This Month In Blastocystis Research (FEB 2014) - The Protease Edition

A few interesting papers on Blastocystis appeared this month on PubMed. I would like to give a great salute to Ron Fayer's group in Maryland who took to investigating faecal samples and tissue sections from naturally infected pigs. Due to the protease theme of this blog post, I won't go into detail with this paper, but only highlight a few points. The researchers found Blastocystis ST5 in faecal samples from all 11 pigs investigated. By examination of tissue sections they found that Blastocystis existed in the lumen of the jejunum, caecum, proximal and distal colon, but not in the duodenum and ileum. Moreover:

"In tissue sections, Blastocystis was found primarily in the lumen usually associated with digested food debris, sometimes in close proximity or appearing to adhere to the epithelium, but no stages were found to penetrate the epithelium or the lamina propria."

So, the authors did a great job to describe Blastocystis tropism in the pig intestine. It is new to me that the parasite can be found in the jejunum; if anything, I would have thought that the ileum might be 'affected', and certainly the caecum and possibly the remainder of the colon. It is also important to note that in these naturally infected pigs (ST5 is probably the most common subtype in pigs), no signs of invasiveness was detected.

Now, moving on to the proteases, there is a paper out by Arutchelvan Rajamanikam and Suresh K Govind called 'Amoebic forms of Blastocystis spp. - evidence for a pathogenic role'. The study links protease activity to amoebic forms of Blastocystis, which the authors found in symptomatic carriers but not in asymptomatic carriers. Amoeboid forms of Blastocystis being associated with symptomatic infections were described already in 2006 by T C Tan and K G Suresh (whom I believe is identical to S K Govind). While the study is small, investigation of Blastocystis proteases has been going on for a while, and I thought it would be useful to go over some of the literature.

Proteases (or proteinases or peptidases) are enzymes that degrade proteins and therefore useful for instance for the mobilisation and storage of proteins (i.e. 'food'), and the general development and differentiation of cells and tissues, but these enzymes may also be vital for for instance pathogen survival and virulence in the human body (i.e. 'defence' and 'invasion'). Proteases exist in all organisms, i.e. in pro- and eukaryotes + viruses. Proteases are classified on the basis of catalytic mechanism, and five known distinct classes are described: metallo, aspartic, cysteine, serine, and threonine. Being enzymes, proteases digest substrates, can be inhibited, and their functions are dependent on pH and temperature. Hence, proteases can be identified by substrate digestion and by intended inhibition by selective inhibitors (for cystein protease such inhibitors include N-ethylmaleimide, iodoacetamide, and para-hydroxymercuribenzoate for instance).

Turning to the intestinal protozoon Entamoeba for a short while, cysteine proteases have been studied in detail and are among the most likely candidates responsible for the differential pathogenocitiy (virulence factors) of morphologically similar species of Entamoeba: Entamoeba histolytica expresses at least 5 types of cysteine proteases (ACP1, ACP2, ACP3, EhCP5, and EhCP112) and can invade host tissue (leading to amoebiasis), while Entamoeba dispar expresses at least three types of cysteine proteases (EdCP1, EdCP2, and EdCP3) without the ability to invade host tissue. Clinical isolates of E. histolytica release 10- to 1,000-fold more cysteine proteinase activity into the supernatant than E. dispar isolates, although  significant day-to-day variability may be seen. Extracellular cysteine proteases cleave immune secretory IgA (facilitating adhesion of the organism (pathogen) to mucosal surfaces), degrade the extracellular matrix, activate complement, and degrade IgG to circumvent the host immune response. The first evidence of amoebic pathology is local depletion of intestinal mucus and disruption of the epithelial barrier as a result of degradation of the extracellular matrix, which occurs in part from the action of cysteine proteases. More than 80% of patients with amoebiasis develop antibodies against cysteine proteases. Please note that E. histolytica is not consistently invasive; only 10% of E. histolytica infections are believed to be invasive.

Importantly, cysteine proteases are critical to host invasion in a number of parasites. Specific inhibitors block invasion in Trypanosoma cruzi, Plasmodium falciparum, Cryptosporidium parvum, and Toxoplasma gondii.

The main reservoir of Blastocystis ST7 appears to include birds.

