Please watch this video abstract co-authored by one of my colleagues, Mark van der Giezen, about the search for suitable drug targets in Blastocystis.
The whole paper can be found here.
Friday, October 26, 2012
Video Abstract on Blastocystis Paper on Search for Drug Targets
Please watch this video abstract co-authored by one of my colleagues, Mark van der Giezen, about the search for suitable drug targets in Blastocystis.
The whole paper can be found here.
The "Flagyl" Poll
For some reason the "Flagyl" poll in the right side bar of this blog was reset; the number of votes was approaching 100. The question was
The interesting thing is that there was a tie between "no improvement" and "transient improvement", and although this poll could have been heavily biased in numerous ways, it is still completely in line with our experience: Many patients report transient alleviation of symptoms, while others have no clinical benefit from Flagyl. Flagyl is an antibiotic targeting a wide range of bacteria and single-celled parasites. It is sometimes successful in terms of eradicating Dientamoeba fragilis, one of the most common parasites in the human intestine, and a parasite which may cause symptoms especially in children (we are currently conducting a randomised control clinical trial at Statens Serum Institut to explore clinical and microbiological effect of metronidazole treatment of children with D. fragilis).
Many people will get diagnosed with Blastocystis without knowing whether they might also be positive for D. fragilis (and vice versa). It is a complex situation, since both parasites are common, they are difficult to detect unless you use PCR or other specialised analyses, and in most labs they are not tested for on a routine basis. And if they happen to be part of the panel of organisms that is tested for, it may be so that insensitive methods are used for their detection, which means that only a fraction of the cases will be detected. So, this is a bit of a conundrum in itself!
So, it's not easy to know what causes the temporary alleviation in some patients. Is it due to parasite recrudescence? Is it due to parasite eradication with subsequent re-infection? And which parasite? Blastocystis? Dientamoeba? Any others? Or, is it due to perturbation of the intestinal flora in a "positive" direction, which is then gradually going back to normal? Placebo effect? There are possibly many more explanations...
However, deep sequencing of faecal samples pre- and post treatment of parasite-positive patients will probably answer many of our questions...
Literature:
Engsbro AL, Stensvold CR, Nielsen HV, & Bytzer P (2012). Treatment of Dientamoeba fragilis in Patients with Irritable Bowel Syndrome. The American journal of tropical medicine and hygiene PMID: 23091195
"For those who have received metronidazole (Flagyl or
Protostat) treatment for Blastocystis, please indicate whether you
experienced no, transient or permanent improvement (or none of the above)"
The interesting thing is that there was a tie between "no improvement" and "transient improvement", and although this poll could have been heavily biased in numerous ways, it is still completely in line with our experience: Many patients report transient alleviation of symptoms, while others have no clinical benefit from Flagyl. Flagyl is an antibiotic targeting a wide range of bacteria and single-celled parasites. It is sometimes successful in terms of eradicating Dientamoeba fragilis, one of the most common parasites in the human intestine, and a parasite which may cause symptoms especially in children (we are currently conducting a randomised control clinical trial at Statens Serum Institut to explore clinical and microbiological effect of metronidazole treatment of children with D. fragilis).
Many people will get diagnosed with Blastocystis without knowing whether they might also be positive for D. fragilis (and vice versa). It is a complex situation, since both parasites are common, they are difficult to detect unless you use PCR or other specialised analyses, and in most labs they are not tested for on a routine basis. And if they happen to be part of the panel of organisms that is tested for, it may be so that insensitive methods are used for their detection, which means that only a fraction of the cases will be detected. So, this is a bit of a conundrum in itself!
So, it's not easy to know what causes the temporary alleviation in some patients. Is it due to parasite recrudescence? Is it due to parasite eradication with subsequent re-infection? And which parasite? Blastocystis? Dientamoeba? Any others? Or, is it due to perturbation of the intestinal flora in a "positive" direction, which is then gradually going back to normal? Placebo effect? There are possibly many more explanations...
However, deep sequencing of faecal samples pre- and post treatment of parasite-positive patients will probably answer many of our questions...
Literature:
Monday, October 8, 2012
Additional Comments on Blastocystis Treatment
I want to thank for the many emails I get! Unfortunately, I
cannot respond to each one of them, in part due to time limits, in part since some
of them are a bit off my topic or very difficult to answer. However, a few words on Blastocystis
treatment (again!), which will hopefully satisfy some of the readers:
Differences in the reported efficacy (microbiological and
clinical cure) of certain drugs or drug combinations may be due to one or more
of the following:
1) Actual differences in efficacy due to differences in pharmacokinetics,
and -dynamics. Some drugs used for treatment of intestinal parasites are
absorbed quickly from the intestine, while others are practically not absorbed
at all (but stay in the intestinal lumen). For instance: Metronidazole is absorbed almost 100% in the proximal part of the intestine and may very well fail to reach Blastocystis, which resides is in the large intestine.
2) Different methods are used for evaluating treatment
efficacy. If insensitive methods are used, the efficacy of any drug will be
overestimated. Culture in combination with PCR is clearly advantageous in terms
of evaluating microbiological efficacy since it will detect viable cells (see previous blog posts).
