Showing posts with label Public Health. Show all posts
Showing posts with label Public Health. Show all posts

Monday, August 1, 2016

This Month in Blastocystis Research - Interactive Edition

What are your thoughts on Blastocystis carriage and age?

More and more data suggest that the prevalence of Blastocystis carriage increases by age - at least to a certain point.

Some intestinal parasites, such as Cryptosporidium, may not be that uncommon in infants/very young toddlers, while they are much less common in older children and adolescents. Other parasites appear to peak in prevalence around the age of 7, e.g., Dientamoeba fragilis.

Meanwhile, Blastocystis appear to increase in prevalence by age until mature adulthood... why is that? And what does it tell us? Please comment! I'm not having all the answers to these questions myself, and if some interesting suggestions pop up, I'll post them! You only need a Google account to be able to comment. If you don't have one, please send your comment using

crs[at]blastocystis.net 

For those interested in Blastocystis carriage in association with age, I have listed a couple of relevant recent studies below.

References:

Forsell J, Granlund M, Samuelsson L, Koskiniemi S, Edebro H, & Evengård B (2016). High occurrence of Blastocystis sp. subtypes 1-3 and Giardia intestinalis assemblage B among patients in Zanzibar, Tanzania. Parasites & Vectors, 9 (1) PMID: 27356981  

Poulsen CS, Efunshile AM, Nelson JA, & Stensvold CR (2016). Epidemiological Aspects of Blastocystis Colonization in Children in Ilero, Nigeria. The American Journal of Tropical Medicine and Hygiene, 95 (1), 175-9 PMID: 27139454

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   

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

Tuesday, December 1, 2015

This Month in Blastocystis Research (NOV 2015) - Persian Gulf Edition

Today is the first time an Airbus A380 will be landing in Copenhagen Airport, Denmark. Flying in from Dubai, it will mark the inauguration of a runway that was recently refurbished to enable accommodation of a plane of this size.

I therefore thought I'd make a tribute to this particular day by dedicating the "This Month in Blastocystis Research" post to studies on Blastocystis recently published by researchers based along the Persian Gulf. Three surveys on Blastocystis from this region recently made it to parasite/microbiology research journals. The studies are important since they represent examples of studies employing molecular tools for screening and molecular characterisation of parasite isolates identified in regions where such data are extremely scarce. Some of these data will enable us to better understand host specificity, differences in geographic distribution, clinical and public health significance, and transmission patterns.

 The first study was on Blastocystis in Qatar and published in Acta Tropica; it was already mentioned in my September blog entry.

I was lucky to be involved in the second study, which was a study carried out in Sharjah, United Arab Emirates, and designed by Ali ElBakri and colleagues. In this study, we screened a total of 133 samples from ex-pats living in Sharjah, subtyping the samples positive for Blastocystis using partial small subunit (SSU) ribosomal RNA gene sequencing. Fifty-nine (44.4%) samples were positive, of which 39 were successfully sequenced and subtyped. The ST distribution was as follows: ST3, 58.9% (23/39); ST1, 28.2% (11/39); and ST2, 7.6% (3/39). This study is the first to provide data on the prevalence of Blastocystis and the distribution of various STs in the UAE. As usual, ST4 was absent, while ST1, ST2, and ST3 were all common in this geographical region; a situation similar to most other regions outside of Europe.

The third study was from the city of Baghmalek in Southwestern Iran, and was published by Khoshnood and colleagues in Jundishapur Journal of Microbiology. This team used microscopy to identify Blastocystis in 1,410 stool samples from patients presumably suffering gastrointestinal symptoms. A very low prevalence was identified, about 3%. This low figure most likely reflects the use of microscopy, which is an extremely insensitive diagnostic method. From Blastocystis-positive samples, DNA was extracted and submitted to PCR and sequencing targeting the (SSU) ribosomal RNA gene. It says in the article that the subtypes identified in the study included "ST3, ST4, ST5, and ST7 with the most prevalent being ST4 (40.9%)", and the main conclusion is that, unlike the situation in other countries in the Middle East, ST4 was identified as the most prevalent subtype.

