This Month In Blastocystis Research (JAN 2014)

Investigations into the 'biodiversity' existing in a given host is interesting for a number of reasons. One of these reasons has to do with how microorganisms present in our bodies may impact each other or the host. Numerous fields of research are developing at the moment at all levels from studies of gut microbes influencing host microRNA response to bacterial infections to epidemiological/public health studies examining the increased morbidity or risk associated with contracting infection/developing disease given that some infection/disease is present already. Sometimes the reverse is seen, namely that co-infection by multiple parasites is associated with a morbidity lower than the morbidity seen when only one of the parasites is present. For instance, a 2012 study saw that children infected by both Plasmodium falciparum (causing malaria) and hookworm had lower odds of anemia and iron deficiency than their counterparts infected with P. falciparum alone. Other authors have recently reviewed what they see as compelling evidence of chronic viral and helminth coinfections capable of modulating deleterious malaria-specific immune responses. Obviously such types of information are critical to the development of control measures against malaria and helminth infections. A Nigerian colleague of mine is currently exploring co-infections in quite a large Nigerian population, and investigating associations between malaria and soil-transmitted helminths; I'm thankful to be involved.

Along these lines, a new paper sees an inverse association between Blastocystis carriage and tuberculosis/Mycobacterium tuberculosis infections (TB). Dr Franke and colleagues carried out a case–control study to examine associations between parasite infection and tuberculosis (TB) in children in Lima, Peru. Logistic regression analyses revealed that Blastocystis infection was strongly associated with a lower risk of TB (P = 0.002). Hence, the study seems to suggest that Blastocystis carriage may protect against TB. The authors hypothesise that a pro-inflammatory/Th1 response potentially elicited by Blastocystis may protect against other infections, such as TB. While we know very little about Blastocystis induced immunity, little seems to suggest that Blastocystis is related to a chronic pro-inflammatory immunological alert. At least the colonic mucosa of patients with Blastocytis appears to be normal by endoscopy. Given the prevalence of Blastocystis that we have found in the aforementioned Nigerian study, I'd suspect very little TB to be present in this cohort...

The authors highlight a major limitation of their finding, namely the one related to the directionality of the association: It might as well be TB 'protecting' against Blastocystis. Maybe the behaviour (and thereby the exposure to Blastocystis) of patients with TB is different from the behaviour of those who do not have TB; I don't think so though.
I could have wished that a similar analysis had been performed using another common micro-eukaryote in children that the authors did not test for, namely Dientamoeba fragilis. It would be useful to know whether the same association could be identified, or whether the association was specific to Blastocystis. In fact, a general analysis of the microbiota (16S/18S) would have been in place here to learn about other factors potentially responsible for the observations.

Btw, there are some ASM conferences coming up this year relevant to the topic:

1. 5th ASM Conference on Beneficial Microbes, September 27-30, 2014, Washington DC.
2. 1st ASM Conference on Polymicrobial Infections, November 13-16, 2014, Washington DC.

On a different note, there is a paper out by Dr Poirier and his colleauges who have been publishing extensively on Blastocystis, and who were the first ever to sequence and annotate a nuclear genome of Blastocystis. In this new article the authors present a new set of primers applicable to all subtypes of Blastocystis found in humans and targeting the DNA of the mitochondrion-like organelle (MLO). This is reminiscent of the primers used for barcoding (18S analysis), and indeed the primers were validated using strains for which 18S data were available.

The paper highlights a variety of interesting topics and discusses the overall applicability of the two methods  (18S barcoding vs. subtyping using the single-copy MLO rDNA). In both of these scenarios the authors cloned the resulting PCR products to compare intra-isolate genetic variability - something which in itself is very interesting. However, cloning of PCR products is something that is not regularly done in most labs due to time and money constraints. Direct sequencing of barcode products reveals the predominating strain in a mixture, given that no other causes of preferential template amplification exist (e.g. selective primers) and hence shows the consensus sequence; sequence traces may partially or completely fail to reflect cases of mixed infection. However, the issue of not detecting mixed ST infections or mixtures of the same subtype is generally recognised.

The authors present a phylogenetic analysis of the MLO rDNA sequences included and come up with a tree topology different to the one usually seen. Usually, ST1, ST2 and ST5 cluster together, ST3, ST4, and ST8 go together, and ST6, ST7, and ST9 go together. Here, ST1 goes with ST2 and ST3. When I studied the MLOs of Blastocystis on genome level, one of my aims was to see if I could identify evolutionary patterns that had so far not been appreciated and that could be exploited in new hypotheses on the epidemiology and clinical significance of the parasite. Also, as the authors suggest here, mitochondrial DNA is haploid and therefore extremely useful when unambiguous base calling is important as it is in typing schemes. However, so far, when doing phylogenetic analysis of MLO nt sequences and even concatenated MLO nad proteins, we have obtained tree topologies identical to that seen when the 18S gene is used. Maybe the different topology seen here may be due to the way the alignment was constructed/edited? In any case, bootstraps are very low, but if confirmed the finding is very interesting.