3) Drugs used in Blastocystis
treatment may have broad spectrum antibiotic activity (e.g. metronidazole) and
thus affect the surrounding microbiota, which again may influence the ability
of Blastocystis to continue
establishment. Hence indirect drug actions may play a role too.
| Could vegetables contribute to Blastocystis transmission? |
4) Diet. What types of food do we eat? I notice that some people
undergoing treatment for “blastocystosis” are cautious about eating carbs, for
instance, and turn to vegetables only or at least non-carb diets, thinking that
by cutting out carbs, they will cut off the "power supply" to Blastocystis. I’m not sure that this
approach is very effective and it’s also important to acknowledge that the
processing and metabolism of the foods that we ingest are complex. I hope to be able to do a blog post once on short-chain fatty acids, for instance. Again, changes in
our diets may influence our bacterial flora which again may have an impact on Blastocystis.
Importantly, we don’t know much about potential transmission of Blastocystis from raw vegetables and
whether this could be a potential source infection (vegetables contaminated
with Blastocystis).
5) Which leads to the next issue: The issue of re-infection. With so many people infected by Blastocystis (probably between 1-2 b people) it is likely that many of us are often exposed to the parasite. If we receive treatment but are not cut off from the source of infection, microbiological and clinical cure will be short-lived if at all possible.
5) Which leads to the next issue: The issue of re-infection. With so many people infected by Blastocystis (probably between 1-2 b people) it is likely that many of us are often exposed to the parasite. If we receive treatment but are not cut off from the source of infection, microbiological and clinical cure will be short-lived if at all possible.
6) Compliance - some drugs have serious adverse effects, and
so, failure to reach microbiological cure may stem from failure to comply with
drug prescriptions.
7) Differences in drug susceptibility. There is evidence from in vitro studies that Blastocystis subtypes exhibit differences in drug susceptibility.
7) Differences in drug susceptibility. There is evidence from in vitro studies that Blastocystis subtypes exhibit differences in drug susceptibility.
In the absence of sound data that take all of the above factors into account, it is not possible for me (or anyone) to predict exactly which drug (combo) that will work and which will not. I think that it is important that GPs or specialists who take an interest in treating Blastocystis collaborate with diagnostic labs that are experts on Blastocystis diagnostics. If any drug or drug combo enabling microbiological cure can be identified, such pilot data can be used to design randomised controlled treatment studies that again will assist us in identifying whether Blastocystis eradication leads to clinical improvement.
I will try and provide some thougths on other future directions for Blastocystis research soon. Stay tuned!
I will try and provide some thougths on other future directions for Blastocystis research soon. Stay tuned!
Friday, September 28, 2012
Brazilian Society of Protozoology - 2012 meeting
It's time to bone up on my Portuguese! Off to
XXVIII Reunião Anual da Sociedade Brasileira de Protozoologia
in Caxambu, Brazil tomorrow.
Giving keynote lecture on 3rd of October. Title of talk: "Blastocystis - friend or foe?"
The lecture is mainly based on thoughts presented in my recent paper: "Thinking Blastocystis Out of The Box" (PMID: 22704911) and output from our most recent studies.
XXVIII Reunião Anual da Sociedade Brasileira de Protozoologia
in Caxambu, Brazil tomorrow.
Giving keynote lecture on 3rd of October. Title of talk: "Blastocystis - friend or foe?"
The lecture is mainly based on thoughts presented in my recent paper: "Thinking Blastocystis Out of The Box" (PMID: 22704911) and output from our most recent studies.
Sunday, September 2, 2012
Bugs Galore!
After
spending more than 8 years in clinical microbiology with special reference to
parasitology, I’ve come to realise that it truly is a bug’s life! Use of nucleic acid-based
methods such as PCR in routine clinical microbiology diagnostic labs have revealed
that single-celled parasites are colonising the intestine of up to 50% of the
Danish population! And so what? Well, this finding has several implications.
Our results reveal that faecal-oral transmission is much more common in Denmark - a highly industrialised country where drinking water comes from waterworks (i.e. no surface water supplies), where outbreaks even due to bacteria are scarce, and where authorities spend 1.2 billion DKK on food safety and control. Today, 90% of dwellings in Denmark (5.6m citizens) are connected to efficient sewage systems, and Denmark has more than 1,400 treatment plants to purify wastewater from households, businesses and institutions. But somewhere the chain pops off… Even in Denmark it is “bugs galore”, which means that faecal exposure is much more common that we would probably like to think. Intestinal protists (primarily Blastocystis and Dientamoeba) are telltales of exposure to faecal contamination and faecal-oral transmission.
Stensvold CR, Lebbad M, & Clark CG (2012). Last of the human protists: the phylogeny and genetic diversity of Iodamoeba. Molecular biology and evolution, 29 (1), 39-42 PMID: 21940643
A couple of
months ago I revisited Why it is a
bugs life by Jörg
Blech (The Guardian (2002)). Speaking of numbers, - I
wonder which one is the most successful eukaryote in terms of numbers? Blastocystis? Dientamoeba? Or any other “Parasite
sp.”? After realising that microscopy methods allow us to see only the very tip
of the iceberg and after adding PCR to our routine diagnostics, we have found a
few examples of “novel” parasitic species and many more may be in store for us.
Morphologically identical organisms, such as those belonging to Iodamoeba bütschlii, may be found in both human and non-human hosts and may differ
genetically across the nucelar small subunit rRNA gene by up to more than 30%! This is quite astonishing given the fact that the difference between human and murine small subunit rDNA is about 1%! Since these
data have been established only recently, obviously no one knows the respective
clinical significance of these morphologically similar but genetically very different lineages, and further studies may
reveal differences in pathogenicity as seen in other amoebic genera. Blastocystis and Entamoeba coli are somewhat similar examples.
