Showing posts with label clinical microbiology. Show all posts
Showing posts with label clinical microbiology. Show all posts

Tuesday, September 26, 2017

Website for 2nd International Blastocystis Conference is now live!

I'm happy to be able to point your attention towards the website for the 2nd International Blastocystis Conference in Bogotá in October 2018. You can access the website here.

On this site, you'll find information about the venue, the preliminary programme, speakers, registration, and sponsors. You will also find some information about Bogotá.

Please visit the website on a regular basis for potential updates/changes.

See you there!

Monday, June 13, 2016

This Month in Blastocystis Research (MAY 2016)

Very much belated, I'm back to give you the MAY entry of the 2016 "This Month in Blastocystis Research" blog series.

I'm basically just going to highlight a few papers and some other interesting things.

Ever since our metagenomics paper came out, it's as if the interest in Blastocystis in a gut microbiota context is exploding. If you put "Blastocystis microbiota" into the search box in PubMed, today you will get 20 hits, most of which papers are extremely interesting and of course very central to this type of research. Given the number of times I've addressed the relevance of studying Blastocystis in relation to gut microbiota diversity on this blog, I'll try not to flog it to death this time!

Over at Gut Microbiota For Health, a blog was posted a week ago summarising the recent findings of Audebert and colleagues and comparing them to data coming out from our lab. You can read the blog here. Using the Ion Torrent PGM sequencing platform, 16S rDNA gene sequencing was performed on genomic DNAs extracted from Blastocystis-positive and - negative stool samples. What Audebert hypothesised was that if Blastocystis is associated to intestinal disease such as for instance diarrhoea, one would expect to find a higher degree of microbiota perturbation (dysbiosis) in Blastocystis carriers than in non-carriers. Meanwhile, and similar to what we have have published, they reported that gut microbiota diversity is higher in Blastocystis carriers than in non-carriers, indicating that Blastocystis is generally a marker of a healthy gut microbiota rather than a perturbed one. Again similar to what we found in the metagenomics paper, Audebert et al. saw that the bacterial families Ruminococcaceae and Prevotellaceae were also more abundant in carriers than in Blastocystis-negative patients, while Enterobacteriaceae were enriched in Blastocystis-negative patients. What is also really interesting is the fact that the genera Faecalibacterium and Roseburia had a significantly higher abundance in Blastocystis-positive patients. These genera contain bacteria that produce butyrate which has a lot of important and beneficial functions. Loss of butyrate producers is seen for instance in patients with inflammatory bowel disease. The group used some of the same methods as we used in our study presented recently at ECCMID, including rarefaction analysis and calculation of Chao1 indices.

Together with colleagues at the Technical University of Denmark, we were lucky to have The European Journal of Clinical Microbiology and Infection publish our novel data on associations between common single-celled intestinal parasites--Blastocystis and Dientamoeba--and groups of intestinal bacteria, as evidenced by qPCR assays. We confirmed the findings from our metagenomics study, by finding a relatively lower abundance of Bacteroides in the parasite-positive samples than in the -negative ones.

By the way, on the Gut Microbiota For Health site you will find an e-learning course on Microbiota provided by the Gut Microbiota and Health Section of the European Society of Neurogastroenterology and Motility (ESNM) and developed for gastroenterologists.

Speaking of e-learning and gastroenterology: For a couple of years, I've had the immense pleasure of being part of the United European Gastroenterology e-learning task force. We host a resource - UEG Education - developed mainly for gastroenterologists, boasting e-learning courses, "Decide-on-the-Spot" series, "Mistakes in..." series, blogs, and other features. I have included a UEG widget in the right side bar of my blog - please click it!

Back to Blastocystis! Graham Clark and I published a personal view on the current status of Blastocystis in Parasitology International, in which we summarise the development and recent advances in Blastocystis research. The article is expected to form part of a special section/issue dedicated to Blastocystis to commemorate last year's 1st International Blastocystis Symposium in Ankara.

My colleague Juan-David Ramirez and his colleauges published data from a subtyping study from South America including 346 samples. More than 85% of the subtypes found belonged to either ST1, ST2, and ST3 as expected, while the rest belonged to ST4, ST5, ST6, ST7, ST8, ST12 and what they call a new subtype. I think this is the first time ST12 has been reported in humans. Despite the fact that the authors accounted for the databases that they used for subtype and allele calling, there is no mention on the criteria by which the subtypes were called in the NCBI database (i.e., in those cases where no hits could be found at the online Blastocystis database). For instance, what level of similarity was used to identify three samples as ST12? On the same note, which level of similarity was used to identify nine samples as belonging to a "novel subtype" (also, - was it the same sequence across the nine samples?). When dealing with a potentially novel subtype, usually the entire SSU rRNA gene is seqeunced and subjected to phylogenetic analysis, and sequences have not yet been made public in GenBank, so there is no possibility to work with the data so as to validate the findings (which are highly accurate, I'm sure). I think this information is critical to interpreting the data. Nontheless, the work that went into the sampling and the lab work should be highly accredited.