If you have half an hour, treat yourself to "A resurgence in Field Research is Essential to Better Understand the Diversity, Ecology, and Evolution of Microbial Eukaryotes".

Literature:

Archambaud C, Sismeiro O, Toedling J, Soubigou G, Bécavin C, Lechat P, Lebreton A, Ciaudo C, & Cossart P (2013). The intestinal microbiota interferes with the microRNA response upon oral Listeria infection. mBio, 4 (6) PMID: 24327339

Franke MF, Del Castillo H, Pereda Y, Lecca L, Fuertes J, Cárdenas L, Becerra MC, Bayona J, & Murray M (2013). Parasite Infection and Tuberculosis Disease among Children: A Case-Control Study. The American Journal of Tropical Medicine and Hygiene PMID: 24379242 

Frosch AE, & John CC (2012). Immunomodulation in Plasmodium falciparum malaria: experiments in nature and their conflicting implications for potential therapeutic agents. Expert Review of Anti-Infective Therapy, 10 (11), 1343-56 PMID: 23241191

Heger TJ, Edgcomb VP, Kim E, Lukeš J, Leander BS, & Yubuki N (2013). A Resurgence in Field Research is Essential to Better Understand the Diversity, Ecology, and Evolution of Microbial Eukaryotes. The Journal of Eukaryotic Microbiology PMID: 24325268

Poirier P, Meloni D, Nourrisson C, Wawrzyniak I, Viscogliosi E, Livrelli V, & Delbac F (2014). Molecular subtyping of Blastocystis spp. using a new rDNA marker from the mitochondria-like organelle genome. Parasitology, 1-12 PMID: 24467909 

Righetti AA, Glinz D, Adiossan LG, Koua AY, Niamké S, Hurrell RF, Wegmüller R, N'Goran EK, & Utzinger J (2012). Interactions and potential implications of Plasmodium falciparum-hookworm coinfection in different age groups in south-central Côte d'Ivoire. PLoS Neglected Tropical Diseases, 6 (11) PMID: 23133691

Blastocystis Microscopy Captured on iPhone

One of my readers sent me some videos of microscopy of some faecal preparations recorded on his iPhone and using a homemade and very cheap adapter (these adapters will otherwise set you back at least $80). I thought that was pretty cool, and one of the videos is quite good and useful to those who'd like to see what Blastocystis looks like through the microscope. It's not always easy  to confirm the presence of a parasite by just looking at a video, but in this case I was pretty sure, although the spherical structures seen in the beginning of the video might as well be fat cells (in direct preparations you don't get rid of faecal fat and debris, which makes detection and identification of parasites much more complicated, and this is - I believe - a direct smear using one drop of iodine and one drop of safranin). As you can see there is a large variation in the size of Blastocystis - something that you commonly see even within the same isolate - and you can also see that some of the cells differ in terms of the uptake of iodine and safranin; cells that have taken up iodine are much darker (brownish) in colour due to iodine staining of starch, and cells that have taken up safranin have red nuclei. Try to pause the video around 1:02, and you'll see these differences quite clearly. Enjoy!

Btw: If you want to make an adaptor yourself, maybe review the following piece of information that I got from the reader:

Adapters are easy to make, you just need an empty tea box (I used this one http://www.dovetalecollections.com/uploads/products/o//D3851.jpg) and then simply draw around the eye pieces on the back and cut them out, then on the front cut out an area above the left hand piece you cut out for the back (so the iPhone camera can see through it). Then simply put a few match sticks in the front of box where you would like the camera to sit. Then just insert the box over both eye pieces (which makes it stable) and put the iPhone on top - easy! The size of the that tea box is perfect as it is the right distance from the eye piece to the camera lens.

2014 Prospects

Happy New Year!

So, what's in store for us in 2014?

Difficult to say, but as least I can try and say a little about what is going on in our lab. Firstly, we are trying to publish what we are think are very interesting data on how gut bacteria may select for Blastocystis colonisation, a hypothesis we have developed based on studies of metagenomic data.

We are also working with the assembly and annotation of mitochondrial and nuclear genomes in collaboration with our international colleagues; something that will definitely take a while, since we have so few people in our lab to do it (literally one-two persons) but oceans of data (!!) - it's a pity that we cannot speed this up, since genomes are expected to hold keys to some of the great gates of Blastocystis enlightenment. Of course, a constant aim is to attract funding that can help us employ one or more PhD students/post docs interested in genomics and parasites. As always, I encourage my readers to come up with suggestions for funding.

Funding-wise we are also going to try and establish a Marie Curie ITN-network on the roles of intestinal microbial eukaryotes in health and disease and we are also awaiting decisions on other applications; hopefully, we will get some money for gut microbiome and immunological host profiling in experimental animals challenged with Blastocytis cysts. There may also be some work in our lab dealing with the impact of Blastocystis on bacterial communities in in-vitro studies.

Epidemiological data are produced as we speak; luckily, quite a few colleagues in different parts of the world are taking an interest in characterisation of Blastocystis in various cohorts so that we will know more about its epidemiology.