Our results reveal that faecal-oral transmission is much more common in Denmark - a highly industrialised country where drinking water comes from waterworks (i.e. no surface water supplies), where outbreaks even due to bacteria are scarce, and where authorities spend 1.2 billion DKK on food safety and control. Today, 90% of dwellings in Denmark (5.6m citizens) are connected to efficient sewage systems, and Denmark has more than 1,400 treatment plants to purify wastewater from households, businesses and institutions. But somewhere the chain pops off… Even in Denmark it is “bugs galore”, which means that faecal exposure is much more common that we would probably like to think. Intestinal protists (primarily Blastocystis and Dientamoeba) are telltales of exposure to faecal contamination and faecal-oral transmission.
 | |||||
| In Denmark, 90% of dwellings are connected to efficient sewage systems, and the country has more than 1,400 treatment plants. |
However, we
might also learn to see these parasites as other types of indicators. In our
experience Danish patients with inflammatory bowel disease (IBD) represent a
cohort of people whose gut flora is remarkably different from that of other
cohorts (patients with irritable bowel syndrome (IBS) and patients with
non-IBD/non-IBS diarrhoea): Apparently IBD patients don’t harbour parasites.
This can in part be explained by the fact that some IBD patients have had bowel
resection, but even IBD patients with in intact bowel system are generally negative for
parasites.
We know that
in highly developed countries the prevalence of helminth infections has gone
down over the past few decades due to improved hygiene measures, but maybe also
due to other reasons, which have not been clarified, but as we have seen, many
of us are still positive for one or more intestinal parasites. However, most IBD
patients do not have any parasites at all. This correlates well with the
hygiene hypothesis, and it may be so that not only helminths, but also amoebae,
which are able to colonise our guts for months and even years, may be
co-responsible for 1) preventing us from developing inflammatory bowel disease
and other autoimmune diseases by immunomodulatory mechanisms, and 2)
maintaining a sound intestinal flora and ecology. Or is it so that these
protists are dependent on a certain gut ecology or gut flora in order to colonise our intestines for a longer period, and in this way,
they can be seen as indicators of a certain gut microbiota? Do they have any modulatory functions or do they happen to "lead their own life"?
As a
parasitologist and worshipper of most things eukaryotic, I was both pleased and
disconcerted after leaving the MetaHIT conference in Paris in March. Pleased,
since the stratification of people into enterotypes and correlation of
enterotypes to disease phenotypes suited my naïve, B/W perception of the world, but disconcerted since all presentations and posters addressed only
bacteria (and virus to a minor extent, - maybe one on archaea even?). But, how
about intestinal yeasts and parasites? Where in the gene catalogues and pools
of metagenomic data could I find information on eukaryotes? Nowhere. Which hopefully
boils down to methodological limitations rather than absence of interest.
The concept
of paving an avenue of new knowledge with metagenomics data is holistic in its
approach, but it currently fails to encompass a common part of the intestinal
microbiota, possibly due to methodological limitations. However, we are
probably facing the imminent inclusion of eukaryotic data in metagenomic
studies, and this will enable us to investigate the potential role of intestinal
protists and maybe yeasts as biomarkers of certain enterotypes and maybe even
disease or health phenotypes.
Further reading:
Stensvold CR (2012). Thinking Blastocystis out of the box. Trends in parasitology, 28 (8) PMID: 22704911
Sunday, August 19, 2012
The Potential Role of Our Microbiome Ecosystems
For those who like these pop-sci articles on the still somewhat conjecture-like but very inspiring theories about the role of our intestinal microbiome in health and disease, here's a link to an article from The Economist (18 AUG 2012):
The Human Microbiome: Me, myself, us
And let me reiterate: We still don't know much about mikro-eukaryotes in all this... do they play a role as well? And how do they cope with different types of microbiomes?
Anyways, enjoy!
The Human Microbiome: Me, myself, us
And let me reiterate: We still don't know much about mikro-eukaryotes in all this... do they play a role as well? And how do they cope with different types of microbiomes?
Anyways, enjoy!
Saturday, August 18, 2012
To Treat or Not To Treat... But How?
In the "To Treat or Not To Treat" series (please look up previous post here), we have come to the "...But How?" episode.
Blastocystis may be susceptible to a number of drugs - in vitro. In vitro is not the opposite of in vivo. In vitro just means that the test has been done on an organism that has been isolated from its usual habitat and tested e.g. in a flask, test tube, etc. In the lab, strains can be challenged and manipulated in multiple ways, but there is no guarantee that the outcome of an in vitro susceptibility test is reproducible in vivo, i.e. when the organism is challenged in its natural habitat and under "natural" conditions. Hence, if you test Blastocystis against metronidazole or any other compound (such as iodine) in vitro, and you observe an effect, you cannot rely on being able to reproduce the effect in vivo. This is due to a variety of reasons including pharmaco-kinetics and pharmaco-dynamics, including the ability of the drug to reach the parasite in its ecological niche, impact of the drug on other micro-organisms, drug interactions, strain-dependent differences in susceptibility (including inherent or acquired resistance), etc.
