Showing posts with label diagnostics. Show all posts
Showing posts with label diagnostics. Show all posts

Saturday, February 28, 2015

This Month in Blastocystis Research (FEB 2015)

Before heading off to visit dear colleagues at the Public Health Agency of Sweden tomorrow morning, I thought I'd do a quick 'This Month...' post.

Tropical Parasitology has published a paper by Elghareeb and colleagues on  'Laboratory Diagnosis of Blastocystis in Diarrheic Patients'. I was asked to do a Guest Commentary on their paper, and if your're interested you can download my comments here for free (html version). The paper by Elghareeb et al. should also be free for download at the website.

I have been very lucky to work together with Dr Prashant K Pandey and his colleauges in Pune, India. Together we just published the first data on Blastocystis subtypes ever to appear in India for what I know. We subtyped Blastocystis in a cohort of healthy Indian individuals, and found ST1 and ST3 in 27/100 adult individuals tested, while other common subtypes, ST2 and ST4, were absent. Remarkably, ST3 was seen in all positive individuals, while ST1 was seen only in mixed infections. The strains (alleles) found in India were no different to those found in for instance Europe.

There is a paper out by Rossen and colleagues from The Netherlands showing that Blastocystis is relatively uncommon in patients with active ulcerative colitis (UC) and significantly less common in UC patients (13.3%) than in healthy individuals (32.5%). This is completely in line with data that we generated in Denmark a couple of years ago. In fact, at two separate occasions we have been able to look into patients with inflammatory bowel disease. In both cases (one study has been submitted for publication), hardly any Blastocystis was found in patients with Crohn's disease, while a few patients with UC were positive; however, mostly patients with inactive disease appeared to have Blastocystis, while those with flare-ups were negative. Therefore, the influence of dysbiosis on Blastocystis colonisation should be subject to further scrutiny.

A lot of action goes on at the official website for the 1st International Blastocystis Symposium in Ankara in May, with exactly three months to go! Why not take a minute to browse the programme for the Pre-Symposium Course and the Scientific Programme for the actual Symposium? Please go here to familiarise yourself with the new content. 
Also, conference abstracts are pouring in, - did you submit yours yet?


Elghareeb AS, Younis MS, El Fakahany AF, Nagaty IM, & Nagib MM (2015). Laboratory diagnosis of Blastocystis spp. in diarrheic patients. Tropical Parasitology, 5 (1), 36-41 PMID: 25709951

Stensvold, C. (2015). Laboratory diagnosis of Blastocystis spp Tropical Parasitology, 5 (1) DOI: 10.4103/2229-5070.149885  

Pandey PK, Verma P, Marathe N, Shetty S, Bavdekar A, Patole MS, Stensvold CR, & Shouche YS (2015). Prevalence and subtype analysis of Blastocystis in healthy Indian individuals. Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases PMID: 25701123

Rossen NG, Bart A, Verhaar N, van Nood E, Kootte R, de Groot PF, D'Haens GR, Ponsioen CY, & van Gool T (2015). Low prevalence of Blastocystis sp. in active ulcerative colitis patients. European Journal of Clinical Microbiology & Infectious Diseases: Official Publication of the European Society of Clinical Microbiology PMID: 25680316

Wednesday, October 29, 2014

This Month in Blastocystis Research (OCT 2014) - Trick or Treat Edition

Over the past 30 days I've hardly had any time to focus on Blastocystis. I've been busy preparing for and attending UEGWeek 2014, preparing abstracts for next year's ECCMID conference in Copenhagen, and I've also put a lot of effort into preparing proposals for this round of grant calls from the Danish Council for Independent Research

Among other things, we are applying for money to develop DNA-probe based diagnostics, including a unique software, for use in the clinical microbiology lab that entirely circumvents the use of PCR (and thereby amplification bias) and that screens sequence data in real-time. An issue with current state-of-the-art in the area is that no software is available to relevantly and reliably handle the tons of sequence data that next/third/fourth generation sequencing devices are capable of producing. The proposed software will have a vast application range, applicable not only to clinical microbiology but also other areas of microbiology, such as food control, water sanitation, and monitoring of microbes in oil, soil, etc.