References:

Andersen LO, Bonde I, Nielsen HB, & Stensvold CR (2015). A retrospective metagenomics approach to studying Blastocystis. FEMS microbiology ecology, 91 (7) PMID: 26130823

Audebert C, Even G, Cian A, Blastocystis Investigation Group, Loywick A, Merlin S, Viscogliosi E, & Chabé M (2016). Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota. Scientific reports, 6 PMID: 27147260  

O'Brien Andersen L, Karim AB, Roager HM, Vigsnæs LK, Krogfelt KA, Licht TR, & Stensvold CR (2016). Associations between common intestinal parasites and bacteria in humans as revealed by qPCR. European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology PMID: 27230509 

Ramírez JD, Sánchez A, Hernández C, Flórez C, Bernal MC, Giraldo JC, Reyes P, López MC, García L, Cooper PJ, Vicuña Y, Mongi F, & Casero RD (2016). Geographic distribution of human Blastocystis subtypes in South America. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 41, 32-5 PMID: 27034056

Stensvold CR, & Clark CG (2016). Current status of Blastocystis: A personal view. Parasitology international PMID: 27247124   

Thursday, May 5, 2016

This Month in Blastocystis Research (APR 2016)

I thought I’d give examples of some of the Blastocystis-related activities in which I was involved in April.

I was lucky to be invited as part of the faculty for this year’s ECCMID conference in Amsterdam. I had an opportunity to give a talk on Detection of protozoans using molecular techniques in routine clinical practice (click link to watch it). I also co-authored a poster with the title Blastocystis colonization correlates with gut bacterial diversity which is one of several studies recently performed by our group that suggest that Blastocystis is a biomarker – or an indicator if you wish – of a healthy gut microbial environment and high gut microbiota diversity. 

This very topic was one of the two major topics of my colleague Lee O’Brien Andersen’s PhD work; Lee just defended his thesis this Friday and being involved in his work is some of the most interesting, rewarding, and challenging professional activities I’ve experienced so far. I will soon provide a link to an electronic version of his thesis here on this site. I hope that we will be able to fund his post doc aiming to expand his work on comparative Blastocystis genomics, since he only just started this work. Also, I hope that we will be able to do much more research on Blastocystis’ impact on host immunity and gut microbiota using in vitro and in vivo models. We need to know much more about to which extent Blastocystis can actually induce changes in bacterial communities and what these changes are. We also need to know whether manipulation of gut bacteria in a Blastocystis carrier can lead to eradication of the organism. 

Last week, I was so fortunate to oversee the production of an e-learning course in faecal microbiota transplantation (FMT) for Unite European Gastroenterology (UEG), which will probably appear online already in June. FMT is currently used primarily for treating recurrent Clostridium difficile infections, but the application range may extend far beyond this. The presentations included both theoretical and live sessions, and it was a lot of fun to do, not only because of the topic, but also because my colleagues at the Agostino Gemelli University Hospital in Rome were extremely professional, enthusiastic and well-organised. The reason why FMT is interesting in a Blastocystis context includes the fact that while there are quite standardized guidelines as to what is not allowed in donor stool, there is no consensus on what is actually allowed in the stool. Obviously, Blastocystis will often be present in donor stool, and when conventional microbiological methods are used to screen donor stool for pathogens, Blastocystis will only rarely be picked up. Hence recipients may receive stool containing Blastocystis. And so of course we would like to know whether to recommend using or excluding stool positive for Blastocystis (and other common parasites such as Dientamoeba) for FMT.

Friday, April 1, 2016

This Month in Blastocystis Research (MAR 2016)

I'm going to dedicate this post entirely to a recent case presented by my wonderful colleague Bobbi Pritt (Mayo Clinic) in collaboration with Blaine Mathison (CDC), whom I have also been so fortunate to meet.

Please go here to see the case.

Creepy Dreadful Wonderful Parasites: Case of the Week 390.

Let me use the opportunity to congratulate Bobbi Pritt on her fantastic work, admirable skills, and dedication to parasitology!

And by the way; why not treat yourself to Bobbi's 2016 parasite calendar available for purchase here.