We recently described a case in which a woman with irritable bowel syndrome (according to the Rome III criteria) had both Blastocystis subtype 9 (ST9) and Dientamoeba fragilis. In order to try and eradicate the parasites and to see whether any eradication would impact on her clinical situation, she received multiple courses of antibiotic treatment:
1. Metronidazole (750 mg x 3/d for 10 days)
2. Tetracycline (500 mg x 4/d for 10 days)
3. Trimethoprim + Sulfamethoxazole (TMP 800 mg + SXT 160 mg x 2/d for 7 days)
4. Mebendazole + Metronidazole (100 mg x 2 separated by 2 weeks; subsequently metronidazole as in 1.)
5. Paromomycin + Metronidazole (PM 500 mg + MZ 170 mg x 3/d for 10 days)
Mebendazole was given to the entire household due to suspicion of pinworm infection running in the family that could be a potential reservoir of D. fragilis (re-)infection.
No clinical alleviation was seen throughout this period.
PCR-based detection of Blastocystis and D. fragilis was used to evaluate faecal samples 5-10 days post-treatment: Microbiological effect was seen only on D. fragilis which was cleared only after treatment with PM + MZ (5).
So, Blastocystis "survived" this series of antimicrobial treatment. In Denmark, no further relevant treatment options are available for general use (actually, even the use of Humatin (PM) needs a special license).
None of the patient's family members or pets were found to be colonised by the same strain, probably indicating that there was no "local" reservoir for ST9, and that the repeated finding of ST9 was not due to re-infection.
It may be so that Blastocystis requires a certain intestinal bacterial flora to establish. However, we expect that substantial perturbations in the intestinal flora must have taken place during the patient's various treatments, and therefore Blastocystis must be able to quickly overcome and adapt to such perturbations. It may add to the conundrum that in this case the woman harboured ST9, which is only very rarely seen in humans, and we might therefore deduce that its presence would be more volatile. No animal/environmental reservoir has yet been identified for ST9.
There is no doubt that microbiomic profiling of the intestinal flora would be of great benefit in a case like this. If data could be achieved on the impact of these drugs on the relative bacterial structure and function by metagenomic approaches, then this would allow us to explore the changes in the general flora that Blastocystis is capable of withstanding. Certainly, none of these drugs had a measurable in-vivo protistocidal effect on Blastocystis when administered as shown.
I re-emphasise that it is far from certain that Blastocystis is capable of inducing disease, directly or indirectly, and hence, we do not know if, and in which situations, we should aim at eradicating it. Suffice it to say, that in our hands and with the compounds that are available for general use in Denmark, it is apparently extremely challenging to eradicate Blastocystis, if at all possible.
Further reading:
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: 22075794Blastocystis may be susceptible to a number of drugs - in vitro. In vitro is not the opposite of in vivo. In vitro just means that the test has been done on an organism that has been isolated from its usual habitat and tested e.g. in a flask, test tube, etc. In the lab, strains can be challenged and manipulated in multiple ways, but there is no guarantee that the outcome of an in vitro susceptibility test is reproducible in vivo, i.e. when the organism is challenged in its natural habitat and under "natural" conditions. Hence, if you test Blastocystis against metronidazole or any other compound (such as iodine) in vitro, and you observe an effect, you cannot rely on being able to reproduce the effect in vivo. This is due to a variety of reasons including pharmaco-kinetics and pharmaco-dynamics, including the ability of the drug to reach the parasite in its ecological niche, impact of the drug on other micro-organisms, drug interactions, strain-dependent differences in susceptibility (including inherent or acquired resistance), etc.
We recently described a case in which a woman with irritable bowel syndrome (according to the Rome III criteria) had both Blastocystis subtype 9 (ST9) and Dientamoeba fragilis. In order to try and eradicate the parasites and to see whether any eradication would impact on her clinical situation, she received multiple courses of antibiotic treatment:
1. Metronidazole (750 mg x 3/d for 10 days)
2. Tetracycline (500 mg x 4/d for 10 days)
3. Trimethoprim + Sulfamethoxazole (TMP 800 mg + SXT 160 mg x 2/d for 7 days)
4. Mebendazole + Metronidazole (100 mg x 2 separated by 2 weeks; subsequently metronidazole as in 1.)
5. Paromomycin + Metronidazole (PM 500 mg + MZ 170 mg x 3/d for 10 days)
Mebendazole was given to the entire household due to suspicion of pinworm infection running in the family that could be a potential reservoir of D. fragilis (re-)infection.
No clinical alleviation was seen throughout this period.
PCR-based detection of Blastocystis and D. fragilis was used to evaluate faecal samples 5-10 days post-treatment: Microbiological effect was seen only on D. fragilis which was cleared only after treatment with PM + MZ (5).
So, Blastocystis "survived" this series of antimicrobial treatment. In Denmark, no further relevant treatment options are available for general use (actually, even the use of Humatin (PM) needs a special license).
None of the patient's family members or pets were found to be colonised by the same strain, probably indicating that there was no "local" reservoir for ST9, and that the repeated finding of ST9 was not due to re-infection.
It may be so that Blastocystis requires a certain intestinal bacterial flora to establish. However, we expect that substantial perturbations in the intestinal flora must have taken place during the patient's various treatments, and therefore Blastocystis must be able to quickly overcome and adapt to such perturbations. It may add to the conundrum that in this case the woman harboured ST9, which is only very rarely seen in humans, and we might therefore deduce that its presence would be more volatile. No animal/environmental reservoir has yet been identified for ST9.
