Brave New World

Using Blastocystis as an example, we have only recently realised the fact that conventional diagnostic methods in many cases fail to detect Blastocystis in faecal samples, which is why we have started using molecular diagnostics for Blastocystis. I was also surprised to realise that apparently no single drug can be used to treat Blastocystis, and that in fact we do not know which combo of drugs will actually consistently eradicate Blastocystis (Stensvold et al., 2010).

There will come a time - and it will be soon - where it will be common to use data from genome sequencing of pathogenic micro-organisms to identify unique signatures suitable for molecular diagnostic assays and to predict suitable targets (proteins) for chemotherapeutic intervention; in fact this is already happening (Hung et al., in press). However, despite already harvesting the fruits of recent technological advances, we will have to bear in mind that the genetic diversity seen within groups of micro-organisms infecting humans may be quite extensive. This of course will hugely impact our ablility to detect these organisms by nucleic acid-based techniques. For many of the micro-eukaryotic organisms which are common parasites of our guts, we still have only very little data available. For Blastocystis, data is building up in GenBank and at the Blastocystis Sequence Typing Databases, but for other parasites such as e.g. some Entamoeba species, Endolimax and Iodamoeba, we have very little data available. We only recently managed to sequence the small subunit ribosomal RNA gene of Iodamoeba, and we demonstrated tremendous genetic variation within the genus; it is now clear that Iodamoeba in humans comprises a species complex rather than "just" Iodamoeba bütschlii (Stensvold et al, 2012).

Cysts of Iodamoeba

Ribosomal RNA is present in all living cells and is the RNA component of the ribosome. We often use this gene for infering phylogenetic relationships, i.e. explaining how closely or distantly related one organism is to another. This again assists us in hypothesising on transmission patterns, pathogenicity, evolution, drug susceptibility and other things. Since ribosomal RNA gene data are available for most known parasites, we often base our molecular diagnostics on such data. However, the specificity and sensitivity of our molecular diagnostic assays such as real-time PCRs are of course always limited by the data available at a given point in time (Stensvold et al., 2011). Therefore substantial sampling from many parts of the world is warranted in order to increase the amount of data available for analysis. In terms of intestinal micro-eukaryotes, we have only seen the beginning. It's great to know data are currently builiding up for Blastocystis from many parts of the world, - recently also from South America (Malheiros et al., 2012) - but the genetic diversity and host specificity of many micro-eukaryotes are still to be explored. It may be somewhat tricky to obtain information, since conventional PCR and sequencing offer significant challenges in terms of obtaining sequence data; such challenges can potentially be solved by metagnomic approaches - today's high throughput take on cloning; however, although the current next generation sequencing technology hype makes us feel that we are almost there, it seems we still have a long way to go - extensive sampling is key!

Cited literature:

Hung, G., Nagamine, K., Li, B., & Lo, S. (2012). Identification of DNA Signatures Suitable for Developing into Real-Time PCR assays by Whole Genome Sequence Approaches: Using Streptococcus pyogenes as a pilot study Journal of Clinical Microbiology DOI: 10.1128/JCM.01155-12

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

Stensvold, C., Lebbad, M., & Clark, C. (2011). Last of the Human Protists: The Phylogeny and Genetic Diversity of Iodamoeba Molecular Biology and Evolution, 29 (1), 39-42 DOI: 10.1093/molbev/msr238  

Stensvold, C., Lebbad, M., & Verweij, J. (2011). The impact of genetic diversity in protozoa on molecular diagnostics Trends in Parasitology, 27 (2), 53-58 DOI: 10.1016/j.pt.2010.11.005

Stensvold, C., Smith, H., Nagel, R., Olsen, K., & Traub, R. (2010). Eradication of Blastocystis Carriage With Antimicrobials: Reality or Delusion? Journal of Clinical Gastroenterology, 44 (2), 85-90 DOI: 10.1097/MCG.0b013e3181bb86ba

Blastocystis network on Facebook

This blog includes everything from updates on Blastocystis research, paper evaluations, polls, links, lab SOPs, to network opportunities and social interaction suggestions for all of us interested in Blastocystis. This time I want to guide your attention towards the Blastocystis network on Facebook. This is a good place to discuss personal experience with e.g. Blastocystis diagnosis and treatment and symptoms. The group is called "Blastocystis sp. (Blastocystis hominis and sp.)". If you have any experience and comments on Flagyl/Protostat (metronidazole), CDD regimens, including Secnidazole, Nitazoxanide, Furazolidone, Septrim (or Bactrim), Diloxanide Furoate, or other agents, please look up the group and share... We need your experience and views.