Tuesday, December 29, 2015

This Month in Blastocystis Research (DEC 2015)

The potential pathogenicity of Blastocystis is something that has kept me preoccupied for more than a decade. Nonetheless, what I find perhaps even more interesting, is the overall role of Blastocystis in both health and disease.

And so, what do I mean by that?

Well, we just published a MiniReview in Journal of Clinical Microbiology (JCM) with the title: "Blastocystis in Health and Disease--Are we Moving from a Clinical to A Public Health Perspective?" I guess we were a bit lucky to get the paper published as a review, since it's probably more likely to be viewed upon as an Opinion paper, and so it would perhaps have been more suitable for a journal such as Trends in Parasitology. However, we would like medical doctors to be aware of our thoughts, and that's one of the reasons why we approached JCM.

Practically all Blastocystis research has focussed on identifying a role for the parasite in disease. Pathogenic properties have been identified for many other intestinal parasites since long; for Blastocystis, however, we still have no rockhard and reproducible evidence of
  • Outbreaks
  • Virulence-assoicated properties including invasiveness, phagocytosis, or adhesion to other cells
  • Symptom relief upon parasite eradication
Meanwhile, no one has really tried to looked into what Blastocystis may tell us about human health. Together with partner labs, our lab has produced data suggesting that Blastocystis carriage is extremely common, and probably also extremely long lasting. We have also shown that the parasite is associated with certain gut microbial communities and that it is more common in healthy individuals than in patients with IBD, IBS, etc. We have even identified intriguing data that suggest that Blastocystis may be less common in obese individuals compared with lean.



These are some of the most important reasons why I think that research into the public health significance of Blastocystis should be supported. We need to know much about what it means physiologically, microbiologically, and immunologically to be colonised, including 'what happens to our intestinal ecosystem when we are exposed to and colonised by Blastocystis?' Can we identify any benefits from colonisation, and if yes, which are these and can this knowledge be exploited with a view to producing drugs/probiotics that mimic any beneficient properties of Blastocystis? What does it mean to become colonised at an early age vs. only later in life?

In this regard, future areas of research could include studies on the ability of Blastocystis to
  • induce changes in bacterial communities in vitro and in vivo
  • assist in the metabolisation of food items (e.g., short-chain fatty acid metabolism)
  • promote stabilisation of gut microbiota
  • produce immunomodulatory and/or pro-/antibiotic substances, etc.

Happy New Year everone!

Reference:

Andersen LO & Stensvold CR (2015). Blastocystis in Health and Disease–Are We Moving from a Clinical to a Public Health Perspective? Journal of Clinical Microbiology PMID: 26677249

Sunday, November 1, 2015

This Month in Blastocystis Research (OCT 2015)

I'm actually going to skip the small review I do each month for a variety of reasons. Instead, I'm just going to upload a presentation I gave in Tilburg, The Netherlands, a bit more than a week ago, before attending the UEG Week in Barcelona.

I uploaded it to Google Drive, hoping that it will be easy to download for everyone interested. I have not included any notes, hoping that the slides will be pretty much self-explanatory.

I think there is even a bit of Danish in there, - hope you don't mind! Also, the preview option does not work very well, so make sure you download it.

If the presentation left you wondering a bit and wish for more, why not look up my publications listed in PubMed? They are available here.  Some of them can be downloaded for free.

Thank you for your attention.

Tuesday, September 1, 2015

This Month in Blastocystis Research (AUG 2015)

I would like to highlight a comment that we published in PLoS Pathogens, - a paper that is free for download here. It gained some attention on Twitter, and it was recently reviewed in the Faculty of 1000.

We basically highlight the tricky situation that we so often encounter in the field of clinical microbiology, namely the one in which all non-fungal organisms isolated from the human intestinal tract are being referred to collectively as 'parasites'. The word 'parasite' has a negative connotation, indicating that the organism exploits the host with detrimental effects on the host. While this is true for some ciliates, for instance Giardia, other ciliates may in fact be mutualists, which means that these organisms have adapted to a life within a host, providing the host with one or more advantages. One such example is seen in herbivores, where ciliates and flagallates break down cellulose.