Those are the seminal things. Of course, there will be some exciting conferences, which I've mentioned before, and I'm also looking forward to putting together a Blastocystis review.

Blastocystis Highlights 2013

For decades man has striven to improve sanitation and protect production animals and crops from infectious agents to prevent diseases in humans, animals and plants. We tend to eat highly processed foods and many children grow up in almost sterile surroundings, for instance without contact to animals. Consequently, in developed parts of the world we are much less exposed to microorganisms and helminths than previously, and the extensive use of antibiotics for prevention, treatment and control of microorganisms is well-known.

As we are now starting to get a much deeper understanding of the role of the human microbiome (whatever that is) in health and disease, we become aware that microbes are to a large extent beneficial to us; for instance we are currently realising that human health is more or less proportional to intestinal biodiversity, including colonisation by parasites. I have previously mentioned the intentional use of eggs of the parasite Trichuris suis to alleviate symptoms and maybe even reduce disease processes in inflammatory bowel disease, a disorder seemingly stemming from immunological processes out of balance. Also in the food sector, steps may now be taken to diminish breeding and gene manipulation to increase crop yield; instead manipulation of microbes (e.g endophytes) may be used to makes crops hardier. Using microbes to combat other microbes and disease processes, thereby reducing the use of antibiotics and other chemical compounds will probably - and hopefully - be central to controlling diseases and increasing production yields.

For us in the 'Blasto business' these trends are particularly intriguing. I'm not the only one who just a couple of years ago thought that functional bowel disorders such as irritable bowel syndrome (IBS) to a large extent might be directly attributable to undiagnosed parasite infections (e.g. Blastocystis and Dientamoeba), something that could be supported by data going out from one of the studies that I carried out during my PhD studies. Over the years, however, we and other groups have produced data showing that patients with IBS are in fact significantly less colonised by the microbial eukaryotes Blastocystis and Dientamoeba than the healthy 'background' population (see this blog post). We also have access to metagenomic data that link the presence of these microbial eukaryotes with high bacterial diversity in the intestine, and I have colleagues confirming that patients suffering post infectious IBS-like symptoms do practically not harbour these parasites. Our hypothesis now is that - generally speaking - Blastocystis and Dientamoeba are proxies for high intestinal microbial diversity and thereby for a healthy gut ecology. This does of course not rule out the possibility of pathogenic strains/subtypes/whatever of microbial eukaryotes, and we are currently increasing our efforts to investigate whether these organisms can cause disease directly or indirectly, - I'll post a few lines on some of the work we have in mind for 2014 in my next post. I believe that one of the things that most Blastocystis researchers are interested in currently, is to increase our knowledge on the diversity and biological characteristics of mitochondrial and nuclear genomes.

It takes time to produce, analyse and interpret genomic data on Blastocystis, simply because we have so little money that can be allocated to PhD students and post docs. Although we try hard to get funding, we only have one PhD student working with Blastocystis genome data... I could have wished for at least one more. One year ago, I thought that 2013 would see at least one more Blastocystis genome paper, but so far, only very little data have emerged and only included in conference abstracts. But so far it appears that the genetic universe of Blastocystis is even bigger than most of us may have thought! To get a very preliminary impression of their data and how much ST1 differs from ST7 (currently the only available genome), you may want to visit a previous blog post. This basically means that even though we know about genes present and expressed by one subtype, the situation may be completely different for other subtypes! Very interesting, but it also means that a lot of work remains to get a clear picture of what Blastocystis actually does and is capable of doing.

This year was also the year where three seminal papers came out from London School of Hygiene and Tropical Medicine, all of them first authored by Dr Mohammed Alfellani, who did a magnificent job collecting, culturing and 'DNA extracting' samples from both humans, non-human primates, and other animals from various geographic regions. I have already given numerous examples of his findings. Dr Alfellani identified new subtypes, and did a great job in identifying remarkable variations in the geographical distribution of subtypes in both humans and animals. There are also some useful tables in Alfellani's papers showing overviews of molecular data produced by him and our colleagues. We definitely hope that his papers will stimulate other colleagues to pursuing the epidemiology of Blastocystis and should definitely hold some useful tools for those who are new to Blastocystis epidemiology research. 

I had the pleasure to write up a paper to Trends in Parasitology together with Dr Pauline Scanlan. The paper was free for download in November, and we received quite a lot of positive feedback, so thank you all who are so supportive of the work! It was also very rewarding to put together a review for Advances in Parasitology together with Drs Alfellani, van der Giezen and Clark on 'Recent Developments in Blastocystis Research'.

2013 was also the year where my SSI colleagues and I published a comment in the ISME Journal on how impatiently we are awaiting data on the human 'eukaryotic' microbiome. To map the microbial eukaryotes of the intestine and to try and characterise their structure and function and the intestinal ecology that accompany these organisms are activities that should receive top priority in my opinion. There is a link to the comment here.