We are applying for about 270.000 Euros, and although this doesn't sound like an awful lot of money, competition is extremely fierce for this type of grant (although I'm not sure that the competition has to do exclusively with the scientific and innovative quality of the proposal...). So, let's see if it's going to be trick or treat!

Earlier this month, I was honoured to give a talk in Padova at the XXX National Congress of the Italian Society of Protistology on Blastocystis and its role in health and disease. I also got the chance to listen to some of the remarkable talks delivered by passionate colleagues of the society. There was quite a lot on endosymbionts of protists. The development of mitochondria in eukaryotic cells is a classical example of endosymbiosis; however, there are numerous examples of e.g. bacteria infecting protists, including the parasitic ones. Legionella, for instance, may be found in Acanthamoebaknown to host a variety of bacterial endosymbionts. Along the same lines, I wish that studies could be made to look up potential endosymbionts in Blastocystis; endosymbionts which may confer disease, and the varying/unstable presence of which might explain the irregularity in symptoms reported by Blastocystis carriers? The question about endosymbionts in Blastocystis is interesting not only from a metabolic and horizontal gene transfer point-of-view, but also in the perspective of Blastocystis potentially serving as a vector, a vehicle for transmission of bacteria and maybe viruses... A nice paper on endosymbiotic associations within protists is available for a free download here. Rickettsia, for instance, are obligate intracellular bacteria found as endosymbionts in different types of eukaryotes, including amoebae, but also in endothelial cells (which are not phagocytic by nature, similar - presumably - to the case of Blastocystis), and some of these rickettsia are  known as causes of spotted fever and typhus. I think that Zierdt is the only one until now who has studied endosymbiosis in Blastocystis...

After the congress in Padova, I got a chance to pay my first visit ever to Venice, which was nothing short of brilliant.

Venice, October 2014.

I did have an hour here and there, however, to look up newest 'releases' on Blastocystis, and I'm just going to highlight a few of them.

Unfortunately in Russian and not available for download on any of the servers that I can access, there's a paper describing the finding of dividing (i.e. alive) forms of Blastocystis in a liver abscess in an immunocompromised woman. The question here is of course, did the parasite end up here by chance (fistula and/or secondary to bacterial invasion?) or by independent invasion? Hope to receive a copy of the paper at some point... and a translation!

There is a paper in a journal called 'Case Reports in Medicine' on what is called a co-infection of Schistosoma and Blastocystis in a 37-year-old male with chronic kidney disease, in whom Blastocystis was speculated to be the cause of chronic IBS-like symptoms. However, there is a number of issues that I would like readers of the paper to focus on: Apparently, the patient had Schistosoma mansoni detected in the urine suggesting schistosomiasis of the bladder. But how was Schistosoma detected? It doesn't say. Was it by microscopy? The patient was ab-positive, but still intestinal schistosomiasis was not ruled out (by e.g. PCR on faecal DNA, microscopy for ova and parasites, rectal biopsy, etc.). The patient responded well to praziquantel treatment and got rid of symptoms, including the intestinal symptoms ascribed to Blastocystis, for which the patient was prescribed metronidazole. We know that Blastocystis is only rarely eradicated by metronidazole alone, and indeed, the article does not provide data on post-treatment stool examination to see whether Blastocystis was still there. I think there is a chance that Blastocystis was an incidental finding and that intestinal symptoms in this case were due to Schistosoma. Given our recent data and improved diagnostic techniques, Blastocystis will more often now than ever become an incidental finding on routine analysis of faecal samples.

There is a paper by Fletcher and colleagues coming out in Journal of Public Health Research studying the prevalence and geographical distribution of enteric protozoan infections in Sydney, Australia, which I haven't had a chance to study in detail. I just want to emphasize that this study found Blastocystis prevalence to be increasing by age, a finding adding support to accumulating data suggesting that Blastocystis is more common in adults than in children, which is interesting from a clinical, epidemiological, and ecological point of view.

Hope to be able to address an interesting and brand new paper on Blastocystis treatment in Faculty of 1000 very soon. 

Happy Halloween!


