysbiosis being a perturbed, imbalanced microbiota and eubiosis being a healthy, balanced gut microbiota. The authors found that individuals with Blastocystis and Entamoeba were characterised by eubiosis, while individuals with Giardia were characterised by dysbiosis. It says that samples (n = 20) were randomly chosen, but even so, the number of samples tested was low, and care should be taken when interpreting the results. The overall approach, however, is interesting, and somewhat resembles the work that we have been doing in our lab (ref). I also recently blogged about another study with a similar aim (go here to view the post).

I would also like to bring your attention to the EMBO Conference "Anaerobic protists: Integrating parasitology with mucosal microbiota and immunology", which will take place in Newcastle upon Tyne, UK in Aug/Sep 2017 (image). I will be there doing my best to deliver a stimulating talk on current knowledge and advances in Blastocystis and Dientamoeba research. You can visit the conference website by folloing this link

References:

Dogruman-Al F, Stensvold CR, & Yoshikawa H (2016). Editorial - PAR INT - special issue on Blastocystis. Parasitology international, 65 (6 Pt B) PMID: 27742000

Iebba V, Santangelo F, Totino V, Pantanella F, Monsia A, Di Cristanziano V, Di Cave D, Schippa S, Berrilli F, & D'Alfonso R (2016). Gut microbiota related to Giardia duodenalis, Entamoeba spp. and Blastocystis hominis infections in humans from Côte d'Ivoire. Journal of infection in developing countries, 10 (9), 1035-1041 PMID: 27694739

Jacob AS, Andersen LO, Pavinski Bitar P, Richards VP, Shah S, Stanhope MJ, Stensvold CR, & Clark CG (2016). Blastocystis mitochondrial genomes appear to show multiple independent gains and losses of start and stop codons. Genome biology and evolution PMID: 27811175

Smith DR (2016). The past, present and future of mitochondrial genomics: have we sequenced enough mtDNAs? Briefings in functional genomics, 15 (1), 47-54 PMID: 26117139

Saturday, January 30, 2016

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