Blastocystis: To Treat or Not to Treat...

This year, Coyle et al. published a Clinical Practice paper in Clinical Infectious Diseases, a journal with a 5-year impact factor of almost 8. It is still difficult to get papers on Blastocystis published in clinical, peer-reviewed journals of major impact, probably due to the fact that evidence of Blastocystis' pathogenicity is so far only indicative, so it is great to see that the authors have managed to get their manuscript past those iron doors!

A few issues have come to my attention. When reading the abstract the reader will get the impression that subtypes are synonymous with genotypes, which is not the case. In the case of Blastocystis, a subtype is equivalent to a species; one of the reasons why we haven't allocated species names to Blastocystis from humans, other mammals and birds yet, is that we do not have sufficient data on genetic diversity and host specificity to come up with relevant names.

It says in the first page (pdf) that Blastocystis subtype (ST) 3 is found only in humans, which is not true. This subtype is common in non-human primates and can be seen in other, larger animals, including dogs, and also birds, if I remember correctly. However, so far, we only have multilocus sequence typing data from human and non-human primates, and these data indicate that ST3 found in non-human primates is often different from ST3 found in humans.

The authors recommend that asymptomatic individuals with few cysts should not be treated. Then what about asymptomatic individuals with many cysts? Also, with the diagnostic short-comings of microscopy of faecal concentrates, the suggested cut-off at 5 organisms per visual field appears arbitrary and, in best case, fortuitous.

In the abstract, the authors state that metronidazole is the drug of choice, although they appear to be quite aware that this drug has limited effect in terms of eradicating Blastocystis. So, why is metronidazole the drug of choice? Blastocystis is a parasite lodged primarily in the large intestine, and therefore we must anticipate that metronidazole often fails to reach the the parasite in sufficient concentrations due to absorption proximally in the gut. Luminal agents, such as paromomycin, are probably more likely to work, maybe in combination with metronidazole, although we have had a case, where even this combination was not effective.

When reviewing studies of treatment, it is important to acknowledge that insensitive methods have been used to evaluate drug efficacy. Culture combined with PCR is in my opinion the best method available in this respect. I prefer adding culture to the test, since culture detects viable Blastocystis (as opposed to PCR which will detect both viable and non-viable cells). Future randomised controlled treatment studies should therefore use culture and PCR to identify carriers both pre- and post-treatment. Whether Blastocystis-positive stool post-treatment is due to recrudescence, resistance or reinfection is not easily evaluated, but some useful information can be achieved by multi-locus sequence typing of isolates pre- and post-treatment.

Literature cited:

Coyle CM, Varughese J, Weiss LM, & Tanowitz HB (2012). Blastocystis: to treat or not to treat... Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 54 (1), 105-10 PMID: 22075794  

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  

Stensvold CR, Smith HV, Nagel R, Olsen KE, & Traub RJ (2010). Eradication of Blastocystis carriage with antimicrobials: reality or delusion? Journal of clinical gastroenterology, 44 (2), 85-90 PMID: 19834337

Blastocystis Sequence Typing Home Page

Last year, we launched the Blastocystis Sequence Typing Home Page, which is a publicly accessible resource including two major facilities: 1) A sequence database and 2) An isolate database.
The databases cover both SSU-rDNA data and Multilocus Sequence Typing (MLST) data. For those interested in MLST, please visit this paper.The rest of this post will be about SSU-rDNA sequences.

The database has a BLAST function. Barcoding sequences (i.e. sequences which include the 500 5'-most bases in the SSU-rDNA) can be submitted individually or in bulks, and the output file will include information on subtype (ST) and allele. The number of alleles in ST3 is huge (currently n=38) compared to other subtypes, for which only 2-3 alleles have been identified (e.g. ST8). In case a sequence is submitted that is not similar to an allele already present in the database, I suggest that you do an individual sequence query, which enables the generation of an alignment, which will show you the polymorphism(s). In case a new allele is identified, I suggest that we submit this new allele to the sequence database.
We not only strongly encourage using this BLAST feature for quick and standardised subtype and allele identification, but also for submitting isolate data, i.e. barcode sequences with provenance data (data on host, symptoms, geographical origin, etc.); again this can be done by contacting the curator (me); please look up the site for more information.