There are at least two conspicuous situations here: The first one pertains to the rather unusual subtype distribution reported, which appears quite dissimilar to the ones reported from neighbouring countries. The next one is even more odd and pertains to the fact that the sequences (AB915194 - AB915214) generated in the study, and from which the subtype data must have been inferred, do not BLAST to other nuclear ribosomal RNA genes in GenBank, of which there are thousands! In fact, AB915194 represents a protein-coding gene, translating into  

S P Y L L S I S T E E S Y T D S H Y Y G E C T T I A Q S I Y H Q S S K S V E A S I W D C V Y Met T L I Y E G V T D L T Y D E M K A S Y T D P V E T L T V L G K Y P G A D I S G I S L D L V F G Y I G R G I P V I S R I N D G R Y V L I V S Y N S E A V R Y Y D P V L D E Q V R K Q

... which is a Clostridium hypothetical protein with a peptidase domain! This may either reflect an error linked to the accession numbers, or it may reflect a situation where for some reason non-ribosomal DNA sequences were uploaded to GenBank. Given the appearance of the phylogeny included in the article, it could easily be suspected that the sequences produced and used were in fact non-Blastocystis DNA sequences, in which case the paper should be retracted. Before this mystery has been solved, the results of the Iranian study cannot be fully appreciated, and the relevance of citing the study appears very limited for now.

The last study highlights the importance of making sequence data publicly available; if these data had not been available for critical appraisal, the conclusions made in this article could easily have been accepted without any further ado!

References:

Abu-Madi M, Aly M, Behnke JM, Clark CG, & Balkhy H (2015). The distribution of Blastocystis subtypes in isolates from Qatar. Parasites & Vectors, 8 PMID: 26384209

AbuOdeh R, Ezzedine S, Samie A, Stensvold CR, & ElBakri A (2015). Prevalence and subtype distribution of Blastocystis in healthy individuals in Sharjah, United Arab Emirates. Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases PMID: 26611823 

Khoshnood S, Rafiei A, Saki J, & Alizadeh K (2015). Prevalence and Genotype Characterization of Blastocystis hominis Among the Baghmalek People in Southwestern Iran in 2013 - 2014. Jundishapur Journal of Microbiology, 8 (10) PMID: 26587213 

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 30, 2014

This Month in Blastocystis Research (SEP 2014)

Before leaving for Venice and Padova to introduce Blastocystis to the XXX National Congress of The Italian Society of Protistology (ONLUS), allow me to kick in just a few words for the September issue of 'This Month in Blastocystis Research'.

I will highlight two papers.

The first is a study from the US (Yes, - US data! How rare is that?). The team investigated the prevalence and subtype distribution of Blastocystis among client-owned and shelter-resident cats and dogs. Studies of Blastocystis in companion animals are actually quite rare. The authors used nested PCR for detection, followed by sequencing of PCR products. Interestingly, Blastocystis was not detected in any of the >100 fecal samples from client-owned animals. By comparison, Blastocystis was detected in 10/103 (9.7%) shelter-resident canines, and 12/103 (11.65%) shelter-resident felines. There was no significant difference in Blastocystis spp. carriage rates between the shelter-resident dogs and cats. It is likely that differences in diet and other types of exposure account for Blastocystis being found in shelter-resident animals and not in domestic animals. As for cats and dogs, we don't really know much about what to expect subtype-wise. These animals harboured ST10 mostly, a subtype that has only been found in artiodactyls, NHPs, and lemurs, so far, and - taking these new data into account - with little apparent host preference.

Viktor - an avid fox hunter (in 2007).
Other subtypes included ST1 and one case of ST3, and one case of what was most likely a new subtype - maybe! But then, few animals were positive, and given the different data on subtypes in cats and dogs, it's much too early to speculate on host specific subtypes... for now, it appears that there are none, and that maybe cats and dogs are not really natural hosts? A study by Wang and colleagues identified a plethora of subtypes in dogs: Among 22 positive dogs, most of which were from India, ST1, ST2, ST4, ST5, and ST6 were found. Again, nested PCR was used, and I might have a slight concern that this type of PCR approach is so sensitive that it will pick up the smallest quantity of Blastocystis, maybe even dead Blastocystis or other stages of Blastocystis not necessarily colonising the host (contamination, etc.). But I don't know. The authors of the US study noted that Blastocystis was unlikely to be associated with disease of the animals and were unable to establish a reservoir for human colonisation/infection in these animals.