Now what do we know about Blastocystis and cysteine proteases? In 2005, Manoj K Puthia from Dr Kevin S W Tan's group in Singapore identified mainly cysteine protease activity in the 'B. hominis B' strain (which is the ST7 strain used in the genome sequencing and annotation study by Denoeud et al. (2011)) and aspartic protease activity in 'B. ratti WR1 strain' (which is a ST4 strain). Lysates and conditioned medium (culture supernatant) from both axenic strain cultures were able to degrade human secretory IgA over 2 h at 37 C, suggesting that Blastocystis actively secrets proteases that - among other things - degrade IgA, thereby potentially evading host mucosal immunity, and enhancing survival opportunities. Along theses lines, in 2006 Sio and colleagues from Tan's group used enzyme digestion (azocasein spectrophotometric assay and gelatin SDS-PAGE analysis), and inhibition assays to characterise proteases from 'B. hominis B' strain. They showed the existence of cysteine proteases with highest activity at neutral pH (the pH of the colon is neutral if even slightly acidic).

Mirza and Tan confirmed that cysteine protease activity was higher in ST7 than in ST4, while inter- and intra-subtype variation in activity was seen over time. In a small study of ST3 positive individuals, Abdel-Hameed and Hassanin were able to detect protease activity in 17/18 symptomatic individuals but only in 2/8 asymptomatic individuals, suggesting intra-subtype differential protease activity. I don't think they tested for protease activity in the culture supernatant.

Cysteine proteases from Blastocystis were reported by Puthia et al. (2008) to enable activation of interleukin 8 (IL-8) gene expression in the human colonic epithelial T84 cell line. IL-8 is a cytokine that attracts PMN and activates monocytes (interestingly, recent results from Olivo-Diaz et al. (2012) suggest that some IL-8 and IL-10 SNPs could change individual susceptibility increasing the relative risk in the development of irritable bowel syndrome (IBS) in Blastocystis carriers).

Gastrointestinal disorders, such as bacterial enteritis, celiac disease, and inflammatory bowel disease, are reported to be associated with a breakdown of epithelial barrier function which is mainly regulated by 'tight junctions'. There is some experimental evidence that Blastocystis may be able to interfere with this regulation and that it may induce host cell apoptosis without attaching to the gut mucosa. Puthia et al. (2006) explain:

"Pathogen invasion and induction of apoptosis are discrete processes, and there are pathogens that can invade but do not induce apoptosis. It appears that induction of apoptosis of host intestinal cells would not be advantageous to a noninvasive parasite like Blastocystis, as it would result in the loss of colonization sites for the parasite. This unintended induction of host cell apoptosis might be a host response against some parasitic factors like proteases which are necessary for the parasite's own life cycle."

Back to the paper by Rajamanikam and Govind: I cannot remember ever seeing amoeboid stages in Blastocystis cultures myself. But then again, in cultures, Blastocystis can take so many forms (some actually resembling the outline of the head of, well, Mickey Mouse (!) and other cuddly creatures (looks like budding off of new cells), and I wouldn't be able to define strict criteria for stratification of organisms into groups. Since we use Jones' Medium also, I do not suspect that it's a 'medium thing'. What we usually see in well-maintained cultures are small, quite inconspicuous and completely spherical cells. Using the aforementioned digestion assays, Rajamanikam and Govind found elevated protease activity related to patient Blastocystis cultures that had a higher percentage of amoebic forms with intense bands representing higher molecular weight proteases (60-100 kDa); the proteases previously described have been of a size of maximum 75 kDa; however, no attempts were made to characterise the proteases in this study. The authors did not include analysis of conditioned medium, and so we do not know whether these proteases were actually secreted. The proteases identified here may be expressed by the amoebic forms only and so they may be responsible for this particular life cycle stage. Knowledge of substrate specificity might have been useful, and it is also possible to actually determine the protein's amino acid sequence and thereby predict it's structure and function using e.g. mass spectrometry (MS) or Edman degradation of peptides.

Just like Ivan Wawrzyniak and colleagues who recently used SDS-PAGE and MS to characterise proteases secreted by the Blastocystis ST7 (B strain). They were able to match two cysteine proteases identified in the culture supernatant to 2 of 22 proteases predicted by in silico analysis of their ST7 B strain genome data, namely Cathepsin B cysteine protease (CBCP) and a Legumain cysteine protease, which the authors speculated to be potentially involved in pathological processes such as mucin degradation. Incidentally, silencing of CBCP has recently been shown to reduce gut penetration in the helminth Faciola hepatica.