Congress-wise, I experienced my first-time-ever Blastocystis session at a conference! It was at the merged meeting arranged by the Scandinavian-Baltic Society for Parasitology and the European Society for Tropical Medicine & International Health. Next year, I will be involved in the session 'Passion for Parasites' at the general ASM 2014 meeting in Boston (morning session,18th of May 2014), where I'll be giving a talk on Blastocystis, and, later next year, I will also be leading a lecture/workshop associated with the ICOPA conference in Mexico on Blastocystis barcoding, - I hope to see a few upcoming Blastocystis geeks there!

I don't think we have ever had better opportunities to do significant research on Blastocystis. More and more people are working with it, our epidemiological data have become a lot stronger, enabling more subtle hypotheses, lots of strains have been sequenced, we have developed protocols for experimental in-vitro and in-vivo studies, so here's to hoping for a vast increase in funding and work in 2014!

Attaching an image of the Nutcracker display currently adorning the facade of the Hotel d'Angleterre in Copenhagen, I wish all of the readers of this blog a Merry Christmas and a Happy New Year!

Literature:

Nicola Jones (2013). Food fuelled with fungi. Nature, 504 DOI: 10.1038/504199a

Molecular Epidemiology: Developing a Language

Initiatives towards standardising diagnostic methods and convening on taxonomy and reference data is extremely important in a world where multiple research teams independently carry out research using molecular markers to identify and differentiate species and genotypes of infectious organisms; such activity is crucial to identify patterns of transmission, differences in virulence, and opportunities for control and intervention. Without such standards, efforts to survey and surveil such organisms would be more or less futile, and so they are the backbone of molecular epidemiology.

Having seen that a variety of morphologically similar but genetically diverse Blastocystis organisms found in humans could in fact colonise a range of different hosts, we realised back in 2006 that all these variants could not all be 'Blastocystis hominis', which was then the species name used for Blastocystis found in humans, and together with colleagues we took to revisiting Blastocystis terminology: We recognised that we did not know enough about host specificity and genetic diversity to be able to come up with relevant species names, and so we invented (or maybe not invented, but at least 'formalised') the subtype system, a sort of a barcode system, where genetically similar (typically 98-100%) organisms are assigned to the same subtype, hence ST1, ST2, ST3, etc., which we today now know so well.

Slapeta now suggests a barcoding system for Cryptosporidium. This single-celled parasite takes a major toll on the health of infants and toddlers in developing countries (in some places surpassed only by norovirus), and may also cause debilitating disease in immunocompromised. The nomenclature for Cryptosporidium is very complicated for those of us who are not experts; for instance, I only recently realised that C. parvum may now only refer to the Mouse I genotype and not the 'common' or 'traditional' C. parvum (which now appears to be C. pestis), which is common in both humans and cattle. However, there is a debate going on as to which taxonomy should be followed, and whether this novel leap in 'Cryptosporidium taxonomy revision' can be endorsed by Slapeta's fellow Crypto experts, remains to be seen. Contentiousness aside, barcoding Cryptosporidium does seem relevant due to the fact that the host specificity of Cryptosporidium is relatively loose; for instance humans and cattle are known to share at least 9 species of Cryptosporidium... 

In his paper, Jan Slapeta lists all the known species of Cryptosporidium (in the 'revised' terminology), and even includes GenBank reference strains for common molecular markers such as actin, HSP70 and COWP1 used for genotyping. Interestingly, he does not include the GP60 marker, a molecular marker for which the terminology is also discordant.

Slapeta moreover includes a file with reference SSU rDNA sequences that enable a standardisation of genetic analyses. This year, we did in fact a similar thing for Blastocystis: Along with our 2013 Protist paper surveying Blastocystis subtypes in animals (including the identification of a couple of new subtypes!), we uploaded a reference alignment consisting of some complete SSU rRNA gene sequences present in GenBank; one or more for each of the now known 17 subtypes; more will be added as more subtypes are discovered. The file can be downloaded when accessing the online version of the paper, and we hope that everyone interested in analysing sequences that represent potentially novel subtypes will use this reference alignment (which has been edited to eliminate regions of ambiguous base alignment); it should be quite helpful. Again, I also bring your attention to the pubmlst Blastocystis database, where fast files obtained by Blastocystis barcoding can be queried in batches for quick analysis of large amounts of sequence data. There's a Youtube video here on Blastocystis barcoding and how to use the pubmlst database.

Consensus on methods, terminology and diagnostic algorithms is essential to developing a common language and understanding of how infectious organisms impact our lives; without it,  confusion wreaks havoc with our efforts.

