Tuesday, November 20, 2012
Saturday, November 17, 2012
Amelioration of Colitis by Parasites - or "An Elliott & Weinstock Special"
Common parasites such as Blastocystis and Dientamoeba fragilis are often incriminated of causing chronic or intermittent diarrhoea or other intestinal symptoms despite the absence of compelling evidence. What most of us probably fail to realise is that parasites may actually prevent and ameliorate intestinal illness, including inflammatory bowel disease, other types of colitis, and other types of autoimmune diseases.
Inflammatory bowel disease (IBD) includes the two most common manifestations ulcerative colitis and Crohn’s Disease and affects more than 2 million people in North America and Europe. They are chronic inflammatory conditions of the gut that usually begin when people are in the second to third decade of life. Although the causes of these inflammatory diseases remain unknown, they are assumed to result from inappropriately aggressive mucosal (i.e. related to our intestinal lining) immune responses to elements or substances in our intestine. IBD is treated with immuno-suppresive drugs.
IBD has emerged primarily in the Western world along with a significant reduction in cases of intestinal helminthiasis due to clean food and water, improved hygiene and sanitation, and the development and use of antibiotics. In Denmark, helminthic infections due to previously common parasitic worms such as Ascaris (roundworm) are now at the point of being almost extinct in the indigenous population.
The hygiene hypothesis proposes that a causal link exists between the adoption of modern hygiene and the increase in the prevalence of immune dysfunctions. The extent of perinatal maturation of the immune system may play a crucial role in terms of our likelihood of developing allergic and autoimmune diseases later in life. The maturation process includes establishment of tolerance to food and harmless microorganisms, but also defence mechanisms against pathogens. If our environment is "too clean", we may fail to give our immune system the best possible opportunity to mature and differentiate appropriately. A robust immune response will protect us from recurrent infections, but if misdirected, it can cause disease.
Part of our immune system is the "adaptive immune system" - or our "immunologic memory" - made up by cells such as lymphocytes (T- and B-cells), macrophages, dendritic cells, etc. plus antibodies and hormone-like substances (eg. cytokines) that are secreted to activate/inactivate or up- and down-regulate these cells. Our immune systems has to be able to recognise a plethora of foreign material such as bacteria, viruses and parasites, and to distinguish "self" from "non-self". IBD may be caused by mal-functions in our own immune system, and so may a lot of other diseases, diseases that we call "autoimmune diseases", and which include coeliac disease, multiple sclerosis, type 1 diabetes, and rheumatoid arthritis.
10,000 years ago, humans were infected by a variety of species of worms that are common in some parts of the world even today and hence humans and parasites have co-evolved over thousands of years. Importantly, most wild animals in their natural habitat are carriers of many types of parasites. A "clever" parasite does little harm to its host. Parasites have developed mechanisms that enable them to survive in their hosts, and also, the human immune system has developed a way to adapt to these common intruders.
How can one explain the amelioration of symptoms due to
colitis by the presence of intestinal nematodes? Helminths appear to induce immune host regulatory cells that suppress
inflammation, and helminth infections are strong inducers of immune
regulatory circuits. The immune system changes in response to helminth colonisation and factors secreted by helminths that can influence immune cell function. It is likely that several immune-regulatory mechanisms are exploited by individual helminths. Otherwise, a helminth could not reliably evade our immune system to reproduce.
A new study has produced data that suggest that treatment of macaques suffering from chronic diarrhoea with eggs of the whipworm Trichuris suis can alleviate symptoms and modulate both the intestinal microbiota and immunoregulatory pathways. Trichuris suis is the whipworm of the pig, and contrary to Trichuris trichiura (image), T. suis appears not to be able to produce disease in primate hosts (including humans). When T. suis ova (TSO) are administered to humans, transient shedding of ova in faeces may be seen after a few weeks, but the individual remains asymptomatic.
Inflammatory bowel disease (IBD) includes the two most common manifestations ulcerative colitis and Crohn’s Disease and affects more than 2 million people in North America and Europe. They are chronic inflammatory conditions of the gut that usually begin when people are in the second to third decade of life. Although the causes of these inflammatory diseases remain unknown, they are assumed to result from inappropriately aggressive mucosal (i.e. related to our intestinal lining) immune responses to elements or substances in our intestine. IBD is treated with immuno-suppresive drugs.