I never got around to checking Viktor (our cat, pictured above) for Blastocystis. Now it's too late.

I would like to move on to another study. This time the data is from a paper that has just appeared in press in Clinical Gastroenterology and Hepatology. We  analysed faecal DNAs from patients diagnosed with irritable bowel syndrome and healthy individuals. The reason for doing this was due to the fact that intestinal parasite have been speculated to play a role in the development of IBS, a disease affecting about 16% of the adult Danish population. And so we thought that the prevalence of common parasites such as Blastocystis and Dientamoeba fragilis might be higher in IBS patients than in healthy individuals. The study was led by Dr Laura R Krogsgaard, who took a quite unusual approach to collecting questionnaires and faecal samples, namely by collaborating with the company YouGov Zapera.  
We obtained faecal samples from 483 individuals, of whom 186 were cases – ie. patients with IBS – and 297 were healthy controls. DNA was extracted directly from the stool using the easyMag protocol, and the faecal DNAs were submitted to real-time PCR based screening for Blastocystis, Dientamoeba, Entamoeba histolytica and E. dispar, Cryptosporidium, and Giardia.



Above you see the results of the various analyses. Blue columns represent healthy individuals, and orange columns represent IBS patients. Fifty percent of the healthy controls were positive for one or more parasites, while this proportion was significantly lower in IBS patients, 36%. Also for each individual parasite, the number of positive cases was higher among controls than among patients with IBS. Dientamoeba was the most common parasite among healthy controls and IBS patients. In terms of Blastocystis subtypes, the distribution of subtypes between the two groups was non-significant (data not shown).We ended up by concluding that our findings indicated that these parasites are not likely to play a direct role in the pathogenesis of IBS. Longitudinal studies are required to understand their role in gastrointestinal health. 

Still, the role of Blastocystis in human health and disease remains ambiguous, although lots of interesting data is emerging. In order to try and understand the theories behind Blastocystis' potential able to generate disease, I would like to point the readers' attention to a new review, developed by Ivan Wawrzyniak and his prolific colleauges.

Ciao!

References

Krogsgaard LR, Engsbro AL, Stensvold CR, Vedel Nielsen H, & Bytzer P (2014). The Prevalence of Intestinal Parasites is not Greater Among Individuals with IBS: a Population-Based Case-Control Study. Clinical Gastroenterology and Hepatology : the official clinical practice journal of the American Gastroenterological Association PMID: 25229421

Krogsgaard LR, Engsbro AL, & Bytzer P (2013). The epidemiology of irritable bowel syndrome in Denmark. A population-based survey in adults ≤50 years of age. Scandinavian Journal of Gastroenterology, 48 (5), 523-9 PMID: 23506174

Ruaux CG, & Stang BV (2014). Prevalence of Blastocystis in Shelter-Resident and Client-Owned Companion Animals in the US Pacific Northwest. PloS One, 9 (9) PMID: 25226285  

Wang W, Cuttell L, Bielefeldt-Ohmann H, Inpankaew T, Owen H, & Traub RJ (2013). Diversity of Blastocystis subtypes in dogs in different geographical settings. Parasites & Vectors, 6 PMID: 23883734

Wawrzyniak I, Poirier P, Viscogliosi E, Dionigia M, Texier C, Delbac F, & Alaoui HE (2013). Blastocystis, an unrecognized parasite: an overview of pathogenesis and diagnosis. Therapeutic Advances in Infectious Disease, 1 (5), 167-78 PMID: 25165551 

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, April 1, 2014

This Month In Blastocystis Research (MAR 2014)

If there's one paper that really made my eye balls pop over the past 30 days, it's the paper appearing a couple of days ago in BMC Infectious Diseases by Safadi et al. on Blastocystis in a cohort of Senegalese children. The paper is open access and can be downloaded here. But I'll be jumping right at it:

A 100% prevalence of Blastocystis in a cohort of 93 Senegalese children! 

The children represented a mixed group of children with and without symptoms. And yes, they were all colonised!

Are Senegalese children obligate carriers of Blastocystis? Image courtesy of whl.travel.
I will not at all try and discuss the potential clinical implications of this. I don't think we currently have the appropriate tools to ascertain to which extent a 100% Blastocystis prevalence is a public health problem. 