There is no doubt that microbiomic profiling of the intestinal flora would be of great benefit in a case like this. If data could be achieved on the impact of these drugs on the relative bacterial structure and function by metagenomic approaches, then this would allow us to explore the changes in the general flora that Blastocystis is capable of withstanding. Certainly, none of these drugs had a measurable in-vivo protistocidal effect on Blastocystis when administered as shown.
I re-emphasise that it is far from certain that Blastocystis is capable of inducing disease, directly or indirectly, and hence, we do not know if, and in which situations, we should aim at eradicating it. Suffice it to say, that in our hands and with the compounds that are available for general use in Denmark, it is apparently extremely challenging to eradicate Blastocystis, if at all possible.
 |
| Microbe Resilience (Source) |
Further reading:
Engsbro AL, & Stensvold CR (2012). Blastocystis: To Treat Or Not To Treat...But How? Clinical infectious diseases : an official publication of the Infectious Diseases Society of America PMID: 22893582
Friday, August 10, 2012
Is This A New Subtype?
To quote one of my colleagues attending the recent IWOP 2012 meeting in Tarrytown, NY, Blastocystis subtyping in humans and animals is becoming 'trendy', and so we keep trying to advocate for a standardisation of the metholodology of Blastocystis subtyping.
We recently changed the title of our page at www.pubmlst.org/blastocystis so that now it is called Blastocystis Subtype (18S) and Sequence Typing (MLST) Databases, and we added some text to front page:
Now, the last bit is extremely important. We have seen examples of researchers (including ourselves!) assigning sequences to a new a subtype in the absence of complete SSU rDNA data (in fact complete sequences for ST10-ST14 are not yet publicly available!). Doing so has a least two major limitations/drawbacks: Far from all SSU rDNA regions have been validated as being representative of the whole SSU rRNA gene in terms of phylogenetic analysis, and therefore phylogenetic inferences based on non-validated regions may have little or at least less support than anticipated. Moreover, if someone analyses e.g. position 600-1600, and phylogenetic analysis based on this region reveals a potentially new subtype, this makes it impossible for his/her colleague who has data covering positions 1-600 from a Blastocystis isolate that may also represent a new subtype to ascertain whether it might be same subtype (see example below)!
Obtaining complete SSU rDNA sequences directly from faecal DNA may be a cumbersome task but is sometimes possible by combining sequence-specific primers with low-specificity primers such as the RD5 and the RD3 primers (Clark, 1997). If a cultured isolate is available, obviously this makes complete SSU rDNA sequencing much easier.
While it appears that the number of subtypes occurring in humans stays around 9, our gut feeling is that we are yet to uncover quite a few subtypes colonising non-human mammals, and it's great to see an increasing number of teams exploring the genetic diversity of Blastocystis. For instance, Dr Ronald Fayer and his group recently published exciting data on a new Blastocystis subtype in cattle, which they named ST14 (Fayer et al., 2012).
Importantly, caution should be taken to avoid creating confusion in subtype terminology. Confusion can arise when independent researchers assign the same new subtype name (e.g. ST14, ST15, etc.) to novel sequences which in fact belong to different ribosomal lineages, or when incomplete SSU rDNA sequence data are used; this situation was seen recently, when Petrasova et al. (2011), assigned a Colobus sequence to ST5, although it was in fact a ST13 sequence (Clark et al., in press); the situation arose, since Petrasova et al. (2011) did not have data covering the region currently available for ST13 (Parkar et al., 2010), and therefore believed that their sequence was a unique ST5 variant. As for ST14, less than 500 bp are currently available, and these 500 bp are not in the barcode region, making it difficult for all teams using barcoding to compare their data. And so we would like to advocate for making complete SSU rDNA sequences publicly available (Genbank) for potentially new subtypes, for at least two reasons:
1. Phylogenetic inferences based on the complete SSU rDNA will be more robust than those obtained from analysing shorter sequence streches.
2. Complete seqeunces are needed for reference since subtype screening typically includes a single round PCR such as barcoding (Scicluna et al., 2006) amplifying about 550 bp; in the situation where complete SSU rDNAs are available for all known subtypes, it will be quick to analyse, whether a sequence may represent a new subtype, since this will be independent on the SSU rDNA region studied.We therefore hope that complete SSU rDNA sequences will soon be made publicly available for ST10-ST14.
So, when does a complete SSU rDNA sequence represent a new subtype? Well, we have a review paper in press in Advances in Parasitology on recent developments in Blastocystis research, which will be published in less than six months probably, and which also touches on this topic; once the paper is published, I will try and make a summary our thoughts on this...
Further reading:
We recently changed the title of our page at www.pubmlst.org/blastocystis so that now it is called Blastocystis Subtype (18S) and Sequence Typing (MLST) Databases, and we added some text to front page:
In terms of genetic markers, the barcode region (Scicluna et al., 2006) is by far the best represented in publicly available sequence databases, and the correct subtype can be identified by BLAST analysis in the sequence database at the present site. Blasting against this database has the added advantages, compared to using GenBank, of automatically assigning allele types to the SSU-rDNA as well as using the consensus subtype nomenclature (unlike GenBank where the subtype is included only if one was part of the accession submission and no attempt to impose a standard nomenclature is made). In case the sequence does not match any of the ones in the database despite full coverage of the region, this indicates that the sequence represents a new allele or maybe even a new subtype depending on the amount of variation. If a new subtype is suspected, we suggest doing PCR and sequencing of the complete SSU rRNA gene with subsequent phylogenetic analysis using reference sequences.