Back in 2007, Jésus Serrano-Luna and colleagues studied proteases from pathogenic Naegleria fowleri (causing primary amoebic meningoencephalitis) and non-pathogenic Naegleria gruberi. They observed cysteine proteases in both species, but more proteases in the N. gruberi than in N. fowleri. Protease activity appeared to depend on pH and temp, and moreover, protease patterns for crude extracts and conditioned medium differed

It's probably fair to assume that the expression of potential virulence genes such as genes encoding cysteine proteases may depend on a multiple factors, most of which are yet to be identified, or at least, confirmed. For now, the marked differences in cysteine protease production/expression between and within Blastocystis STs together with experimental evidence highlighting a variation in pathophysiological effects and immunological responses to Blastocystis subtypes isolated from symptomatic and asymptomatic carriers, could be seen as supporting the hypothesis that cysteine proteases may be essential virulence factors responsible for variation in disease symptoms observed across carriers. For more on this, why not look up this paper (free in PubMed Central). However, it is also tempting to think that differential protease expression is merely reflecting various stages in the parasite's life cycle. Things would have been so much easier if we had access to a strain in culture capable of invasion or isolated from an outbreak of Blastocystis infection. But, contrary to parasites of 'acknowledged clinical significance', we do not have such a strain, and neither invasion nor outbreaks of Blastocystis have been reported of, at least not convincingly, I think; please correct me, if I'm wrong. I think it's time for a coffee...

Literature:

Abdel-Hameed DM, & Hassanin OM (2011). Proteaese activity of Blastocystis hominis subtype 3 in symptomatic and asymptomatic patients. Parasitology Research, 109 (2), 321-7 PMID: 21279383

Denoeud F, Roussel M, Noel B, Wawrzyniak I, Da Silva C, Diogon M, Viscogliosi E, Brochier-Armanet C, Couloux A, Poulain J, Segurens B, Anthouard V, Texier C, Blot N, Poirier P, Ng GC, Tan KS, Artiguenave F, Jaillon O, Aury JM, Delbac F, Wincker P, Vivarès CP, & El Alaoui H (2011). Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite. Genome Biology, 12 (3) PMID: 21439036 

Fayer R, Elsasser T, Gould R, Solano G, Urban J Jr, & Santin M (2014). Blastocystis tropism in the pig intestine. Parasitology Research PMID: 24535732

McGonigle L, Mousley A, Marks NJ, Brennan GP, Dalton JP, Spithill TW, Day TA, & Maule AG (2008). The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration. International Journal for Parasitology, 38 (2), 149-55 PMID: 18048044

Mirza H, & Tan KS (2009). Blastocystis exhibits inter- and intra-subtype variation in cysteine protease activity. Parasitology Research, 104 (2), 355-61 PMID: 18846388

Olivo-Diaz A, Romero-Valdovinos M, Gudiño-Ramirez A, Reyes-Gordillo J, Jimenez-Gonzalez DE, Ramirez-Miranda ME, Martinez-Flores WA, Martinez-Hernandez F, Flisser A, & Maravilla P (2012). Findings related to IL-8 and IL-10 gene polymorphisms in a Mexican patient population with irritable bowel syndrome infected with Blastocystis. Parasitology Research, 111 (1), 487-91 PMID: 22287022

Poirier P, Wawrzyniak I, Vivarès CP, Delbac F, & El Alaoui H (2012). New insights into Blastocystis spp.: a potential link with irritable bowel syndrome. PLoS Pathogens, 8 (3) PMID: 22438803

Puthia MK, Vaithilingam A, Lu J, & Tan KS (2005). Degradation of human secretory immunoglobulin A by Blastocystis. Parasitology Research, 97 (5), 386-9 PMID: 16151742

Puthia MK, Sio SW, Lu J, & Tan KS (2006). Blastocystis ratti induces contact-independent apoptosis, F-actin rearrangement, and barrier function disruption in IEC-6 cells. Infection and Immunity, 74 (7), 4114-23 PMID: 16790785

Que X, & Reed S L (2000). Cysteine Proteinases and the Pathogenesis of Amebiasis. Clinical Microbiology Reviews, 13 (2), 196-206 DOI: 10.1128/CMR.13.2.196-206.2000

Rajamanikam A, & Govind SK (2013). Amoebic forms of Blastocystis spp. - evidence for a pathogenic role. Parasites & Vectors, 6 (1) PMID: 24499467

Serrano-Luna J, Cervantes-Sandoval I, Tsutsumi V, & Shibayama M (2007). A biochemical comparison of proteases from pathogenic Naegleria fowleri and non-pathogenic Naegleria gruberi. The Journal of Eukaryotic Microbiology, 54 (5), 411-7 PMID: 17910685

Sio SW, Puthia MK, Lee AS, Lu J, & Tan KS (2006). Protease activity of Blastocystis hominis. Parasitology Research, 99 (2), 126-30 PMID: 16518611 

Wawrzyniak I, Texier C, Poirier P, Viscogliosi E, Tan KS, Delbac F, & El Alaoui H (2012). Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite. Parasitology International, 61 (3), 437-42 PMID: 22402106 

'Save the Date's + Resources

Some 'Save the Date's:

1. ASM Meeting, Boston, MAY 2014:
Speaker: Christen Rune Stensvold 
Session Title: Passion for Parasites! Current Topics in Medical Parasitology 
Session Date/Time:  5/18/2014 8:00:00 AM 
Presentation Title: Blastocystis Clinical Relevance: More Common and Important than You Think


2. ICOPA, Mexico City, AUGUST 2014:

UNU-BIOLAC has accepted to fund the MOLECULAR PARASITOLOGY WORKSHOP-ICOPA 2014 to be held in Mexico city August 7-10! pic.twitter.com/YbTjUI0MFt
— Chr. Rune Stensvold (@Eukaryotes) February 22, 2014

 
3. 1st International Blastocystis Symposium, Ankara, 28-29 MAY 2015:

Please go here for more information.