However, technically and scientifically, I'm extremely pleased to see a study like this one. My group and some of my colleagues have somewhat similar data in the pipeline, and it's great to see this next generation of survey data emerging from different regions of the world, based on the use of highly sensitive molecular tools to screen for Blastocystis. I cannot emphasise the importance of this too much.

The authors hoovered faecal samples from the children for Blastocystis-specific DNA using both PCR + sequencing (barcode region) and real-time PCR. Importantly, quite a few samples negative by barcoding were positive by real-time PCR, and so if the authors had included only PCR + sequencing, the prevalence would have been only 75% or so. It may be not very surprising that barcoding PCR did not pick up all cases of Blastocystis, but then again, it has always been known that the barcoding PCR is not diagnostic - one of the primers, RD5, is a general eukaryotic primer, while the other one, BhRDr is Blastocystis-specific. Also, the PCR product is about 600 bp; diagnostic PCRs should preferably be designed to produced much smaller amplicons (100 bp or so) for a variety of reasons.

The research team subtyped all samples, and found ST3 to be the most prevalent subtype - colonising about 50% of the children. ST1 and ST2 were also common, while ST4 was found in only 2 children and only in mixed infections. Mixed subtype infections was seen in 8 cases. Note the small fraction of ST4. This subtype is very common in Europe but seems to be rare in most other regions.

There is no doubt that we with molecular tools are now starting to obtain data that represent a more precise snapshot of reality than before when tools of low sensitivity and unable to give strain information were used. And while qPCR can take us a long way in terms of precisely distinguishing positive from negative samples, we still have an amplification step that may interfere with the DNA information that we obtain. The French group involved in this study has over multiple studies done  an admirable job in terms of pursuing the extent of mixed subtype infections. Whether the data are based on sequencing of PCR products amplified by genus-specific primers, or whether real-time PCR  using genus-specific primers is used, it can still be argued that these methods have limitations due to application of genus-specific primers in both cases. It is going to be interesting to compare the evidence that we have collected from subtyping over the past few years with analysis of metagenomics data, which are independent of PCR amplification, and thus not subject to potential bias. 

A 100% prevalence means that transmission pressure is massive. Three subtypes are common. Still, mixed infections are present in less than 10%. If this is indeed a realistic picture, this may imply that once established, a Blastocystis strain is capable of keeping other strains at bay? In keeping with waht I said above, it is also possible that the extent of mixed infections is higher, and that the PCR methods only detect the more predominant strain, making the prevalence of mixed ST infection seem low.

It's tempting to believe that such a high prevalence of Blastocystis compared to Europe is due to exposure to contaminated water, but how does this explain a whopping 30% Blastocystis prevalence in the background population in Denmark, a country characterised by supreme hygienic standards and 'perfect plumbing' with all potable water being pumped up from the ground (ie. hardly no surface water)? Have all individuals positive for Blastocystis in Denmark been out traveling to more exotic countries with less well controlled water infrastructures? Or is Blastocystis just highly transmissible through e.g. direct contact? And will all who are exposed develop colonisation? What are the determinants? It's probably not fair to dismiss the idea of Blastocystis being waterborne (as one of the modes of transmission) due to the fact that Blastocystis has not been cause of waterborne outbreaks. If Blastocystis is non-pathogenic, it can easily be transmitted by water. In fact, if Blastocystis is waterborne and never gives rise to outbreaks, what does this tell us about it's pathogenic potential? Well, acute disease such as that seen for some bacteria, viruses, and Cryptosporidium, Giardia and microsporidia is probably not something that is associated with the organism.

I could have wished for allele analysis of the subtypes detected. It should be possible in all cases where barcode sequences were available, - simply and easy using this online tool. But the data is available in GenBank so everyone interested can have a look. 

There is plenty of interesting things to address, but for now I'll leave it here, and on behalf of all of us interested in Blastocystis research just thank the people behind the paper for publishing this important study!

And nope, this is no April Fool!

Literature:

El Safadi D, Gaayeb L, Meloni D, Cian A, Poirier P, Wawrzyniak I, Delbac F, Dabboussi F, Delhaes L, Seck M, Hamze M, Riveau G, & Viscogliosi E (2014). Children of Senegal River Basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infectious Diseases, 14 (1) PMID: 24666632

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

Stensvold CR (2013). Blastocystis: Genetic diversity and molecular methods for diagnosis and epidemiology. Tropical Parasitology, 3 (1), 26-34 PMID: 23961438

Friday, November 22, 2013

Do IBS Patients Lack Blastocystis and Dientamoeba??