In the clinical microbiology lab we face different types of organisms when dealing with stool samples: Giardia, Cryptosporidium and Entamoeba histolytica are considered true parasites, i.e. organisms benefitting from the environment of a host, at the expense of the host, and symptoms such as diarrhoea may develop, indicating host damage. Parasites such as Cryptospordium are usually infecting an individual for a short while, with immunity developing. Meanwhile, we also encounter eukaryotic organisms that are known to be able to colonise the intestine for a very long time, - decades, without being expelled by the host; Blastocystis belong to this group. For some reason it is as if the body 'tolerates' the presence of the organism. Maybe Blastocystis is good at evading local immune responses, or maybe the body wishes to 'keep' Blastocystis for some reason and so  developed a way to tolerate it... as I've hinted at before on this blog, maybe Blastocystis may assist us in one or more metabolic processes, for instance, either directly or indirectly, maybe by selecting for or influencing bacterial communities. Indeed, we recently found evidence of Blastocystis being specifically related to certain groups of bacteria, which, if confirmed, opens up for a whole new line of research, including the use of Blastocystis as a probiotic.

I know that this last sentence may sound harsh in some people's ears; nevertheless, most research involving Blastocystis so far has been quite static and unimaginative, and it's about time that food microbiologist and the like start taking an interest in the micro-eukaryotes that tend to be common and stable conolisers of our guts.

If YOU take an interest in this topic, I suggest you look up the articles cited below.

References and further reading:

Andersen LO, Bonde I, Nielsen HB, & Stensvold CR (2015). A retrospective metagenomics approach to studying Blastocystis. FEMS Microbiology Ecology, 91 (7) PMID: 26130823

Lukeš J, Stensvold CR, Jirků-Pomajbíková K, & Wegener Parfrey L (2015). Are Human Intestinal Eukaryotes Beneficial or Commensals? PLoS Pathogens, 11 (8) PMID: 26270819

Parfrey LW, Walters WA, & Knight R (2011). Microbial eukaryotes in the human microbiome: ecology, evolution, and future directions. Frontiers in Microbiology, 2 PMID: 21808637

Scanlan PD, Stensvold CR, Rajilić-Stojanović M, Heilig HG, De Vos WM, O'Toole PW, & Cotter PD (2014). The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota. FEMS Microbiology Ecology, 90 (1), 326-30 PMID: 25077936

Wednesday, February 4, 2015

This Month in Blastcystis Research - JAN 2015

I'm going to dedicate this blog post entirely to the upcoming 1st International Blastocystis Symposium.

I'm not sure how much advertising there is for this congress (our budgets are limited), but the fact that we are already receiving abstracts is a good sign! Abstacts may be submitted until April 1st, 2015. Please note that the 'early bird' registration discount expires at the 15th of February.

You will find the online abstract submission form here.

If you think about going but have not paid a visit to the official conference website, I recommend you to do so, clicking this link. You'll hopefully find most if not all the information that you'd be looking for, and there's a lot to be learned. Please also make sure to browse the social programme in order to be able to make appropriate arrangments.

It's a two-day symposium, running from the 28th to the 29th of May, 2015. Moreover, on the 27th, there will be an all-day workshop on various diagnostic and molecular epidemiological aspects, including a barcoding (subtyping) course. There will be more info on that very soon, - please keep an eye on the website.

We are doing all we can to attract scientists with vast experience in Blastocystis research to cover the floor with exciting and stimulating talks, and I think we're doing more than OK. Some of the speakers will be writing up reviews on their respective topics, and these reviews will appear in a special themed issue in Parasitology International.

There will be a quite a few prizes for best talks and posters, etc., thanks to ELSEVIER among others.

It will be one-track symposium, and the first day will focus mostly on some fundamental topics, such as genomics and biochemistry, while the next day will include talks on clinical and diagnostic data.

It's my clear impression that main organiser Dr Funda Dogruman-Al is working 25 hours a day to make everything come together, and Dr Hisao Yoshikawa has also already invested a lot of energy.

Again: please note that early registration will close at the 15th of February, and abstract submission deadline has been extended to April 1st, 2015.

Looking very much forward to seeing you there!

Thursday, May 1, 2014

This Month In Blastocystis Research (APR 2014)

Due to all sorts of activities I have not been able to update myself with 'novelties' in the scientific Blastocystis literature lately.

Instead, I would like to highlight two review/opinion papers on the use of PCR-based methods for diagnosis of intestinal parasitic infections in the clinical microbiology laboratory.

Both papers have been published very recently (actually one is still 'in press'). The first is co-authored by Jaco J Verweij and myself, and appears in the April issue of 'Clinical Microbiology Reviews'. This paper aims to provide a relatively systematic review of the extent and relevance of PCR- and sequencing-based methods for diagnosis and epidemiology studies of intestinal parasites, and is as such an inventory of all sorts of DNA-based diagnostic and typing modalities for individual protists and helminths.