Literature:

Alfellani MA, Taner-Mulla D, Jacob AS, Imeede CA, Yoshikawa H, Stensvold CR, & Clark CG (2013). Genetic diversity of Blastocystis in livestock and zoo animals. Protist, 164 (4), 497-509 PMID: 23770574

Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, Faruque AS, Zaidi AK, Saha D, Alonso PL, Tamboura B, Sanogo D, Onwuchekwa U, Manna B, Ramamurthy T, Kanungo S, Ochieng JB, Omore R, Oundo JO, Hossain A, Das SK, Ahmed S, Qureshi S, Quadri F, Adegbola RA, Antonio M, Hossain MJ, Akinsola A, Mandomando I, Nhampossa T, Acácio S, Biswas K, O'Reilly CE, Mintz ED, Berkeley LY, Muhsen K, Sommerfelt H, Robins-Browne RM, & Levine MM (2013). Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet, 382 (9888), 209-22 PMID: 23680352

Šlapeta J (2013). Cryptosporidiosis and Cryptosporidium species in animals and humans: a thirty colour rainbow? International Journal for Parasitology, 43 (12-13), 957-70 PMID: 23973380  

Stensvold CR, Suresh GK, Tan KS, Thompson RC, Traub RJ, Viscogliosi E, Yoshikawa H, & Clark CG (2007). Terminology for Blastocystis subtypes--a consensus. Trends in Parasitology, 23 (3), 93-6 PMID: 17241816

Striepen B (2013). Parasitic infections: Time to tackle cryptosporidiosis. Nature, 503 (7475), 189-91 PMID: 24236315

Xiao L, Ryan UM, Fayer R, Bowman DD, & Zhang L (2012). Cryptosporidium tyzzeri and Cryptosporidium pestis: which name is valid? Experimental Parasitology, 130 (3), 308-9 PMID: 22230707 

This Month in Blastocystis Research (NOV 2013)

Few commercial kits are available for detection of Blastocystis. One of them is the ParaFlorB kit developed by Boulder Diagnostics which uses a monoclonal antibody to detect Blastocystis-specific antigen. We are currently testing this kit in our lab, and I hope to be able to get back with a summary of our experience once the evaluation has finished. Another kit is the EasyScreenTM Enteric Parasite Detection Kit (Genetic Signatures, Sydney, Australia), which was recently evaluated by some of my Australian colleagues (Stark et al., 2013). In this case, Blastocystis has been included in a panel testing for 5 parasitic genera, the other ones being Giardia, Cryptosporidium, Entamoeba, and Dientamoeba, which makes it interesting in a clinical microbiology context, - at least for research purposes.

It can certainly be discussed whether both Dientamoeba and Blastocystis should be part of routine screening for single-celled intestinal parasites. For some years, we have included Dientamoeba in a PCR panel also consisting of Cryptosporidium, Giardia and Entamoeba, but we are about to remove it from this panel. This does not mean that we will not be testing for Dientamoeba; it only means that we will offer testing for Dientamoeba as a separate analysis, in line with our tests for Blastocystis.

According to the study, the kit performs quite well with the only major impediment being the fact that it does not enable differentiation between pathogenic and apathogenic species of Entamoeba; another drawback is the fact that it does not enable detection of the more rare protozoa, such as Cystoisospora and Cyclospora (and I would also mention microsporidia and maybe Balantidium coli). Also, I might be a little worried that the kit will not pick up all species and genotypes of Cryptosporidium, - in fact little was done to challenge the kit in the evaluation. Regarding Cryptosporidium, only C. hominis and C. parvum were tested. In Sweden, at least 10% of all human cryptosporidiosis is due to non-hominis and non-parvum species and genotypes. This is an observation that has led me to revisit our own Cryptosporidium real-time PCR. With help from Welsh and Swedish colleagues I managed to establish quite a broad panel of different Cryptosporidium species and genotypes, and much to my surprise, our 'old' real-time PCR failed to detect the vast majority of these... which means that this Cryptosporidium PCR was far from genus-specific. So, I set out to design a genus-specific PCR which is now being integrated with our Giardia real-time PCR in a duplex assay.
Anyway, similar to Cryptosporidium, many species of Blastocystis - the so-called subtypes - can colonise and infect humans. In the evaluation of the EasyScreen kit, only subtypes 1, 3, and 4 were used to challenge the kit, and so, it is not known whether the kit also detects other subtypes found in humans (ST2, ST5, ST6, ST7, ST8, and ST9).

For those interested in these diagnostic multiplex systems, please also visit a previous blog post.

Anastasios Tsaousis and his Canadian group in Halifax had a paper out just now in Eukaryotic Cell expanding their work on the evolution of the cytosolic iron/sulfur cluster assembly machinery in Blastocystis spp. and other microbial eukaryotes. This type of work is crucial for obtaining a deeper understanding of the metabolism of Blastocystis and to understand how it has evolved and how it potentially differs from other eukaryotes.  Apparently, Iron-sulfur cluster-containing proteins and their biosynthetic machinery in single-celled parasites are remarkably different from those in their mammalian hosts and they therefore represent a potentially relevant target for the development of novel chemotherapeutic and prophylactic agents against parasite infections. For those interested in iron-sulfur clusters in protists in general, a review was published in Advances in Parasitology some weeks ago (please see cited literature).

There is paper out on fasciolosis and co-infections, including Blastocystis, and in that paper it appears that nitazoxanide may be able to eradicate Blastocystis. However, only three persons were treated, and I'm not sure that the diagnostic tests used would have picked up light infections of Blastocystis.