Our goal is to produce a database which accommodates large sets of data that can be submitted to scrutiny by everyone. The isolate database currently holds almost 700 isolates with 118 unique alleles - I hope this can be expanded much, much more. Also, data extracts can be done at all times, and below is a random example of an extract from human and non-human data from France downloaded from GenBank:

The colours indicate different alleles in different hosts (see legend to the right). A file with all alleles in fasta format is available here. You can paste them into the search field here for a total list of alleles currently in the database; try clicking on a couple to familiarise yourself with the system... One of the things that we can see here is that alleles 34, 36, 37 (ST3) and allele 4 (ST1) are the most common alleles in humans in France. It may seem a bit confusing to speak of both subtypes AND alleles. However, alleles are a good proxy for MLST data, and hence, looking at alleles is useful, e.g. in terms of transmission studies.

There are many other ways of extracting and visualising data from the isolate database. For more information on barcoding, subtypes, alleles, and the databases, please do not hesitate to contact me. I emphasise that the database only works with sequences that include the barcode region; mutliple SSU-rDNA targets have been used for subtyping, but due to the fact that this database is based on barcode data, we recommend that subtyping be done by barcoding (see references).

Useful literature:

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  

Scicluna SM, Tawari B, & Clark CG (2006). DNA barcoding of Blastocystis. Protist, 157 (1), 77-85 PMID: 16431158

1,000 page views!

This blog was put up not even a month ago, and I'm happy to see that the blog has had more than 1,000 page views already! Main interest seems to come from the USA, Australia, Canada and the UK.
I celebrate the "anniversary" by enabling Google Translate - so now this blog should be available in multiple languages. Thanks for stopping by!

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

Blastocystis Facts Sheet

I've tried to summarise a few facts here:

• Blastocystis is a single-celled, microbial parasitic protist colonising mainly the large intestine of man and other mammals, birds, reptiles, and other animals, even insects.
• The parasite is extremely common in humans, and possibly the most common microbial non-fungal eukaryote in the human intestine. More than one billion people may be colonised.
• Blastocystis comprises many ribosomal lineages, most or all of which are comparable to separate species; they are currently known as subtypes (ST).
• Humans are common hosts of ST1, ST2, ST3 and ST4, whereas other subtypes such as ST6, ST7 and ST8 are seen occasionally. ST5 and ST9 are very rare in humans. 
• Almost all subtypes found in humans are also found in animals; however, zoonotic transmission is probably uncommon, at least for the most common subtypes (ST1—ST4).
• Most carriers do probably not experience more intestinal symptoms than the average individual.
• We do not know when to seek to eradicate Blastocystis and there are no valid treatment guidelines. The effect of metronidazole may be very limited.
•  ST3 is probably the most common subtype in humans.
• ST4 may be more much more common in Europe than outside Europe. 
• ST4 has been seen frequently in patients with different types of diarrhoea or other intestinal problems, but appears uncommon in healthy individuals.
• Blastocystis is best detected by (real-time) PCR and culture; conventional parasitological techniques have generally poor sensitivity.

·         Ongoing epidemiological studies seek to analyse subtype distributions in various cohorts, e.g. IBS patients and the background population. We also continuously explore the genetic variation and host specificity of Blastocystis. Genome studies seek to unravel virulence genes that may be involved in pathogenesis, but only the genome for ST7 has been sequenced so far.

Intestinal Symptoms

For over a century, the clinical significance of Blastocystis has puzzled medical doctors scientists. After realising the extensive genetic diversity in Blastocystis, one of the current main hypotheses is that Blastocystis subtypes differ in terms of clinical significance. In other words: Symptoms, such as diarrhoea or other intestinal upset, may be associated only with one or more subtypes, while other subtypes are strict commensals.