Now, the last bit is extremely important. We have seen examples of researchers (including ourselves!) assigning sequences to a new a subtype in the absence of complete SSU rDNA data (in fact complete sequences for ST10-ST14 are not yet publicly available!). Doing so has a least two major limitations/drawbacks: Far from all SSU rDNA regions have been validated as being representative of the whole SSU rRNA gene in terms of phylogenetic analysis, and therefore phylogenetic inferences based on non-validated regions may have little or at least less support than anticipated. Moreover, if someone analyses e.g. position 600-1600, and phylogenetic analysis based on this region reveals a potentially new subtype, this makes it impossible for his/her colleague who has data covering positions 1-600 from a Blastocystis isolate that may also represent a new subtype to ascertain whether it might be same subtype (see example below)!
Obtaining complete SSU rDNA sequences directly from faecal DNA may be a cumbersome task but is sometimes possible by combining sequence-specific primers with low-specificity primers such as the RD5 and the RD3 primers (Clark, 1997). If a cultured isolate is available, obviously this makes complete SSU rDNA sequencing much easier.
While it appears that the number of subtypes occurring in humans stays around 9, our gut feeling is that we are yet to uncover quite a few subtypes colonising non-human mammals, and it's great to see an increasing number of teams exploring the genetic diversity of Blastocystis. For instance, Dr Ronald Fayer and his group recently published exciting data on a new Blastocystis subtype in cattle, which they named ST14 (Fayer et al., 2012).
Importantly, caution should be taken to avoid creating confusion in subtype terminology. Confusion can arise when independent researchers assign the same new subtype name (e.g. ST14, ST15, etc.) to novel sequences which in fact belong to different ribosomal lineages, or when incomplete SSU rDNA sequence data are used; this situation was seen recently, when Petrasova et al. (2011), assigned a Colobus sequence to ST5, although it was in fact a ST13 sequence (Clark et al., in press); the situation arose, since Petrasova et al. (2011) did not have data covering the region currently available for ST13 (Parkar et al., 2010), and therefore believed that their sequence was a unique ST5 variant. As for ST14, less than 500 bp are currently available, and these 500 bp are not in the barcode region, making it difficult for all teams using barcoding to compare their data. And so we would like to advocate for making complete SSU rDNA sequences publicly available (Genbank) for potentially new subtypes, for at least two reasons:
1. Phylogenetic inferences based on the complete SSU rDNA will be more robust than those obtained from analysing shorter sequence streches.
2. Complete seqeunces are needed for reference since subtype screening typically includes a single round PCR such as barcoding (Scicluna et al., 2006) amplifying about 550 bp; in the situation where complete SSU rDNAs are available for all known subtypes, it will be quick to analyse, whether a sequence may represent a new subtype, since this will be independent on the SSU rDNA region studied.We therefore hope that complete SSU rDNA sequences will soon be made publicly available for ST10-ST14.
So, when does a complete SSU rDNA sequence represent a new subtype? Well, we have a review paper in press in Advances in Parasitology on recent developments in Blastocystis research, which will be published in less than six months probably, and which also touches on this topic; once the paper is published, I will try and make a summary our thoughts on this...
Further reading:
Clark CG (1997). Extensive genetic diversity in Blastocystis hominis. Molecular and biochemical parasitology, 87 (1), 79-83 PMID: 9233675
Fayer R, Santin M, & Macarisin D (2012). Detection of concurrent infection of dairy cattle with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and microscopic methods. Parasitology research PMID: 22710524
Parkar U, Traub RJ, Vitali S, Elliot A, Levecke B, Robertson I, Geurden T, Steele J, Drake B, & Thompson RC (2010). Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers. Veterinary parasitology, 169 (1-2), 8-17 PMID: 20089360
Scicluna SM, Tawari B, & Clark CG (2006). DNA barcoding of blastocystis. Protist, 157 (1), 77-85 PMID: 16431158
Thursday, July 19, 2012
Micro-Eukaryotic Diversity in The Human Intestine
While we’re currently being flooded by papers on the
intestinal microbiome, we still have very few dealing with the intestinal “micro-eukaryome”
(forgive me my "badomics", I should have known better after reading this piece by Dr Eisen).
Hamad et al., just published their work on “Molecular Detection of Eukaryotes in a Single Human Stool Sample from Senegal” in PLoS One. They used a panel of 22 broad-specificity eukaryotic primers on genomic DNA extracted directly from faeces, cloned PCR products and did a blast search of the resulting sequences. They found about 18 micro-eukaryotic species in this particular faecal sample, most of which were fungi, and only two of which were “parasites”, namely Blastocystis sp. (subtype not given) and Entamoeba hartmanni, a so-called non-pathogenic amoebic species.They used both culture and culture-independent methods (PCR directly on genomic DNA from faeces) for the detection of intestinal fungi.
The study is interesting for a number of reasons:
1) It is one of the few papers out there on micro-eukaryotic diversity in faecal samples (other ones are listed in the reading list below), and we still know very little about micro-eukaryotes' potential interaction with the host and their ecological niche.