Fletcher S, Caprarelli G, Merif J, Andresen D, Hal SV, Stark D, & Ellis J (2014). Epidemiology and geographical distribution of enteric protozoan infections in Sydney, Australia. Journal of Public Health Research, 3 (2) PMID: 25343139

Nagel R, Bielefeldt-Ohmann H, & Traub R (2014). Clinical pilot study: efficacy of triple antibiotic therapy in Blastocystis positive irritable bowel syndrome patients. Gut Pathogens, 6 PMID: 25349629

Nowack EC, & Melkonian M (2010). Endosymbiotic associations within protists. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 365 (1541), 699-712 PMID: 20124339

Prodeus TV, Zelia OP, Khlebnikova TA, & Pikul' DA (2014). [Extraenteric infection caused by Blastocystis spp. in a female patient with liver abscess]. Meditsinskaia Parazitologiia i Parazitarnye Bolezni (2), 6-9 PMID: 25296418

Young CR & Yeo FE (In Press). Blastocystis and Schistosomiasis coinfection in a patient with chronic kidney disease. Case Reports in Medicine 

Thursday, May 1, 2014

This Month In Blastocystis Research (APR 2014)

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

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

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

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

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

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


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

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

Wednesday, September 4, 2013

Yes, we do take orders!

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

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

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

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

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

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

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

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

Relevant articles on molecular diagnostics for Blastocystis detection and subtyping:

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

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

Friday, August 30, 2013

This Month In Blastocystis Research (AUG 2013)

Quite a few papers relevant to Blastocystis research have made it to PubMed over the past month! Therefore, the August version of 'This Month in Blastocystis Research' is more like a list of papers + short descriptions/comments, rather than one or two actual paper reviews.

Dr Aldert Bart and his Dutch colleagues have published a study that confirms data emerging from other parts of Europe. Using microscopy (fixed faecal smears) and PCR, they found an almost 40% prevalence of Blastocystis in returning travelers with symptoms, and a prevalence of 18% in patients referred for other reasons. The distribution of subtypes found in the study population was quite similar to what has been found elsewhere in Europe with ST3 predominating (42%) and the rest of the subtypes attributable to ST1 (22%), ST2 (22%), ST4 (12%), ST6 (1%) and ST7 (1%).

The Tropical Parasitology theme issue on Blastocystis has now gone live. You’ll find a link to the editorial and the three papers included in the symposium here.

In my previous post I referred to a new study from Colombia which includes subtyping of Blastocystis isolates from humans, and a variety of animals, including birds. The paper is interesting for a number of reasons, but first and foremost it confirms the virtual absence of ST4 in humans in S America. Moreover, the study included 70 Blastocystis positive samples from asymptomatic carriers, 40 positive samples from patients with diarrhoea, and 15 positive samples form patients with IBS. Remarkably, all samples from healthy carriers were typed as ST1, those from patients with diarrhoea belonged to ST2, and those from IBS patients to ST3. Such a clear-cut distribution of subtypes across cohorts is unprecedented and of course warrants confirmation and further investigation. In Europe, ST4 is very common in humans, while it appears rare in humans in many other parts of the world. ST4 also appears rare among non-human primates (NHPs), our closest living relatives, and while NPHs and humans otherwise tend to share the same major subtypes (ST1, ST2, and ST3), this suggests that while subtypes 1, 2 and 3 have probably co-evolved with primates, ST4 has only recently entered the primate population with a preference for humans! I have hinted at this many times by now, but I find it extremely interesting which is why I keep repeating it.

There is a paper out by Santos and Rivera from the Philippines comparing microscopy of direct faecal smear with culture and PCR for detection of Blastocystis. They ended up concluding that culture was the best diagnostic modality, but it should be noted that the PCR used in the study targets a 1.8 kbp product (complete SSU rRNA gene!), and much smaller products are usually targeted in diagnostic PCR assays. The Blastocystis real-time PCR developed by me and my colleagues targets a sequence stretch of ~120 bp, securing optimum test sensitivity. The results of the Philippine study should be interpreted with this in mind.

Li et al., have published data on experimental infection of ST1 in Sprague-Dawley rats. Animals belonging to this species appeared susceptible to a ST1 strain isolated from a diarrhoeic patient that had been kept in culture and for which induction of cysts had been performed with a view to infecting the rats. The study confirms that Blastocystis is mainly a parasite of the coecum and colon. The authors found evidence of Blastocystis invasion into the lamina propria in one of the animals, and signs of inflammation in all animals challenged. While it is great to see that experimental models can be sustained and that encystation can be induced in vitro, at least two important factors must be kept in mind to fully comprehend the study: Although cysts were isolated by gradient centrifugation prior to inoculation, it is unlikely that all bacteria have been removed from cyst suspensions; in other words, the cyst preparation is not likely to be 'sterile', and any effect of the potentially accompanying bacterial flora is difficult to determine. Moreover, rats may not be natural hosts of ST1 (very few data available on the topic!), and so, the pathology caused in the rats may be an unlikely finding in humans, who are indeed natural hosts of ST1 and may have developed a high degree of tolerance to this subtype.