IBD has emerged primarily in the Western world along with a significant reduction in cases of intestinal helminthiasis due to clean food and water, improved hygiene and sanitation, and the development and use of antibiotics. In Denmark, helminthic infections due to previously common parasitic worms such as Ascaris (roundworm) are now at the point of being almost extinct in the indigenous population.
The hygiene hypothesis proposes that a causal link exists between the adoption of modern hygiene and the increase in the prevalence of immune dysfunctions. The extent of perinatal maturation of the immune system may play a crucial role in terms of our likelihood of developing allergic and autoimmune diseases later in life. The maturation process includes establishment of tolerance to food and harmless microorganisms, but also defence mechanisms against pathogens. If our environment is "too clean", we may fail to give our immune system the best possible opportunity to mature and differentiate appropriately. A robust immune response will protect us from recurrent infections, but if misdirected, it can cause disease.
Part of our immune system is the "adaptive immune system" - or our "immunologic memory" - made up by cells such as lymphocytes (T- and B-cells), macrophages, dendritic cells, etc. plus antibodies and hormone-like substances (eg. cytokines) that are secreted to activate/inactivate or up- and down-regulate these cells. Our immune systems has to be able to recognise a plethora of foreign material such as bacteria, viruses and parasites, and to distinguish "self" from "non-self". IBD may be caused by mal-functions in our own immune system, and so may a lot of other diseases, diseases that we call "autoimmune diseases", and which include coeliac disease, multiple sclerosis, type 1 diabetes, and rheumatoid arthritis.
10,000 years ago, humans were infected by a variety of species of worms that are common in some parts of the world even today and hence humans and parasites have co-evolved over thousands of years. Importantly, most wild animals in their natural habitat are carriers of many types of parasites. A "clever" parasite does little harm to its host. Parasites have developed mechanisms that enable them to survive in their hosts, and also, the human immune system has developed a way to adapt to these common intruders.
 |
| Egg of Trichuris trichiura. Courtesy of Dr Marianne Lebbad. |
A new study has produced data that suggest that treatment of macaques suffering from chronic diarrhoea with eggs of the whipworm Trichuris suis can alleviate symptoms and modulate both the intestinal microbiota and immunoregulatory pathways. Trichuris suis is the whipworm of the pig, and contrary to Trichuris trichiura (image), T. suis appears not to be able to produce disease in primate hosts (including humans). When T. suis ova (TSO) are administered to humans, transient shedding of ova in faeces may be seen after a few weeks, but the individual remains asymptomatic.
Gene expression profiling of colonic biopsies from the macaques treated with TSO revealed up-regulation of genes typically involved in the so-called Th1-type immuno-response prior to TSO challenge, while induction of the Th2-type response followed after the TSO challenge; the Th2-type response resulted in mucosal repair, probably by increasing mucus production and turnover of epithelial cells, which again led to a reduction of bacterial attachment to the gut lining and a restoration of microbial diversity.
Briefly, a Th1-type response is generally a pro-inflammatory response that, among many other things, is responsible for microbicidal actions and perpetuating autoimmune responses. Excessive pro-inflammatory responses can lead to uncontrolled tissue damage, so there needs to be a mechanism to counteract this. The Th2-type response includes the secretion of the anti-inflammatory cytokines, co-responsible for a general anti-inflammatory response. In excess, Th2-type responses will counteract the Th1-mediated microbicidal action. The optimal scenario would therefore seem to be that humans should produce a well balanced Th1- and Th2-type response, suited to the immune challenge.
On top of the immunoregulatory impact, there is emerging evidence that helminths promote the growth and expansion of groups of bacteria that are beneficial or "probiotic" to the host. In the study of the macaques, the TSO induced a change in the intestinal microbiota.
While variation in160 genes in the human genome or more have been associated with increased risk of developing IBD, no specific gene variant that is sufficient or required for dysregulated mucosal inflammation as occurs in Crohn's disease or ulcerative colitis has been identified so far. There is a field of thought now saying that - over thousands of years - the human gut flora, including helminths, drove the development of variations in genes orchestrating various immune response pathways, and such genetic variations selected to operate under the influence of helminth infection could cause disease when operating without that influence.
So, the take home message here is that infestation by intestinal parasites may be a double-edged sword: While on one hand they may cause symptoms, they may on the other hand prevent us from developing inflammatory bowel disease and other autoimmune or allergic manifestations. Hence, helminths, although parasites, may contribute something in return to their hosts, and the loss of helminths removes a natural governor that helped to prevent disease due to immune regulation. Of course, more trials are needed before "helminth therapy" can actually be standardised, commercialised and used in the prophylaxis and treatment of IBD and gut allergic conditions. Once a good mechanistic understanding of how helminths alter immunity is available, it may even be possible to apply identified factors individually or in combination to treat disease.