I feel like sharing data from a poster created by one of my colleagues, Dr Laura Rindom Krogsgaard who works at Køge Sygehus, Denmark. She presented the poster last month at the United European Gastrointestinal (UEG) Week in Berlin.

Laura is currently doing a very interesting survey on IBS and IBS-like symptoms in Danish individuals. Her first publication was on the epidemiology of IBS in Denmark (see literature list below). She performed a web-based survey, using YouGov Zapera, and questionnaires were emailed to a web panel (n = 19,567) representative of the general Danish population aged 18-50 years containing info on gender, age, geography and type of intestinal symptoms (if any). IBS and subtypes were estimated by the Rome III criteria. Of 6,112 responders, 979 (16%) fulfilled the Rome III criteria for IBS and had no organic diagnosis likely to explain their symptoms. IBS subtypes detected included  mixed IBS (36%), IBS with diarrhea (33%), IBS with constipation (18%), and unsubtyped IBS (11%).

At the Laboratory of Parasitology, we helped Laura analyse stool samples from survey participants for parasites. Not surprisingly, Blastocystis and Dientamoeba were by far the most common parasites detected; however, it appeared that individuals with IBS symptoms were less likely to be colonised by these parasites than their controls! Which means that we have a situation reminiscent of that seen in IBD patients, only less pronounced. 

Laura was able to survey symptom developement over 1 year and compare this to the incidence of Blastocystis and Dientamoeba, and none of the parasites (indvidually or in co-infection) were linked to symptom development.

Indeed, Laura's data are in line with the general tendency that we see for Blastocystis (see figure below). Blastocystis appears to be rare in individuals with perturbation of the intestinal microbiota (due to antibiotic treatment, inflammation, infection, diet, etc.), while common in healthy individuals, most of whom are probably characterised by high gut microbial diversity and thereby - apparently - the right substrate/growth conditions for Blastocystis.


Literature:

Krogsgaard LR, Engsbro AL, & Bytzer P (2013). The epidemiology of irritable bowel syndrome in Denmark. A population-based survey in adults ≤50 years of age. Scandinavian Journal of Gastroenterology, 48 (5), 523-9 PMID: 23506174

The entire poster "Dientamoeba fragilis and Blastocystis: Two parasites the irritable bowel might be missing" presented at the UEGweek can be viewed here via SlideShare.


Friday, April 26, 2013

This Month in Blastocystis Research (APR 2013)

I've been extremely bored all day writing up my evaluation of a (not so interesting) PhD thesis, and I thought I'd spice up my day by introducing a new series of posts on this blog inspired by so many other blogs, namely: This Month in Blastocystis Research! A place for me to go through some of the most recent papers on Blastocystis.

There is paper out by Gould and Boorom who look at the stability of Blastocystis surface antigen over time. They show that detection of Blastocystis by an immunofluorescense assay (IFA) is not hampered after1 year of storage of faecal material in formalin compared to results immediately after the sampling point. Detection of Blastocystis by IFA is something that is not often used (that's my impression, anyway), but makes sense in cases where laboratory analyses can be performed only weeks-months after sample collection (e.g. during field work), in which case samples need to be preserved. We usually, however, recommend storing faecal material in (70%) ethanol (in the relationship 1 part faecal sample to 4 parts of ethanol), where the sample is mixed with the ethanol initially by vortexing the tube (typically a 2 mL Eppendorf tube) for 5-10 min, and subsequently keeping the tubes away from light until further processing. Importantly, in contrast to formalin-fixed stool, ethanol-fixed stool can be made highly suitable for PCR by just washing the samples x3 in PBS prior to DNA extraction. An example of this methodology can be seen in our study of Blastocystis in members of the Tapirapé tribe in Mato Grosso, Brazil (go here for a free download).

I'd wish that Gould and Boorom had validated their findings by running a PCR on the samples too (specificity and sensitivity testing). The IFA assay was also used in a publication from 2010 by Dogruman-Al et al., who found a diagnostic sensitivity of the IFA assay of 86.7% compared to culture; also here, adding PCR would have been relevant to better determine the diagnostic qualities of the IFA assay.