Are dogs, wolves, and other canids natural hosts of Blastocystis?

When visiting Australia earlier this month, I had the pleasure of meeting Wenqi Wang and Tawin Inpankaew, both PhD students working at School of Veterinary Science, The University of Queensland Gatton Campus and supervised by Dr Rebecca Traub. One of the foci of this group is to study Blastocystis in animals, for instance in households where animals are kept as pets. Recently, a paper emerged from this group on diversity of Blastocystis subtypes in dogs in different geographical settings, hence domestic/pound dogs from Brisbane, Australia, semi-domesticated dogs from a village in Cambodia, and stray dogs from Mumbai and other Indian cities. Using sensitive PCR methods they observed that almost one fourth of the Indian dogs were infected, while dogs in the Cambodian village and in Queensland remained largely uninfected. Coprophagy and access to Blastocystis-positive stool from different hosts may account for the relatively high prevalence in stray dogs in India, although one might assume that the prevalence would then be even much higher? The team used nested PCR in their study and found four different subtypes in the Indian dogs, including ST1, ST4, ST5 and ST6. Whether all of their data collectively indicate that dogs are not natural hosts of Blastocystis is a matter of debate and remains to be more thoroughly investigated. Indeed, prevalence and subtype data from studies of samples from wild life canids (dingos, jackals, wolves, coyotes, but also foxes and raccoon dogs) would shed further light on this topic.

Finally, for those interested in how Blastocystis deals with oxidative stress and related metabolic issues, there is a paper out on iron-sulphur cluster biogenesis in protozoan parasites by Ali and Nozaki citing works by Tsaousis (2012), Denoeud (2011), Long (2011), and Stechmann (2008).


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

Bart A, Wentink-Bonnema EM, Gilis H, Verhaar N, Wassenaar CJ, van Vugt M, Goorhuis A, van Gool T. Diagnosis and subtype analysis of Blastocystis sp. in patients in a hospital setting in the Netherlands. BMC Infectious Diseases, 13:289.

Li J, Deng T, Li X, Cao G, Li X, & Yan Y (2013). A rat model to study Blastocytis subtype 1 infections. Parasitology Research PMID: 23892480 DOI: 10.1007/s00436-013-3536-7

Parija SC (2013). Blastocystis: Status of its pathogenicity. Tropical Parasitology, 3 (1) PMID: 23961433

Parija SC, & Jeremiah S (2013). Blastocystis: Taxonomy, biology and virulence. Tropical Parasitology, 3 (1), 17-25 PMID: 23961437 

Ramírez JD, Sánchez LV, Bautista DC, Corredor AF, Flórez AC, & Stensvold CR (2013). Blastocystis subtypes detected in humans and animals from Colombia. Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases PMID: 23886615

Sekar U, & Shanthi M (2013). Blastocystis: Consensus of treatment and controversies. Tropical Parasitology, 3 (1), 35-9 PMID: 23961439

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

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

Thursday, March 21, 2013

LUMINEX xMAP Technology in Parasite Diagnostics

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

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

Saturday, November 10, 2012

How Hard Can It Be?

