Showing posts with label taxonomy. Show all posts
Showing posts with label taxonomy. 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.

Literature:

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, 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.

References:

SYMPOSIUM

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

OTHER:

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

Thursday, April 26, 2012

What is Blastocystis?

Intestinal parasites of humans can be divided into mainly helminths ('worms' including cestodes, nematodes and trematodes), and single-celled eukaryotic organisms. Most single-celled intestinal parasites belong to one of four main groups:
  • Archamoebae or Amoeboids (e.g. Entamoba, Iodamoeba, Endolimax)
  • Ciliates (e.g. Balantidium)
  • Sporozoa (e.g. Cryptosporidium, Cyclospora, Cystoisospora; even microsporidia)
  • Flagellates (e.g. Giardia, Chilomastix, Enteromonas, Pentatrichomonas, Retortamonas, Dientamoeba (unflagellated flagellate!))
Traditionally, these four groups have been referred to as protozoa.

However, the most common, single-celled intestinal parasitic eukaryote, Blastocystis, does not belong in any of these four categories. Taxonomically, Blastocystis belongs to the heterogeneous group of Stramenopiles, which includes slime nets, diatoms, water moulds and brown algae. Most stramenopiles are free-living organisms. Blastocystis is an atypical stramenopile not only as this group is named for the straw-like tubular hairs on the flagella and sometimes the cell body - Blastocystis has no flagella and lacks any tubular hairs - but also due to its parasitic nature.

Often, Blastocystis is referred to as a 'protozoon', although 'protist' is more appropriate. Protists can be defined basically as any eukaryote that is not a plant, an animal or a fungus.

One of the closest relatives of Blastocystis identified to date is Proteromonas lacertae, a parasite of reptiles.

Interestingly, Proteromonas does have flagella and hairs on the cell body. For comparison, the image below shows Blastocystis (culture) - appearing almost amoeboid, only with very limited morphological hallmarks (note examples of binary fission and the eccentrically located nuclei and mitochondrion-like organelles).

Blastocystis is one of two Stramenopiles known to infect humans, the other being Pythium insidiosum, which has been associated with keratitis and dermatological lesions mainly in SE Asia.

Other organisms with close relation to Blastocystis include Karotomorpha, Cepedea, Protoopalina and Opalina.

For further information, please visit

Silberman, J., Sogin, M., Leipe, D., & Clark, C. (1996). Human parasite finds taxonomic home Nature, 380 (6573), 398-398 DOI: 10.1038/380398a0  

HOEVERS, J., & SNOWDEN, K. (2005). Analysis of the ITS region and partial ssu and lsu rRNA genes of Blastocystis and Proteromonas lacertae Parasitology, 131 (2), 187-196 DOI: 10.1017/S0031182005007596  

Kostka, M., Cepicka, I., Hampl, V., & Flegr, J. (2007). Phylogenetic position of Karotomorpha and paraphyly of Proteromonadidae Molecular Phylogenetics and Evolution, 43 (3), 1167-1170 DOI: 10.1016/j.ympev.2006.11.002

Friday, April 6, 2012

Why "Blastocystis sp." and not "Blastocystis hominis"?

Blastocystis identified in humans used to be referred to as "Blastocystis hominis". However, after the advanced use of nucleic acid-based tools in the 90s and 00s it became clear that

1) morphologically identical Blastocystis can be genetically extremely diverse
2) Blastocystis in humans comprises at least 9 species (or, perhaps more correctly, ribosomal lineages), 8 of which can be found in other animals as well.

This means that host origin is not a reliable indicator of organism identity.

Blastocystis appears to exhibit only moderate host specificity - at least at subtype level - , and until a more substantial sampling from various hosts has been carried out, we will have to go with "Blastocystis sp." followed by an appropriate subtype (ST) number (according to species/ribosomal lineage), e.g. "Blastocystis sp. ST3", which is one of the 4 subtypes commonly found in humans.

In order to make subtype analysis very easy, we have created a site (together with Keith Jolley, Oxford University), where a bulk of sequences can be assigned to subtype in few seconds. Single sequence entries are also possible.

To sum up: Blastocystis hominis is a misleading and currently an invalid taxon.

(Read more about this in our Blastocystis consensus paper from 2007 in Trends in Parasitology)