The second one is authored solely by Jaco J Verweij and is currently in the 'first online' section in the journal 'Parasitology'. This paper offers a discussion of the application of PCR-based method as a supplementary tool or a substitute for conventional methods (microscopy, antigen detection, etc.). Dr Verweij deals with central questions such as 'Is Molecular Detection Good Enough?' and 'Is Molecular Detection Too Good To Be True?'.

And so these two papers complement each other quite well. For those interested in the very low prevalence of intestinal helminth infections in the Western world, the latter paper has a table which summarizes some quite stunning data.

Although DNA-based methods currently in use do have quite a few limitations, I do believe that for a long while the application of species- and genus-specific PCR methods (real-time PCR, conventional PCR + sequencing, etc.) will appear relevant and state-of-the-art. Dr Verweij, I and a few of our colleagues around the world are currently discussing to which extent next generation sequencing methods can be used to
  • generate data that can assist us in identifying the role of pro- and eukaryote microbial communities in health and disease
  • serve as a tool to generate sequences that can be processed by designated software and thereby identify patterns of microbial communities associated with various disease and health conditions
To this end, at the Laboratory of Parasitology, Statens Serum Institut, we are currently assisting in the development of a software called BIONmeta. BION meta is an open-source package for rRNA based pro- and eukaryote community analysis. Like Qiime and Mothur it is open source but with a growing number of advantages. The package has so far been developed mostly by Niels Larsen (DK), one of the original Ribosomal Database Project authors. It is as yet unpublished, but has been selected for in-house trial-use by companies and institutions that also partly sponsor its development.When relevant, I'll post more information on this software.

References:

Verweij JJ, & Stensvold CR (2014). Molecular testing for clinical diagnosis and epidemiological investigations of intestinal parasitic infections. Clinical Microbiology Reviews, 27 (2), 371-418 PMID: 24696439

Verweij, JJ. (2014). Application of PCR-based methods for diagnosis of intestinal parasitic infections in the clinical laboratory Parasitology, 1-10 DOI: 10.1017/S0031182014000419

Tuesday, November 5, 2013

A Shift of Paradigm in Blastocystis Research? Free paper in Trends in Parasitology!

As mentioned earlier, Dr Pauline Scanlan and I put together an opinion for Trends in Parasitology. This paper may possibly be one in a string of research and review papers heralding a shift of paradigm in Blastocystis research. I hope that it will stimulate the debate about the clinical significance of Blastocystis, and it can be downloaded for free all of this month; please go here for a free copy (go and look in the right side bar in the 'Featured Articles' section; there you will also find a free copy of the review on foodborne parasites mentioned in my previous blog post!).

Reference:

Scanlan PD, & Stensvold CR (2013). Blastocystis: getting to grips with our guileful guest. Trends in Parasitology PMID: 24080063

Sunday, September 8, 2013

Fellowships in Blastocystis Research

We are continually looking into the opportunity for funding for research in Blastocystis and we are on the lookout for young researchers with a MSc or PhD degree who want to spend at least a couple of years in Blastocystis research. Right now, taking an omics approach to studying the clinical significance of Blastocystis is extremely relevant of course, given the amount of genetic diversity of the parasite, its apparent association to groups of bacteria/bacterial richness, its varied distribution across different cohorts, and the general availability of ngs technology and data pipelines.

I'm going to focus my next funding application on the integration of metagenomics, metabolomics, comparative genomics, and transcriptomics, and I'm hoping to find one or two persons with track records documenting extensive experience with one or more of these disciplines and who take an interest in parasites/parasitic protists in general and/or in Blastocystis in particular.

Please note that this is NOT a job offer, but merely an invitation to get into some sort of a dialogue. What we can offer is access to samples, strains, technology, and a Blastocystis-centred network.

Please do not answer in the comments section, but contact me directly (mail/phone) for further info + expression of interest. You'll find a link to my contact details in the previous blog post. Thanks.

Wednesday, September 4, 2013

Yes, we do take orders!

I get an increasing amount of requests for Blastocystis testing (and testing for other parasites as well, for instance Dientamoeba fragilis). Initially, I was happy to do this for free, but now the requests are so regular that I need to add a fee to the tests.

And yes, we do take orders! As the regular reader of this blog would know by now, I run the part of our  Parasitology lab at Statens Serum Institut, Copenhagen, that deals with Blastocystis diagnostics and diagnostics for intestinal parasites in general. I have been developing and optimising molecular Blastocystis diagnostics for years, something which is also witnessed by my scientific production. Please note that we take orders only from health authorities. This means that if you want to have samples tested in our lab, you should contact your GP/specialist/whatever, and have him/her put the order through.