How strange the world of clinical microbiology is when you compare the fields of mycology, parasitology, bacteriology and virology to each other. Such different possibilities, opportunities, limitations, and diagnostic challenges! The 3 month mortality rate of invasive aspergillosis, a disease mainly caused by Aspergillus fumigatus and seen in mainly patients with haematological malignancies, patients undergoing allogenic HSCT and patients in ICUs, may be as high as 60%, and therefore a quick and reliable diagnosis is mandatory to secure timely therapeutic intervention. But, - Aspergillus fumigatus happens to be ubiquitous, and contamination of patient samples, whether blood or airway samples, may always be a potential cause of false-positive test results, and one of the reasons why the use of PCR as a first line diagnostic tool in routine mycology labs is still limited. Antigen tests, such as the Galactomannan antigen test, which also allow quick diagnosis can also be false-positive, not only due to sample contamination, but also due to galactomannan residues in medical compounds, such as the widely applied antibiotic Tazocin (piperacillin-tazobactam), which means that patients who have been given this drug and who submit a blood sample for galactomannan testing may test slightly positive even in the absence of an Aspergillus infection.
These are only some classical examples. In the field of mycology, positive predictive values (PPV; i.e. what is the probability of disease given a positive test result) are sometimes unacceptably low, and the lower the prevalence of the disease, the lower the PPV. This means that you need a lot of experience and knowledge on pre-test-probability + data from clinical and diagnostic work-ups, including anamnestic details, to determine whether or not the patient should receive therapy, such as treatment with voriconazole, -  a relatively expensive drug.

Aspergillus fumigatus - the most common cause of invasive aspergillosis - on blood agar.

In the parasitology lab, however, things are quite different. Contamination of patient samples is rarely an issue, and in most cases not possible at all (disregarding DNA contamination of course). Specificity of microscopy is very often very high (close to 100%), which means that the PPV is very high even in cases where the disease is rare. Hence, if cysts of Giardia have been detected in your stool, it's due to the presence of the parasite in your body. It's a bit more tricky with PCR-based analyses, where the specificity does not rely on your ability to visually distinguish between e.g. Giardia and non-Giardia elements, but where it's all about designing oligos that anneal only to Giardia-DNA.
While in the mycology lab we struggle with low PPVs, one of the biggest challenges for me and my colleagues in the parasitology lab is to optimise the negative predictive value (NPV) of a faecal parasite diagnostic work-up - how can we rule out parasitic disease by cost-effectively putting together a panel of as few tests as possible?

There are many other differences. For instance, you can grow bacteria and fungi in the lab very easily, in fact, culture of bacteria and fungi is an essential diagnostic tool, which also allows you to submit the strain to antibiotic or antimycotic susceptibility testing and molecular characterisation/MALDI-TOF analysis in case you are not sure about the species ID. So, you have the strains right there in front of you, on agar plates, and they grow and grow, and you can keep them for as long as you like, - clean, non-contaminated strains on selective media.
You can't really do that with parasites, not nearly to the same extent and as easily, that is. For instance, you can culture Blastocystis directly from stool for sure (go here for the protocol), but only in the presence of bacteria (some of my colleagues do actually now and then manage to grow strains of Blastocystis in the absence of bacteria, they obtain what is called "axenic" cultures, but I believe that they cannot do it consistently and in limited time.). And it's a pity, since there is so much you can do when you have "clean" patient strains. Apart from susceptibility testing (which would actually be a bit difficult since Blastocystis is strictly anaerobic, so you can't really have it in microtiter plates or on RPMI plates on the table in front of you, but the strains could be challenged in the growth tubes), you can also extract DNA, and you would know that all the DNA that you extract from the isolate is from that particular strain, and not from bacterial contaminants. You can use the strain for production of antigens which can be used in ELISAs and used to generate mono- and polyclonal antibodies... Sequencing genomes of various subtypes would be a lot easier and quicker, and so on...

So, what appears obvious in one field of microbiology is not as obvious in another field, and vice versa. I wish Blastocystis was much easier to isolate. Dientamoeba too. Dientamoeba is probably as common as Blastocystis, and not rarely seen in co-infections. It is strange to contemplate that a parasite infecting hundreds of millions of people has not yet had its genome sequenced? We have no clue when it comes to effector proteins in Dientamoeba, and also for this parasite, what we know about its clinical significance relies mainly on epidemiological data.

There is no doubt that concerted efforts of experienced scientists should make it possible to develop appropriate and relevant culture protocols for these parasites. It does, however, require a lot of resources and time to get to know these common, but oh so fragile and reclusive little creatures...