Blastocystis is very difficult to eradicate and colonisation is chronic. Do symptoms caused by potentially  pathogenic subtypes persist or do they develop initially only to diminish after host immunological adaptation? Do fluctuations in symptoms reflect fluctuations in parasite load? Such issues ire important when interpreting results generated from cross-sectional surveys of subtypes in various cohorts.
Moreover, intestinal symptoms are difficult to define. Diarrhoea may be defined by 3 stool passages per day or more, while many other symptoms can be very difficult to define, if at all possible. Irritable bowel syndrome (IBS) and - to some extent - food allergy may both be considered differential diagnoses of symptomatic Blastocystis infections.

IBS diagnosis is currently defined by the Rome III criteria, and there are at least three types of IBS, namely IBS with diarrhoea, IBS with constipation and IBS with a mixture of diarrhoea and constipation.

Symptoms may be experienced differently from person to person. While abdominal cramping is perceived mostly as a symptom and something unpleasant, flatulence may by many be seen as a sign of a "healthy tummy" (e.g. due to consumption of a high fibre diet), although "inconvenient". Some individuals may very well tolerate intermittent intestinal symptoms and do not consult their GPs or other health care professionals, while others may be much more sensitive to any changes in for instance stool patterns.

What some people do not realise is that many methods fail to detect Blastocystis. PCR and culture are the most sensitive methods, but are still only rarely used. Moreover, PCR is also suitable for the detection of Dientamoeba fragilis, which is a parasite often seen in co-infection with Blastocystis. These two parasites are probably the most common single-celled eukaryotes in the human intestine.

This means that complete and accurate microbiological make-ups are far from always performed. And so, incomplete microbiological examination coupled with differential diagnostic challenges, potential immunological adaptation and the very subjective components of symptom presentation renders our quest for clear-cut associations extremely challenging. Blastocystis will often be seen as the culprit of symptoms, possibly simply to the reason that it is the only potential microbial pathogen that has been demonstrated in a stool sample. Cohort studies using sensitive diagnostic methods for pathogen surveillance are expensive, but may be one of the few only ways forward with regard to epidemiological studies that can assist us in resolving the clinical significance of Blastocystis.

Blastocystis Subtyping in Routine Microbiology Labs

When I speak to colleagues in and outside Europe and visit research portals and social media, including Facebook groups, I get the impression that Blastocystis subtyping is something that is still very rarely done, despite the fact that most clinical microbiologists and biologists acknowledge that subtypes may differ in terms of clinical significance and in other respects. We get new data on Blastocystis subtypes in various cohorts from time to time from research groups around the world, but all reports are characterised by relatively small sample sizes and subtyping methodology has not yet been standardised. This type of research is moreover challenged by the fact that Blastocystis is common in healthy individuals (i.e. people not seeing their GPs for gastrointestinal problems), and this makes it extremely difficult to identify the subtype distribution in the "background" population.

Although we recommend barcoding (see one of my previous posts) as the subtyping method of choice, there is no "official report" identifying the Blastocystis subtyping gold standard. Therefore, I'm currently setting up a lab project that is going to help us compare the most common methods used for subtyping in order to identify the one most suitable. I emphasise that the best method used for subtyping is not the PCR that should be used for diagnostic purposes, mostly due to the fact that PCRs for subtyping amplify 300-600 bp, which are much longer amplicons than the one we go for in diagnostic PCRs (typically 80-100 bp). We therefore recommend our novel TaqMan-based real-time PCR for initial diagnosis, or culture, which is inexpensive and relatively easy and provides you with a good source of cells for DNA extraction.
I hope that we will be able to come up with some robust data soon that will allow us to recommend the most suitable approach and hope to publish our results in a clinical microbiology journal of high impact, and I hope that this will prompt Blastocystis subtyping in many labs. Once this report has been published, I intend to upload a protocol here at the site where lab procedures for diagnosis and subtyping will be described in detail. Stay tuned!

Blastocystis @ EzineArticles

I realise that output from my research ends up being read/noticed by only relatively few people compared to the number of individuals colonised by Blastocystis (!) This is due to the fact that research results are published in a language and context not always easily understood by laymen, and that few of my articles are available online free of charge.

This is part of why I created this blog. And  now I also have the pleasure of recommending a Blastocytis article on EzineArticles: Blastocystis - Everyone's Parasite?

Please share.