Speaking of treatment: Another paper has appeared from the highly productive team in Sydney, - this time on treatment failure in patients with chronic Blastocystis infection and first-authored by Ms Tamalee Roberts, whom I was so fortunate to spend some time with during the recent congress in Copenhagen. The paper is a little difficult to follow, particularly since nothing is mentioned in the Materials and Methods section on the choice of treatment and treatment strategies in general, but then again, the paper is based on a string of individual (groups of) cases with different kinds of treatment approaches and various backgrounds. I really like the fact that the authors are looking at multiple cases and also that have included a few patients receiving the Triple Therapy (nitazoxanide, furazolidone, secnidazole), which appears to have no major clinical efficacy. The paper also confirms the uselessness of metronidazole when it comes to eradicating Blastocystis. What I could have wished for is that the authors had been able to pursue the microbiological effect of treatment in each of the cases; only in some cases do we get to know about clearance/persistence of Blastocystis. Also, here at the SSI we sometimes wonder, whether persistence of symptoms after treatment may in some cases reflect adverse effects of the treatment (including perturbation of intestinal flora), in which case even randomised controlled treatment (RCT) studies are difficult to design and interpret, unless very clear case definitions and inclusion criteria are available. Hence, for RCT studies I think it is pertinent only to include patients with very similar symptoms (and possibly microbiomes!); given the prevalence of Blastocystis, this shouldn't be too difficult.

Regarding my most recent blog post, I have noticed that it caused quite a stir! I did anticipate some kerfuffle though. But fact is that we have gradually been able to collect so much data from different, independent studies, and the trend appears clear. We now need to investigate what this means, and whether this is something that can be exploited.

There will be no DEC 2013 version of 'This Month in Blastocystis Research' - instead I plan on doing a 'Blastocystis Highlights in 2013' post in line with last year's. Suggestions for significant papers/contributions are welcome!

Cited literature:

Ali V, & Nozaki T (2013). Iron-sulphur clusters, their biosynthesis, and biological functions in protozoan parasites. Advances in Parasitology, 83, 1-92 PMID: 23876871

Roberts T, Ellis J, Harkness J, Marriott D, & Stark D (2013). Treatment failure in patients with chronic Blastocystis infection. Journal of Medical Microbiology PMID: 24243286

Stark D, Roberts T, Ellis JT, Marriott D, & Harkness J (2013). Evaluation of the EasyScreen™ Enteric Parasite Detection Kit for the detection of Blastocystis spp., Cryptosporidium spp., Dientamoeba fragilis, Entamoeba complex, and Giardia intestinalis from clinical stool samples. Diagnostic Microbiology and Infectious Disease PMID: 24286625

Tsaousis AD, Gentekaki E, Eme L, Gaston D, & Roger AJ (2013). Evolution of the Cytosolic Iron/Sulfur cluster Assembly machinery in Blastocystis sp. and other microbial eukaryotes. Eukaryotic Cell PMID: 24243793
 

Zumaquero-Ríos JL, Sarracent-Pérez J, Rojas-García R, Rojas-Rivero L, Martínez-Tovilla Y, Valero MA, & Mas-Coma S (2013). Fascioliasis and intestinal parasitoses affecting schoolchildren in atlixco, puebla state, Mexico: epidemiology and treatment with nitazoxanide. PLoS Neglected Tropical Diseases, 7 (11) PMID: 24278492

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.

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

This Month in Blastocystis Research (OCT 2013)

Thanks to Google Scholar and PubMed feeds I can keep myself relatively up-to-date with emerging Blastocystis data and 'breaking news' in the field.

One of things that have caught my attention recently, is the string of foodborne outbreaks in Sweden, due to Cryptosporidium, Cyclospora and microsporidia stemming from (presumably) imported produce. A few of my colleagues (Robertsen et al., in press) have just published a large review on the impact of globalisation on foodborne parasites - a resource that has been a long time coming, and which I hope will be read and contemplated by many. The review includes a table on parasites isolated from fresh produce (for some reason the Swedish data was not included), and among these is Blastocystis, which was identified in fresh produce from Saudi Arabia (original data published by Al-Binali et al., 2006). Apart from this, hardly any data is out there on Blastocystis in the environment, and we therefore still know very little about potential sources of transmission and how we are exposed.

In Clinical Microbiology and Infection there is a paper out by Mass et al. on detection of intestinal protozoa in paediatric patients with gastrointestinal symptoms by multiplex real-time PCR. Not surprisingly, the study is from The Netherlands, the cradle of real-time PCR-based parasite diagnostics in clinical microbiology. It's a great paper despite all its limitations, but I couldn't figure out which Blastocystis PCR they used for the study, - I think the authors failed to provide a reference for it. Anyway, the authors found 30% of the children colonised by Blastocystis, while Dientamoeba fragilis was found in a staggering 62%, which is more or less equivalent to what we see in Denmark in this type of cohort (please refer to previous blog post on Dientamoeba fragilis). It appeared that symptom resolution was just as common in patients who were treated with different antibiotics as in patients who were not treated, and the authors end up by highlighting the fact that it is still difficult to know whom and when to test for these parasites, and when to treat them.