For general screening, I recommend real-time PCR analysis. For evaluation of treatment I recommend adding Blastocystis culture (a positive culture means ongoing Blastocystis infection, while DNA-based tools such as our real-time PCR will detect both dead and live organisms). We also perform subtyping of Blastocystis upon request.

In cases where colleagues want to outsource diagnostic work related to research, we are currently opening up for the possibility of testing large panels of faecal samples (fresh, frozen, or ethanol-preserved) for Blastocystis, Dientamoeba fragilis or other parasites by molecular assays (including DNA extraction) - and - if requested - in combination with traditional microscopy of faecal concentrates.

A selection of our analyses for parasites can be viewed here.

Our parasitology lab is merged with the mycology lab, and therefore we have plenty of opportunity to test the same stool sample for parasites and yeasts (e.g. Candida), if requested. As a new feature, Blastocystis+Dientamoeba+Candida analyses can now be requested in combination as a 'package' with a discount. We are happy to send out test tubes and transport envelopes, but I repeat that charges will apply.

Research-wise, we are currently taking different approaches to detecting and differentiating non-human eukaryotic DNA/RNA in human faecal samples, among these the GUT 18S approach.

For further inquiries and information, please do not hesitate to contact me (contact details can be found here).

Relevant articles on molecular diagnostics for Blastocystis detection and subtyping:

Stensvold CR, Ahmed UN, Andersen LO, & Nielsen HV (2012). Development and evaluation of a genus-specific, probe-based, internal-process-controlled real-time PCR assay for sensitive and specific detection of Blastocystis spp. Journal of Clinical Microbiology, 50 (6), 1847-51 PMID: 22422846

Stensvold CR (2013). Comparison of sequencing (barcode region) and sequence-tagged-site PCR for Blastocystis subtyping. Journal of Clinical Microbiology, 51 (1), 190-4 PMID: 23115257

Friday, May 10, 2013

Cell Symposium: Microbiome & Host Health - Lisbon 2013

My colleagues from Statens Serum Institut and I are heading to Lisbon, Portugal, tomorrow morning to attend the Cell Symposium on Microbiome and Host Health (link may be really busy now).

We are bringing a poster displaying some of our work related to our GUT18S project: A Novel Approach For Eukaryotic Phylogenetic Interrogation Of Clinical Samples Using Next Generation Sequencing Of SSU rRNA Genes; a pdf version of the poster can be downloaded here.

The GUT18S work is partly funded by the Marie Curie Actions (FP7) program.

Sunday, May 5, 2013

More on 'Bugs as Drugs'

This morning, I was doing a lazy ramble through my favourite blogs and found a post by Carl Zimmer on 'Bugs as Drugs' - primarily on probiotics. And I just came to realise that there is a very interesting tendency these years of using bugs as drugs in a variety of fields.

We are all very much aware of the worries about the increase in antibiotic resistance in bacterial and other pathogens. Moreover, it appears that sometimes antibiotic treatment leads to imbalance in the intestinal microbiota (dysbiosis); a well-known example is intractable Clostridium difficile infections which can potentially lead to pseudomembranous colitis.

C. difficile infection can lead to pseudomembranous colitis
Earlier this year, an article appeared in the renowned The New England Journal of Medicine on a randomised, controlled treatment study on duodenal infusion of donor faeces for recurrent C. difficile. The researchers found that the infusion of donor faeces was significantly more effective for the treatment of recurrent C. difficile infection than the use of vancomycin, the drug usually recommended in this situation. In fact 15/16 patients had resolution of C. difficile-associated diarrhoea upon first or second infusion; however, it might be worthwhile 'shopping around' for the right donor.

And so, how are these faecal transplants developed and administered? Well, it appears that donors are volunteers who have been through a selection process based on a questionnaire on risk factors of infectious diseases. Then donor faeces is screened for parasites (including Blastocystis and Dientamoeba - yes, it warms my heart to see this so explicitly spelled out in the paper... but I wonder which methods were used - it doesn't say!) and enteropathogenic bacteria. Moreover, blood samples from donors are screened for e.g. HIV, hepatitis and antibodies against e.g. Entamoeba histolytica and Strongyloides. Next, a donor pool is created with repeated screening every 4 months. On the day of infusion, faeces is collected by the donor and immediately brought to the hospital, where it is diluted with 500 mL of sterile saline. The solution is stirred, and the supernatant strained and poured in a sterile bottle. Within 6 h after collection of the faecal sample by the donor, the solution is infused through a nasoduodenal tube (2 to 3 mintues per 50 mL). Patients are subsequently monitored for 2 h. Apparently, this is how it works!