2) Many fungal species were detected by cloning of PCR products obtained by various primer pairs. It is possible that many of these are fungi stemming from the environment and diet, and not actually fungi colonizing the intestinal tract of this person; indeed the primers were able to pick up eukaryotic DNA such as that from tomatoes and common hop, stemming from the person’s diet. This is also one of the draw-backs of studies of fungi in stool samples: Even for mycologists it may prove difficult to determine which fungi are likely to be colonisers rather than fungi in transit due to environmental exposure, including diet. Analysis of consecutive samples from the same individual(s) (similar to the approach by Scanlan and Marchesi (2008)) will assist in identifying which fungi are stable and probable colonisiers. Similar to other studies, the investigators highlight the disparate findings resulting from the use of culture-dependent and culture-independent analyses; culture may be a way of identifying which ones of the many fungi detected by PCR that are actual colonisers.
3) We still don’t know much about what to expect when we take an approach like this. In the present study, multiple primer pairs were put into use, and 11 primer pairs yielded PCR products. The primer pairs amplified products of different lengths (some of them covering the complete SSU rDNA (18S)), and large products can sometimes be difficult to amplify and/or sequence for a variety of reasons; also preferential amplification may be a limiting factor. What would sometimes be useful is an in-silico analysis of the spectrum of organisms covered – at least theoretically - by each set of primers. In the papers I’ve seen so far aiming to display the eukaryotic diversity in human stool, Blastocystis has been a consistent finding, while Dientamoeba fragilis, which, at least in Denmark is almost as prevalent as Blastocystis (in some cohorts even more prevalent) and can be seen in co-infection, has not been reported so far. When you are presented with a list like the one presented by Hamad et al., you are inclined to believe that this list is exhaustive, but I think in-silico analysis data on such broad-specificity primers used for the detection of eukaryotic DNA would help us validate the use of these primers. Another approach to test the applicability of this methodology is to construct samples of DNA from known organisms in different ratios... and then test how the primers and cloning perform. What is also important is the very method of DNA extraction... obviously, our ability to detect DNA from any organism relies on our ability to extract DNA from it.
4) The study of micro-eukaryotes and their roles in health and disease includes first and foremost knowledge about which species and lineages that can be found and which ones that are the most common. Molecular methods are needed to identify the organisms in our intestine, since for instance parasites that look the same (morphological identity) can be genetically diverse with differing abilities to cause disease. We know from studies of micro-eukaryotes in ruminants that for instance some ciliates can be directly beneficial to the host, while others - such as cryptosporidia - are virtually obligate pathogens causing watery diarrhoea. Moreover, some organisms, including micro-eukaryotes, may be extremely difficult to culture even short-term, and also microscopy has limitations.
While we are still searching for virulence genes and other effector proteins in common micro-eukaryotes such as Blastocystis and Dientamoeba fragilis which could potentially cause disease directly, we also need to look for more indirect effects. Although much lower in numbers than our bacteria, (some) micro-eukaryotes may predate on beneficial bacteria to an extent where dysbiosis is reached. "Defaunation" of the intestine is speculated to be associated not only with impaired absorption of nutrients, but also with the development of severe disesases such as colon cancer and if micro-eukaryotes are able to skew our flora, this may have indirect impact on our health; many of our commensal bacteria are essential to some of our vital body functions, - indeed our intestinal flora can be viewed as a separate organ (see previous blog posts).
In the era of "omics" and "ngs" tools, it is interessesting to see a paper on global microbiotic diversity using a "conventional" cloning and sequencing approach in 2012. It may be one of the last papers of its kind?
To sum up: it is clear that a healthy intestine may be populated by a variety of micro-eukaryotes and future studies of the structure and function of the intestinal microbiome including micro-eukaryotes will help us understand their role in health and disease.
Let me end this post by uploading an image depicting "A Tree of Eukaryotes" (including Blastocystis) from an excellent protist blog by a colleague - my rendition here is practically useless, but I hope it might tease you to go and look at it in detail on "Welcome to the Ocelloid" by Psi Wavefunction.
Further reading:
Hamad I, Sokhna C, Raoult D, & Bittar F (2012). Molecular detection of eukaryotes in a single human stool sample from senegal. PloS one, 7 (7) PMID: 22808282
Pandey PK, Siddharth J, Verma P, Bavdekar A, Patole MS, & Shouche YS (2012). Molecular typing of fecal eukaryotic microbiota of human infants and their respective mothers. Journal of biosciences, 37 (2), 221-6 PMID: 22581327
Scanlan PD, & Marchesi JR (2008). Micro-eukaryotic diversity of the human distal gut microbiota: qualitative assessment using culture-dependent and -independent analysis of faeces. The ISME journal, 2 (12), 1183-93 PMID: 18670396
Hamad et al., just published their work on “Molecular Detection of Eukaryotes in a Single Human Stool Sample from Senegal” in PLoS One. They used a panel of 22 broad-specificity eukaryotic primers on genomic DNA extracted directly from faeces, cloned PCR products and did a blast search of the resulting sequences. They found about 18 micro-eukaryotic species in this particular faecal sample, most of which were fungi, and only two of which were “parasites”, namely Blastocystis sp. (subtype not given) and Entamoeba hartmanni, a so-called non-pathogenic amoebic species.They used both culture and culture-independent methods (PCR directly on genomic DNA from faeces) for the detection of intestinal fungi.