In Mexico, Sanchez-Aguillon and colleagues have documented a very nice study on parasitic infections in a Mexican HIV/AIDS cohort. Quite a few of the patients had Cryptosporidium, Cyclospora or Cystoisospora, the presence of which was - not surprisingly - associated with diarrhoea. Table 1 in the paper is a bit confusing, but I believe that Blastocystis was found in about 30%; of note, only ST1 and ST3 were found, adding further support to the hypothesis that ST1 and ST3 are common in most parts of the world, while especially ST4 exhibits vast differences in geographic 'affinity'. The authors end their paper by saying

"Other molecular markers for Blastocystis ST should be studied to elucidate the complexity of this heterogeneous genus and its role in human disease."

Let me just add that subtype identification is a valid proxy for intra-generic diversity in Blastocystis, - we have been looking at mitochondrial genomes and found that analyses based on mitochondrial markers and ribosomal genes reveal similar phylogenetic relationships. So, in terms of transmission and epidemiology in general, the subtyping system ('barcoding') is highly applicable and robust. It is true, however, that we need to see if we can identify specific genes potentially responsible for pathogenesis. The Mexican paper can be accessed here.

There's a very nice paper out now from the Swiss Tropical and Public Health Institute and University of Basel on differential diagnoses of common dermatological problems in returning travellers. Blastocystis has been included in the list (in the section on allergic skin reactions/urticaria) together with a plethora of other infectious agents. Lots of informative images there, and the paper has a nice structure.

Despite loads of daily feeds, a lot of papers relevant to Blastocystis research still escape my attention. I realise that there was a paper out in PLoS Genetics in June on Saprolegnia parasitica (an oomocyte parasitising on fish) which appears to be a good and interesting read. Maybe I'll come back to this one!

For me personally, this month in Blastocystis research has been a month of putting together grant proposals - more so now than usual -, many initiatives are being taken, networks are being expanded, and interesting data are accumulating from various projects... I hope to be back with details on some of this soon!

Literature:

Maas L, Dorigo-Zetsma JW, de Groot CJ, Bouter S, Plötz FB, & van Ewijk BE (2013). Detection of intestinal protozoa in paediatric patients with gastrointestinal symptoms by multiplex real-time PCR. Clinical Microbiology and Infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases PMID: 24131443

Fabiola Sanchez-Aguillon, Eduardo Lopez-Escamilla, Francisco Velez-Perez, Williams Arony Martinez-Flores, Patricia Rodriguez-Zulueta, Joel Martinez-Ocaña, Fernando Martinez-Hernandez, Mirza Romero-Valdovinos, Pablo Maravilla (2013). Parasitic infections in a Mexican HIV/AIDS cohort. The Journal of Infection in Developing Countries PMID: 24129632 

Neumayr A, Hatz C, Blum J. In Press. Not be missed! Differential Diagnoses of Common Dermatological Problems in Returning Travellers. Travel Medicine and Infectious Disease. http://dx.doi.org/10.1016/j.tmaid.2013.09.005

Robertson LJ, Sprong H, Ortega YR, van der Giessen JW, Fayer R. In Press. Impact of globalisation on foodborne parasites. Trends in Parasitology  http://dx.doi.org/10.1016/j.pt.2013.09.005

Dying to know about Dientamoeba?

It's difficult to say 'Blastocystis' without saying 'Dientamoeba fragilis'. Both parasites tend to be extremely common in countries where other intestinal parasites (e.g. Entamoeba, Giardia, Cryptosporidium) are of low endemic occurrence, and they are often seen together in patient samples. It is only due to the recent introduction of DNA-based diagnostic methods (PCR) that we now know that these parasites are much more common than previously anticipated.

So, while I'm trying to encourage guest bloggers, I thought I'd introduce a 'guest star' - Dientamoeba!

Dientamoeba fragilis trophozoites with the characteristic binucleated feature.

The parasite belongs to the trichomonads, which also comprise parasites such as Histomonas meleagridis (the cause of 'blackhead disease' in turkeys) and - more distantly - Trichomonas vaginalis.

At our Parasitology Lab at Statens Serum Institut in Copenhagen we have been using real-time PCR for specific detection of Dientamoeba fragilis in faecal samples from patients with gastrointestinal symptoms for quite a few years now. In the period of 2008-2011 we analysed 22,484 stool samples for D. fragilis. The overall prevalence of the parasite in these samples was 43% but depended mainly on age (Figure 1). D. fragilis prevalence appears to fluctuate dramatically depending on the age group. Highest prevalence was seen among 7-year-olds, and a second 'peak' is seen in the parental age suggesting that infected children pass on infections to their parents. 

Figure 1:  Prevalence of D. fragilis as a function of age. (For more information, see Röser et al., 2013b).