As always, things are much more complex than presented here, but this post gives an impression of some of the fields of thought. Not all autoimmune diseases are driven by excessive Th1-type responses; some types of asthma may be driven by Th2-type response, but even here, helminths may favourably modulate immunoregulatory pathways.
Obviously, it would be interesting to explore how other parasitic infections impact on our immune system and gut flora. Interestingly, one helminth species appears to have "survived" in our "sterile" environment, - the pinworm (Enterobius)... and as pointed out in one of my recent blog posts (go here), many of us are definitely exposed to parasites that persist in our intestines for months, maybe years. What's their role in all of this?
Further reading:
Dirtying Up Our Diets - go here.
Parasitic Worm Eggs Ease Intestinal Ills By Changing Gut Microbiota - go here.
Jostins L, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature, 491 (7422), 119-24 PMID: 23128233
On top of the immunoregulatory impact, there is emerging evidence that helminths promote the growth and expansion of groups of bacteria that are beneficial or "probiotic" to the host. In the study of the macaques, the TSO induced a change in the intestinal microbiota.
While variation in160 genes in the human genome or more have been associated with increased risk of developing IBD, no specific gene variant that is sufficient or required for dysregulated mucosal inflammation as occurs in Crohn's disease or ulcerative colitis has been identified so far. There is a field of thought now saying that - over thousands of years - the human gut flora, including helminths, drove the development of variations in genes orchestrating various immune response pathways, and such genetic variations selected to operate under the influence of helminth infection could cause disease when operating without that influence.
So, the take home message here is that infestation by intestinal parasites may be a double-edged sword: While on one hand they may cause symptoms, they may on the other hand prevent us from developing inflammatory bowel disease and other autoimmune or allergic manifestations. Hence, helminths, although parasites, may contribute something in return to their hosts, and the loss of helminths removes a natural governor that helped to prevent disease due to immune regulation. Of course, more trials are needed before "helminth therapy" can actually be standardised, commercialised and used in the prophylaxis and treatment of IBD and gut allergic conditions. Once a good mechanistic understanding of how helminths alter immunity is available, it may even be possible to apply identified factors individually or in combination to treat disease.
As always, things are much more complex than presented here, but this post gives an impression of some of the fields of thought. Not all autoimmune diseases are driven by excessive Th1-type responses; some types of asthma may be driven by Th2-type response, but even here, helminths may favourably modulate immunoregulatory pathways.
Obviously, it would be interesting to explore how other parasitic infections impact on our immune system and gut flora. Interestingly, one helminth species appears to have "survived" in our "sterile" environment, - the pinworm (Enterobius)... and as pointed out in one of my recent blog posts (go here), many of us are definitely exposed to parasites that persist in our intestines for months, maybe years. What's their role in all of this?
Further reading:
Dirtying Up Our Diets - go here.
Parasitic Worm Eggs Ease Intestinal Ills By Changing Gut Microbiota - go here.
Jostins L, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature, 491 (7422), 119-24 PMID: 23128233
Broadhurst, MJ., et al.Therapeutic helminth infection of macaques with idiopathic chronic diarrhoea alters the inflammatory signature and mucosal microbiota of the colon PLoS Pathogens (PLoS Pathog 8(11): e1003000. doi:10.1371/journal.ppat.1003000).
Saturday, November 10, 2012
How Hard Can It Be?
How strange the world of clinical microbiology is when you compare the fields of mycology, parasitology, bacteriology and virology to each other. Such different possibilities, opportunities, limitations, and diagnostic challenges! The 3 month mortality rate of invasive aspergillosis, a disease mainly caused by Aspergillus fumigatus and seen in mainly patients with haematological malignancies, patients undergoing allogenic HSCT and patients in ICUs, may be as high as 60%, and therefore a quick and reliable diagnosis is mandatory to secure timely therapeutic intervention. But, - Aspergillus fumigatus happens to be ubiquitous, and contamination of patient samples, whether blood or airway samples, may always be a potential cause of false-positive test results, and one of the reasons why the use of PCR as a first line diagnostic tool in routine mycology labs is still limited. Antigen tests, such as the Galactomannan antigen test, which also allow quick diagnosis can also be false-positive, not only due to sample contamination, but also due to galactomannan residues in medical compounds, such as the widely applied antibiotic Tazocin (piperacillin-tazobactam), which means that patients who have been given this drug and who submit a blood sample for galactomannan testing may test slightly positive even in the absence of an Aspergillus infection.