Thursday, March 21, 2013

LUMINEX xMAP Technology in Parasite Diagnostics

Over the past few years nucleic acid based methods have revolutionised parasite diagnostics in modern clinical microbiology (CM) labs. Real-time PCR is really gaining a foothold in CM labs, but despite the opportunity for plexing, mostly only up to 6 DNA targets can be included in each assay (due to the number of available channels).

LUMINEX xMAP technology used for detection of specific nucleic acids (Dunbar, 2006) bypasses this limit, and up to 100 DNA targets can be included in one single assay in a 96-well plate format. You can read about the technology here.


Saturday, February 2, 2013

Blog Feedback

I'm very thankful for all the positive feedback I get from readers across the globe, mostly by email. Due to time limits I can only respond to 5-10% of the mail, and I'm sorry for not getting back to the rest of you.

Meanwhile, this blog currently holds more than 60 posts, and you will also find a lot of key words in the right side bar, so take your time and browse a few posts or look up a few relevant key words, -  you might find an answer to one or more  of your questions.

Having said that, I try to read all my email, and I am listening! The feedback and questions that I get are vital for our work and help us identify the avenues that we need to take to unveil the many mysteries of Blastocystis.

And let me just say this for now: A proper microbiological work-up (by state-of-the-art methods, including PCR for intestinal parasites), is something that is offered on a routine basis in only very few laboratories, and also the number of clinically orientated Blastocystis research centres can be counted on one hand, I believe. Subtyping of Blastocystis is currently done mostly in epidemiological surveys (as part of research projects), and I suspect that our lab is one of the very few labs in the world doing subtyping on a routine basis.

Oh, and I've been asked by some readers about how to get blog updates. It's easy: You can follow this blog by email, - just scroll down and find "follow by email" in the right side bar and enter your email address. You can also subscribe to posts via atom (go to the very bottom of the page).

And then here's a little something about stomach acidity and intestinal microbiota from Scientific American, - but make sure to read the comments underneath the post too!
 

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:
Clark CG, & Diamond LS (2002). Methods for cultivation of luminal parasitic protists of clinical importance. Clinical microbiology reviews, 15 (3), 329-41 PMID: 12097242

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
 
Munasinghe VS, Stark D, & Ellis JT (2012). New advances in the in-vitro culture of Dientamoeba fragilis. Parasitology, 139 (7), 864-9 PMID: 22336222

Friday, October 26, 2012

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

"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:
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

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, 55 (10), 1431-2 PMID: 22893582

Saturday, June 2, 2012

Blastocystis and Microbiomology

Speaking of metagenomics: The July 2012 issue of one of the most prestigious journals in the field of clinical microbiology, Clinical Microbiology and Infection (CMI – published by European Society of Clinical Microbiology and Infectious Diseases), focuses entirely on recent advances in metagenomics, including its implications on clinical microbiology. Several of the keynote speakers from the MetaHIT conference in Paris (March, 2012) have contributed with papers, including Rob Knight, Willem M. de Vos and Paul W. O’Toole. In his editorial, Didier Raoult, puts emphasis on mainly two things: 1) that we need to be patient with data obtained from studies using metagenomics, since currently some conclusions are pointing in different directions and data are still scarce, and 2) that metagenomic studies should be independent of financial support from commercial sources, such as the industry of antibiotics and probiotics.

Although it may be too early to make b/w inferences from data already published, I think that the pioneers in metagenomics teach us to re-think or at least modify several hypotheses about the role of intestinal microbes in gastrointestinal health and disease and pursue new and exciting trajectories. In this blog post I would like to highlight a few things that may be interesting to people who are not familiar with metagenomics, but who are interested in our gut flora and how it may impact our lives.

So, what is metagenomics? Well, only a few years ago, microbiologists were used to looking at one single organism at a time, when exploring the potential role of an organism in health and disease. They were dependent on isolating the organism, for instance by culture, in order to have sufficient material for molecular studies, and in order to avoid mix-up of data from contaminating organisms. However, the human intestinal microbiome (gut flora) is made up by a plethora of organisms, mainly prokaryotes (bacteria), but also to some extent eukaryotes (parasites and fungi), archaea and viruses. Metagenomics, facilitated by massive high-throughput parallel sequencing of nucleic acids extracted from human faecal samples, allows us to get a holistic picture of the entire gut flora of a person. I.e.: We move from examining one single species or organism at a time, to be analysing entire eco-systems. We get to know not only the composition of microbic species inhabiting our gut, but also how they impact our body physiology: Interestingly, Gosalbes et al. (2012) describe how the composition of the intestinal flora may differ significantly from person to person, but later shows that the active intestinal flora is fairly similar among healthy individuals. So, what’s the active flora? Briefly: while metagenomics analyses the DNA (16s rDNA) from the microbiome and hence provides us with data on the mere composition of microbes, including a quantification, metatranscriptomics looks at RNA communities by looking at 16S rRNA and mRNA transcripts. In this way, we get to know the function of the intestinal microbiota and can temporarily ignore the part of the microbial community that is in “stand-by” mode only. The collective genome of the intestinal microbes vastly surpasses the coding capacity of the human genome with more than 3 million genes - in comparison the human genome comprises 20,000-25,000 protein-coding genes.