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

Verweij PE, Kema GH, Zwaan B, & Melchers WJ (2012). Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatus. Pest management science PMID: 23109245

Stensvold, C., Jørgensen, L., & Arendrup, M. (2012). Azole-Resistant Invasive Aspergillosis: Relationship to Agriculture Current Fungal Infection Reports, 6 (3), 178-191 DOI: 10.1007/s12281-012-0097-7

Maertens J, Theunissen K, Verhoef G, & Van Eldere J (2004). False-positive Aspergillus galactomannan antigen test results. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 39 (2), 289-90 PMID: 15307045
Munasinghe VS, Stark D, & Ellis JT (2012). New advances in the in-vitro culture of Dientamoeba fragilis. Parasitology, 139 (7), 864-9 PMID: 22336222

Sunday, July 1, 2012

Do I Get Diagnosed Correctly?

I can tell especially from Facebook discussions that people across the globe wanting to know about their "Blastocystis status" are worried that they are receiving false-negative results from their stool tests, and that many Blastocystis infections go unnoticed. And I think I should maybe try and say a few things on this (please also see a recent blog post on diagnosis, - you'll find it here). I might try and simplify things a bit in order not to make the post too long.

Below, you'll find a tentative representation of the life cycle of Blastocystis. It is taken from CDC, from the otherwise quite useful website DPDx - Laboratory Identification of Parasites of Public Health Concern.

Proposed life cycle of Blastocystis.
I don't know how useful it is, but what's important here is the fact that we accidentally ingest cysts of Blastocystis, and we shed cysts that can be passed on to other hosts. The cyst stage is the transmissible stage, and the way the parasite can survive outside the body; we don't know for how long cysts can survive and remain infective. In our intestine and triggered by various stimuli, the cysts excyst, transiting to the non-cyst form, which could be called the trophozoite / "troph" stage, or to use a Blastocystis-specific term, the "vacuolar stage" (many stages have been described for Blastocystis, but I might want to save that for later!). This is possibly the stage in the life cycle where the parasite settles, thrives, multiplies, etc. You can see a picture of vacuolar stages in this blog post. Many protozoa follow this simple life cycle pattern, among them Giardia and most species of Entamoeba. If the stool is diarrhoeic and you are infected by any one or more of these parasites, it may be so that only trophozoites, and, importantly, no cysts, are shed! This has something to do with reduced intestinal transit time and, maybe more importantly, the failure of the colon to resorb water from the stool which means that the trophozoites do not get the usual encystation stimuli. Importantly, trophozoites are in general non-infectious.

There is documentation that once colonised with Blastocystis, you may well carry it with you for years on end, and as already mentioned a couple of times, no single drug or no particular diet appears to be capable of eradicating Blastocystis - this is supported by the notion that Blastocystis prevalence seems to be increasing by age, although spontaneous resolution may not be uncommon, - we don't know much about this. Now, although day-to-day variation in the shedding of Blastocystis has been described, it is my general impression that colonised individuals may shed the parasite with each stool passage, and well-trained lab technicians/parasitologists will be able to pick up Blastocystis in a direct smear (both cysts and trophs may be seen). To do a direct smear you simply just mix a very small portion of the stool with saline or PBS on a slide, put a cover slip over it and do conventional light microscopy at x200 (screening) or x400 (verification). Very light infections may be difficult to detect this way, and if you don't have all the time in the world, a direct smear may not be the first choice.

The "king" of parasitological methods, however, is microscopy of faecal concentrates (Formol Ethyl Acetate Concentration Technique and any variant thereof), which is remarkable in its ability to detect a huge variety of parasites. Especially cysts of protozoa (e.g. Giardia and Entamoeba) and eggs of helminths (e.g. tapeworm, whipworm and roundworm) concentrate well and are identified to genus and species levels based on morphology. The method is not as sensitive as DNA-based methods such as PCR, but as I said, has the advantage of picking up a multitude of parasites and therefore good for screening; PCR methods are targeted towards particular species (types) of parasites. A drawback of the concentration method is that it doesn't allow you to detect trophzoites (i.e. the fragile, non-cystic stage), and, as mentioned, diarrhoeic samples may contain only trophozoites and no cysts...

In many countries it is very common for people to be infected by both protozoa and helminths, and in those countries microscopy of faecal concentrates is a relevant diagnostic choice. In Denmark and many Western European countries, the level of parasitism is higher than might be expected (from a hygiene and food safety point of view) but due to only few parasitic species. Paradoxically, the intestinal parasites that people harbour in this part of the world are parasites that do not concentrate well. They are mainly:

1) Blastocystis
2) Dientamoeba fragilis
3) Pinworm (Enterobius vermicularis)

Only troph stages have been described for Dientamoeba fragilis and it may be transmitted by a vector, such as pinworm (look up paper by Röser et al. in the list below for more information); this mode of transmission is not unprecedented (e.g. Histomonas transmission by Heterakis). Eggs of pinworm may be present in faeces, but a more sensitive method is the tape test.