Just found out that out of 1065 Blastocystis papers in PubMed, 269 are can be downloaded for free! If you enter 'Blastocystis' in the search box, you'll see the 1065 or so hits, but if you go to the right side bar, you have the option of having the Free Full Text (269) display.

I have disabled Google+ comments for now due to repetitive abuse. However, it is still possible to comment on blog posts, only now comments will be reviewed and potentially moderated by me prior to publishing.

In case there should be readers who think that I'm trying to propagate the view that Blastocystis is pathogenic, I hope that after going through my blog posts they will realise that I'm not; in fact, I'm much more trying to be the devil's advocate: Blastocystis is 'innocent' unless proven otherwise. In my opinion we have very little clinical evidence of pathogenicity. And at our lab, we generally do not recommend treating patients with Blastocystis. In fact, we really don't know HOW to treat Blastocystis, - and maybe that's one of the most fundamental issues in Blastocystis research. I know that many treatment regimens are currently in use for Blastocystis despite the absence of clinical guidelines, and some of them are used systematically at various clinics it seems, but off the top of my head I cannot think of one single randomised controlled treatment study that have explored the microbiological and clinical effect of treatment. Such studies are critical to our understanding of  the role of the parasite in health and disease, although even this type of studies have limitations such as non-specific drug actions that will blur our ability to point out Blastocystis as the culprit, and also some drugs may have adverse effects that mimic symptoms potentially caused by Blastocystis, including symptoms related to intestinal dysbiosis. I hope that those who have extensive experience with Blastocystis treatment will soon take to sharing their knowledge.

But I guess that what we are currently trying in various fields is to get a differentiated view of Blastocystis - for instance: can colonisation turn into infection, and is there any such thing as a Blastocystis infection at all? Can, and if so, when does Blastocystis carriage lead to pathology/disease? Which are the interactions between Blastocystis and the remaining microbiota? What host factors may be responsible for potential differences in Blastocystis-mediated disease susceptibility?

Don't miss the February issue of 'This Month In Blastocystis Research' which will be available in a week or so.

Announcement of The 1st International Blastocystis Symposium

It is a pleasure for me to be able to announce that Dr. Funda Dogruman-Al and I have started planning

'The 1st International Blastocystis Symposium' 

to be held in Ankara, May 28-29 in 2015 under the auspices of Gazi University, Faculty of Medicine, Ankara, Turkey.

We have received very positive responses so far from quite a few of the leading scientists in Blastocystis research expressing their interest in the symposium, and obviously our goal is to be able to raise significant and relevant funding to cover at least some of the expenses related to the meeting. Already, a total of € 6,000 has been raised, but we hope for a lot more.

It may seem like a long time ahead, but we believe that by starting this early, we should stand a fair chance of arranging and completing a meeting that will enable a strengthening of global cooperative networks so as to increase the sharing of ideas, data, expertise, and best practices in this field to the mutual benefit of the researchers and organisations involved. 

The past few years have seen a substantial surge in Blastocystis research in many areas, and so the main foci of this symposium will be:

• Diagnosis and molecular characterisation
• Epidemiology
• Evolutionary biology and advances in genomics
• Intestinal ecology
• Clinical significance including potential relationship to irritable bowel syndrome (IBS)

The symposium will also serve as an initial forum to address and discuss critical questions including: 

• What is the evidence of Blastocystis pathogenicity?
• If Blastocystis may be pathogenic, what then are the factors involved? Which mechanisms could potentially be involved in the development of disease?
• Could specific host factors be responsible for the development of symptomatic Blastocystis infections, and if so, how can these be identified?
• How can Blastocystis infections be controlled?
• Is there any correlation between the human intestinal microbiota and Blastocystis carriage?
• How can novel technology and multi-disciplinary approaches assist in answering fundamental questions concerning the clinical significance of Blastocystis?

We hope that the symposium will attract not only experts on Blastocystis, but also biologists and medical doctors in the fields of clinical microbiology, infectious diseases, gastroenterology, evolutionary biology, and microbial ecology. Certainly, those who have expressed interest so far cover a wide spectrum of these research areas.

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