These are only some classical examples. In the field of mycology, positive predictive values (PPV; i.e. what is the probability of disease given a positive test result) are sometimes unacceptably low, and the lower the prevalence of the disease, the lower the PPV. This means that you need a lot of experience and knowledge on pre-test-probability + data from clinical and diagnostic work-ups, including anamnestic details, to determine whether or not the patient should receive therapy, such as treatment with voriconazole, - a relatively expensive drug.
 |
| Aspergillus fumigatus - the most common cause of invasive aspergillosis - on blood agar. |
In the parasitology lab, however, things are quite different. Contamination of patient samples is rarely an issue, and in most cases not possible at all (disregarding DNA contamination of course). Specificity of microscopy is very often very high (close to 100%), which means that the PPV is very high even in cases where the disease is rare. Hence, if cysts of Giardia have been detected in your stool, it's due to the presence of the parasite in your body. It's a bit more tricky with PCR-based analyses, where the specificity does not rely on your ability to visually distinguish between e.g. Giardia and non-Giardia elements, but where it's all about designing oligos that anneal only to Giardia-DNA.
While in the mycology lab we struggle with low PPVs, one of the biggest challenges for me and my colleagues in the parasitology lab is to optimise the negative predictive value (NPV) of a faecal parasite diagnostic work-up - how can we rule out parasitic disease by cost-effectively putting together a panel of as few tests as possible?
There are many other differences. For instance, you can grow bacteria and fungi in the lab very easily, in fact, culture of bacteria and fungi is an essential diagnostic tool, which also allows you to submit the strain to antibiotic or antimycotic susceptibility testing and molecular characterisation/MALDI-TOF analysis in case you are not sure about the species ID. So, you have the strains right there in front of you, on agar plates, and they grow and grow, and you can keep them for as long as you like, - clean, non-contaminated strains on selective media.
You can't really do that with parasites, not nearly to the same extent and as easily, that is. For instance, you can culture Blastocystis directly from stool for sure (go here for the protocol), but only in the presence of bacteria (some of my colleagues do actually now and then manage to grow strains of Blastocystis in the absence of bacteria, they obtain what is called "axenic" cultures, but I believe that they cannot do it consistently and in limited time.). And it's a pity, since there is so much you can do when you have "clean" patient strains. Apart from susceptibility testing (which would actually be a bit difficult since Blastocystis is strictly anaerobic, so you can't really have it in microtiter plates or on RPMI plates on the table in front of you, but the strains could be challenged in the growth tubes), you can also extract DNA, and you would know that all the DNA that you extract from the isolate is from that particular strain, and not from bacterial contaminants. You can use the strain for production of antigens which can be used in ELISAs and used to generate mono- and polyclonal antibodies... Sequencing genomes of various subtypes would be a lot easier and quicker, and so on...
So, what appears obvious in one field of microbiology is not as obvious in another field, and vice versa. I wish Blastocystis was much easier to isolate. Dientamoeba too. Dientamoeba is probably as common as Blastocystis, and not rarely seen in co-infections. It is strange to contemplate that a parasite infecting hundreds of millions of people has not yet had its genome sequenced? We have no clue when it comes to effector proteins in Dientamoeba, and also for this parasite, what we know about its clinical significance relies mainly on epidemiological data.
There is no doubt that concerted efforts of experienced scientists should make it possible to develop appropriate and relevant culture protocols for these parasites. It does, however, require a lot of resources and time to get to know these common, but oh so fragile and reclusive little creatures...
Further reading:
Verweij PE, Kema GH, Zwaan B, & Melchers WJ (2012). Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatus. Pest management science PMID: 23109245
Stensvold, C., Jørgensen, L., & Arendrup, M. (2012). Azole-Resistant Invasive Aspergillosis: Relationship to Agriculture Current Fungal Infection Reports, 6 (3), 178-191 DOI: 10.1007/s12281-012-0097-7
Maertens J, Theunissen K, Verhoef G, & Van Eldere J (2004). False-positive Aspergillus galactomannan antigen test results. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 39 (2), 289-90 PMID: 15307045
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
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