So far, metagenomic studies have focused mainly on bacteria, and hence we know very little about how intestinal parasites directly or indirectly impact the remaining gut flora and the host, and, importantly, how the bacterial flora influences the presence and activity of parasites. This is due in part to methodological limitations, but mainly to the fact that the bacterial microbiome can be viewed as an organ of the human body (Baquero et al., 2012) taking care of vital and irreplaceable functions that the host is not otherwise capable of, ranging from energy and vitamin metabolism to epithelial barrier integrity and immune modulation (Salonen et al., 2012). Like any other organ, the microbiome has physiology and pathology, and the individual (and collective?) health might be damaged when its collective population structure is altered (Baquero et al, 2012). This is one of the reasons why studies of host-gut flora interactions have focused on bacteria.

One of the striking findings in metagenomic studies is that humans can be more or less successfully stratified into three enterotypes based on their intestinal flora (Arumugam et al., 2011):


We see that the three enterotypes are dominated by mainly three different types of bacteria (Bacteroides, Prevotelia and Ruminocoocus, respectively). However, as mentioned earlier, functional analysis (and probably a lot more sampling) is required to understand microbial communities. One of the interesting topics in this respect is how enterotypes correlate to different health/disease phenotypes; i.e. whether people with a certain gut flora are more prone to (a) certain type(s) of disease(s).There is preliminary evidence that variations in the microbiota are linked to diseases including bowel dysfunction and obesity.

In terms of parasites, I believe that in the near future we will see data revealing to which extent - if any - common intestinal micro-eukaryotes such as Blastocystis and Dientamoeba correlate with these enterotypes or other subsets of bacteria which will enable us to generate hypotheses on the interaction of micro-eukaryotes and the bacterial flora, which in turn may impact host physiology. I will expand a little more on this in an upcoming letter in Trends in Parasitology (article in press).

Interested in more: Why not have a look at Carl Zimmer's article in The New York Times about gut flora transplantation, or read about modulating the intestinal microbiota of older people to promote enhanced nutrition utilisation and to improve general health (O'Toole et al., 2012)... Also, have a look at my most recent blog post.

Literature:

O’Toole, P. (2012). Changes in the intestinal microbiota from adulthood through to old age Clinical Microbiology and Infection, 18, 44-46 DOI: 10.1111/j.1469-0691.2012.03867.x  

Gosalbes, M., Abellan, J., Durbán, A., Pérez-Cobas, A., Latorre, A., & Moya, A. (2012). Metagenomics of human microbiome: beyond 16s rDNA Clinical Microbiology and Infection, 18, 47-49 DOI: 10.1111/j.1469-0691.2012.03865.x  

Baquero, F., & Nombela, C. (2012). The microbiome as a human organ Clinical Microbiology and Infection, 18, 2-4 DOI: 10.1111/j.1469-0691.2012.03916.x  

Salonen, A., Salojärvi, J., Lahti, L., & de Vos, W. (2012). The adult intestinal core microbiota is determined by analysis depth and health status Clinical Microbiology and Infection, 18, 16-20 DOI: 10.1111/j.1469-0691.2012.03855.x

Arumugam, M., Raes, J., Pelletier, E., Le Paslier, D., Yamada, T., Mende, D., Fernandes, G., Tap, J., Bruls, T., Batto, J., Bertalan, M., Borruel, N., Casellas, F., Fernandez, L., Gautier, L., Hansen, T., Hattori, M., Hayashi, T., Kleerebezem, M., Kurokawa, K., Leclerc, M., Levenez, F., Manichanh, C., Nielsen, H., Nielsen, T., Pons, N., Poulain, J., Qin, J., Sicheritz-Ponten, T., Tims, S., Torrents, D., Ugarte, E., Zoetendal, E., JunWang, ., Guarner, F., Pedersen, O., de Vos, W., Brunak, S., Doré, J., Consortium, M., Weissenbach, J., Ehrlich, S., & Bork, P. (2011). Enterotypes of the human gut microbiome Nature, 474 (7353), 666-666 DOI: 10.1038/nature10187