The study is interesting for a number of reasons:
1) It is one of the few papers out there on micro-eukaryotic diversity in faecal samples (other ones are listed in the reading list below), and we still know very little about micro-eukaryotes' potential interaction with the host and their ecological niche.
2) Many fungal species were detected by cloning of PCR products obtained by various primer pairs. It is possible that many of these are fungi stemming from the environment and diet, and not actually fungi colonizing the intestinal tract of this person; indeed the primers were able to pick up eukaryotic DNA such as that from tomatoes and common hop, stemming from the person’s diet. This is also one of the draw-backs of studies of fungi in stool samples: Even for mycologists it may prove difficult to determine which fungi are likely to be colonisers rather than fungi in transit due to environmental exposure, including diet. Analysis of consecutive samples from the same individual(s) (similar to the approach by Scanlan and Marchesi (2008)) will assist in identifying which fungi are stable and probable colonisiers. Similar to other studies, the investigators highlight the disparate findings resulting from the use of culture-dependent and culture-independent analyses; culture may be a way of identifying which ones of the many fungi detected by PCR that are actual colonisers.
3) We still don’t know much about what to expect when we take an approach like this. In the present study, multiple primer pairs were put into use, and 11 primer pairs yielded PCR products. The primer pairs amplified products of different lengths (some of them covering the complete SSU rDNA (18S)), and large products can sometimes be difficult to amplify and/or sequence for a variety of reasons; also preferential amplification may be a limiting factor. What would sometimes be useful is an in-silico analysis of the spectrum of organisms covered – at least theoretically - by each set of primers. In the papers I’ve seen so far aiming to display the eukaryotic diversity in human stool, Blastocystis has been a consistent finding, while Dientamoeba fragilis, which, at least in Denmark is almost as prevalent as Blastocystis (in some cohorts even more prevalent) and can be seen in co-infection, has not been reported so far. When you are presented with a list like the one presented by Hamad et al., you are inclined to believe that this list is exhaustive, but I think in-silico analysis data on such broad-specificity primers used for the detection of eukaryotic DNA would help us validate the use of these primers. Another approach to test the applicability of this methodology is to construct samples of DNA from known organisms in different ratios... and then test how the primers and cloning perform. What is also important is the very method of DNA extraction... obviously, our ability to detect DNA from any organism relies on our ability to extract DNA from it.
4) The study of micro-eukaryotes and their roles in health and disease includes first and foremost knowledge about which species and lineages that can be found and which ones that are the most common. Molecular methods are needed to identify the organisms in our intestine, since for instance parasites that look the same (morphological identity) can be genetically diverse with differing abilities to cause disease. We know from studies of micro-eukaryotes in ruminants that for instance some ciliates can be directly beneficial to the host, while others - such as cryptosporidia - are virtually obligate pathogens causing watery diarrhoea. Moreover, some organisms, including micro-eukaryotes, may be extremely difficult to culture even short-term, and also microscopy has limitations.
While we are still searching for virulence genes and other effector proteins in common micro-eukaryotes such as Blastocystis and Dientamoeba fragilis which could potentially cause disease directly, we also need to look for more indirect effects. Although much lower in numbers than our bacteria, (some) micro-eukaryotes may predate on beneficial bacteria to an extent where dysbiosis is reached. "Defaunation" of the intestine is speculated to be associated not only with impaired absorption of nutrients, but also with the development of severe disesases such as colon cancer and if micro-eukaryotes are able to skew our flora, this may have indirect impact on our health; many of our commensal bacteria are essential to some of our vital body functions, - indeed our intestinal flora can be viewed as a separate organ (see previous blog posts).
In the era of "omics" and "ngs" tools, it is interessesting to see a paper on global microbiotic diversity using a "conventional" cloning and sequencing approach in 2012. It may be one of the last papers of its kind?
To sum up: it is clear that a healthy intestine may be populated by a variety of micro-eukaryotes and future studies of the structure and function of the intestinal microbiome including micro-eukaryotes will help us understand their role in health and disease.
Let me end this post by uploading an image depicting "A Tree of Eukaryotes" (including Blastocystis) from an excellent protist blog by a colleague - my rendition here is practically useless, but I hope it might tease you to go and look at it in detail on "Welcome to the Ocelloid" by Psi Wavefunction.
Further reading:
Hamad I, Sokhna C, Raoult D, & Bittar F (2012). Molecular detection of eukaryotes in a single human stool sample from senegal. PloS one, 7 (7) PMID: 22808282
Pandey PK, Siddharth J, Verma P, Bavdekar A, Patole MS, & Shouche YS (2012). Molecular typing of fecal eukaryotic microbiota of human infants and their respective mothers. Journal of biosciences, 37 (2), 221-6 PMID: 22581327
Thursday, July 12, 2012
10,000 views and a Blastocystis Salute!
I've been writing this blog since late March, and today this blog was visited by viewer #10,000. I think that calls for a salute!
And what could be more appropriate than a piece of music attributed to Blastocystis? Please click here and read and listen for yourselves! And by the way: This is not the only orchestral work that has been written with Blastocystis in mind...
(Please note that this blog can now be accessed simply via http://blastocystis[dot]net - thanks for visiting!)
And what could be more appropriate than a piece of music attributed to Blastocystis? Please click here and read and listen for yourselves! And by the way: This is not the only orchestral work that has been written with Blastocystis in mind...
(Please note that this blog can now be accessed simply via http://blastocystis[dot]net - thanks for visiting!)
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