Intestinal protozoa are transmitted faecal-orally and most of them have a cyst stage. However, a few protozoa appear not to have a cyst stage, among them D. fragilis. There is a lot of evidence that Histomonas meleagridis is transmitted by eggs of Heterakis gallinae, a nematode of galliform birds. Conspicuously, we recently demonstrated the presence of D. fragilis DNA in surface-sterilised eggs of Enterobius vermicularis (pinworm). The implications of this finding are unclear but could suggest a similar vector-borne transmission of D. fragilis.

As in so many other situations it is not possible to dish out simple guidelines as to when to test for and treat D. fragilis. It is clear that many carriers experience few or no symptoms at all, but there are several case reports demonstrating symptom relief in patients eradicated of D. fragilis. We published one such case recently in 'Ugeskrift for Læger' - the journal of the Danish Medical Association. Basically, the report describes lasting symptom relief after documented eradication of D. fragilis using high dose metronidazole. However, the patient's symptoms returned after a year, and  real-time PCR revealed D. fragilis positive stools. Eradication was achieved using paromomycin (250 mg x 3 for nine days).

Contrary to Blastocystis, this parasite exhibits remarkably limited genetic diversity. We recently analysed three different genetic loci (18S, actin, elongation factor 1-alpha), and we confirmed that only 2 genotypes exist, one of which is very rare. Genetically, however, the two genotypes are quite different, and it will be interesting to compare the nuclear genomes of the two, once they have become available.

Dientamoeba has been speculated to be a neglected cause/differential diagnosis of irritable bowel syndrome (IBS). We once found a statistical significant association between IBS and Dientamoeba; however, other more recent and more targeted studies (one of which is ongoing) have not confirmed this association. However, multiple factors could interact and analysing only simple associations such as symptoms related to parasite presence/absence may be a limiting approach; for instance, infection load/intensity may play a role, and other factors such as host genetics/susceptibility and microbiota ecology may be significant factors influencing on clinical outcome as well. On that note, we have observed some very low Ct values in our real-time PCR results for some of our D. fragilis positive patients, suggesting massive infections. D. fragilis infections are probably often long lasting (months), and if symptoms appear in the initial phase of infection only, cross-sectional studies of prevalence and clinical presentation will be potentially misleading. Large longitudinal cohort studies of pre-school children with monitoring of incidence of pinworm and D. fragilis infections would be extremely informative.

Dr Dennis Röser here at the SSI is currently finishing a randomised controlled treatment trial of D. fragilis in children, testing the clinical efficacy of metronidazole treatment versus placebo. Results are expected next year, so watch out for a 'D. fragilis special' by Dr Röser in 2014! It appears a lot easier to eradicate D. fragilis than Blastocystis - at least on a short term basis with metronidazole having an efficacy of about 70% or so (unconfirmed).

A couple of reviews free for download are available; please see literature list below or go here and here.

Suggested 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, 87 (6), 1046-52 PMID: 23091195   

Johnson EH, Windsor JJ, & Clark CG (2004). Emerging from obscurity: biological, clinical, and diagnostic aspects of Dientamoeba fragilis. Clinical Microbiology Reviews, 17 (3) PMID: 15258093

Ogren J, Dienus O, Löfgren S, Iveroth P, & Matussek A (2013). Dientamoeba fragilis DNA detection in Enterobius vermicularis eggs. Pathogens and Disease PMID: 23893951  

Röser D, Nejsum P, Carlsgart AJ, Nielsen HV, & Stensvold CR (2013a). DNA of Dientamoeba fragilis detected within surface-sterilized eggs of Enterobius vermicularis. Experimental Parasitology, 133 (1), 57-61 PMID: 23116599   

Röser D, Simonsen J, Nielsen HV, Stensvold CR, & Mølbak K (2013b). Dientamoeba fragilis in Denmark: epidemiological experience derived from four years of routine real-time PCR. European Journal of Clinical Microbiology & Infectious Diseases : official publication of the European Society of Clinical Microbiology, 32 (10), 1303-10 PMID: 23609513  

Stark DJ, Beebe N, Marriott D, Ellis JT, & Harkness J (2006). Dientamoebiasis: clinical importance and recent advances. Trends in Parasitology, 22 (2), 92-6 PMID: 16380293  

Stark D, Barratt J, Roberts T, Marriott D, Harkness J, & Ellis J (2010). A review of the clinical presentation of dientamoebiasis. The American Journal of Tropical Medicine and Hygiene, 82 (4), 614-9 PMID: 20348509

Stensvold CR, Clark CG, & Röser D (2013). Limited intra-genetic diversity in Dientamoeba fragilis housekeeping genes. Infection, Genetics and Evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 18, 284-6 PMID: 23681023

Stensvold CR, Lewis HC, Hammerum AM, Porsbo LJ, Nielsen SS, Olsen KE, Arendrup MC, Nielsen HV, & Mølbak K (2009). Blastocystis: unravelling potential risk factors and clinical significance of a common but neglected parasite. Epidemiology and infection, 137 (11), 1655-63 PMID: 19393117