I was lucky to be involved in field work in the Lao PDR in 2003 conducted by regional WHO authorities; preserving and analysing faecal samples for parasites by microscopy (Kato Katz) and - later - PCR was what we did!

Adding to the endless row of cross-sectional prevalence papers, there is an article out just now by Abdulsalam et al. (2013) on Prevalence, predictors and clinical significance of Blastocystis sp. in Sebha, Libya (free for download here). The study used culture (Jones' medium) as diagnostic modality and confirmed the existence of frequent asymptomatic carriage. The authors used questionnaire info and multivariate statistical analysis to identify risk factors for Blastocystis carriage among 380 individuals aged 1-75, and what I find really interesting is that they found that participants aged > 18 years were much more prone to having Blastocystis than participants < 18 years (P < 0.001). This is something that we see in Denmark too, and I'm currently trying to collect "sufficient proof"! Whether this is an age accumulation effect due to the chronicity of colonisation remains to be investigated. The authors also found that carriers were more likely to experience symptoms than those who were not carriers (P < 0.001), mainly abdominal pain (P < 0.001), but notably not diarrhoea (P = 0.117).
It's a pity that molecular data was not included the study from Libya. Incidentally, our group recently published subtype data from Sebha, Libya, and it appears that Blastocystis found in humans in Libya mainly belongs to ST1, whereas ST3 is often the most common subtype in most other countries, and what is more: ST4 appears virtually absent in Libya and the rest of Africa... But let's see: The investigators might have more data up their sleeve waiting to be published...

May I also again draw your attention to our recent paper on Blastocystis in non-human primates, in which we find that despite the fact that there is a great overlap of subtypes in human and non-human primates, it appears that ST1 and ST3 strains found in non-human primates differ genetically from those found in humans, indicating cryptic host specificity. We have additional data supporting the theory that Blastocystis in humans is a result of human-to-human transmission (anthroponotic) rather than animal-to-human (zoonotic) transmission. Which is really interesting, since the theory of zoonotic transmission of Blastocystis has been heavily (I dare not say purported, so I'll say) propagated. Having said that, I think we still need to look much deeper into barcoding of Blastocystis from pets and other synanthropic animals before we can make more poignant conclusions.

And, finally, yet another add for our recent review on Recent Development in Blastocystis Research!

Please note that I'm happy to take suggestions for future posts, and I'd also like to encourage guest blogging!

Suggested reading:

Abdulsalam AM, Ithoi I, Al-Mekhlafi HM, Khan AH, Ahmed A, Surin J, & Mak JW (2013). Prevalence, predictors and clinical significance of Blastocystis sp. in Sebha, Libya. Parasites & Vectors, 6 PMID: 23566585

Alfellani MA, Jacob AS, Perea NO, Krecek RC, Taner-Mulla D, Verweij JJ, Levecke B, Tannich E, Clark CG, & Stensvold CR (2013). Diversity and distribution of Blastocystis sp. subtypes in non-human primates. Parasitology, 1-6 PMID: 23561720

Alfellani MA, Stensvold CR, Vidal-Lapiedra A, Onuoha ES, Fagbenro-Beyioku AF, & Clark CG (2013). Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Tropica, 126 (1), 11-8 PMID: 23290980

Clark CG, van der Giezen M, Alfellani MA, & Stensvold CR (2013). Recent developments in Blastocystis research. Advances in Parasitology, 82, 1-32 PMID: 23548084

Dogruman-Al F, Simsek Z, Boorom K, Ekici E, Sahin M, Tuncer C, Kustimur S, & Altinbas A (2010). Comparison of methods for detection of Blastocystis infection in routinely submitted stool samples, and also in IBS/IBD Patients in Ankara, Turkey. PloS One, 5 (11) PMID: 21124983 

Gould R, & Boorom K (2013). Blastocystis surface antigen is stable in chemically preserved stool samples for at least 1 year. Parasitology research PMID: 23609598

Malheiros AF, Stensvold CR, Clark CG, Braga GB, & Shaw JJ (2011). Short report: Molecular characterization of Blastocystis obtained from members of the indigenous Tapirapé ethnic group from the Brazilian Amazon region, Brazil. The American Journal of Tropical Medicine and Hygiene, 85 (6), 1050-3 PMID: 22144442