Now, Blastocystis often disintegrates in the faecal concentration process, and while you might be lucky to pick up the parasite in a faecal concentrate, you shouldn't count on it, and hence the method is not reliable, unless the faecal sample was fixed immediatley after being voided. This is key, and also why fixatives are used for the collection of stool samples in many parts of the world - to enable the detection of fragile stages of parasites. There are many fixatives, e.g. SAF (sodium acetate-acetic formalin), PVA (poly-vinyl alcohol) and even plain formalin will do the trick if it's just a matter of preserving the parasite in the sample. If SAF or PVA is used, this allows you to do permanently stained smears of faecal concentrates, and you will be able to pick up not only cysts of protozoa, but even trophozoites. Trichrome and iron-haematoxylin staining are common methods and are sensitive but very time-consuming and may be related to some health hazards as well due to the use of toxic agents. But this way of detecting parasites is like good craftmanship - it requires a lot of expertise, but then you get to look at fascinating structures with intriguing nuclear and cytoplasmatic diagnostic hallmarks. Truly, morphological diagnosis of parasites is an art form! Notably, samples preserved in such fixatives may be useless for molecular analyses.

Iron-haematoxylin stain of trophozoites of Entamoeba coli
(note the "dirty" cytoplasm characteristic of E. coli).

At our lab we supplement microscopy of faecal concentrates with DNA-based detection of parasites. For some clinically significant parasites, we do a routine screen by PCR, since this is more sensitive than microscopy of faecal concentrates and because this is a semi-automated analysis that involves only DNA extraction, PCR and test result interpretation, which are all things that can be taught easily. Major drawbacks of diagnostic PCR is that you cannot really distinguish between viable (patent infection) and dead organisms (infection resolving, e.g. due to treatment). This is why, in the case of Blastocystis, you may want to do a stool culture as well (at least in post-treatment situations), since only viable cells will be able to grow, obviously.

Two diagnostic real-time PCR analyses have been published, one using CYBR Green and one using a TaqMan probe.

Now, it certainly differs from lab to lab as to which method is used for Blastocystis detection. Some labs apparently apply thresholds for number of parasites detected per visual field, and only score a sample positive if more than 5 parasites per visual field have been detected. I see no support for choosing a threshold, since 1) we do not know whether any Blastocystis-related symptoms are exacerbated by parasite intensity, 2) the number of parasites detected in a faecal concentrate may depend on so many things which have nothing to do with the observer (fluctuations in shedding for instance), and 3) the pathogenic potential of Blastocystis may very well depend on subtype.

If Blastocystis was formally acknolwedged as a pathogen, like Giardia, standardisation of methods would have happened by now. Meanwhile, we can only advocate for the use of PCR and culture if accurate diagnosis of Blastocystis is warranted, while permanent staining of fixed faecal samples constitutes a very good alternative in situations where PCR is not an option.

I have the impression that some labs do DNA-based detection of microbes, including protozoa, and that a result such as "taxonomy unknown" is not uncommon. I don't know how these labs have designed their molecular assays, and therefore I cannot comment on the diagnostic quality and relevance of those tests... it also depends on whether labs do any additional testing as well, such as the more traditional parasitological tests. However, we do know that there is a lot of organisms in our intestine, for which no data are available in GenBank, which is why it is sometimes impossible to assign a name to e.g. non-human eukaryotic DNA amplified from a stool sample.

* More than 1 billion people may harbour Blastocystis.
* Blastocystis is found mainly in the large intestine.
* 95% of humans colonised by Blastocystis have one of the following subtypes: ST1, ST2, ST3, ST4.
* DNA-based detection combined with culture ensures accurate detection of Blastocystis in stool samples and enables subtyping and viability assessment.

Further reading:

Poirier P, Wawrzyniak I, Albert A, El Alaoui H, Delbac F, & Livrelli V (2011). Development and evaluation of a real-time PCR assay for detection and quantification of blastocystis parasites in human stool samples: prospective study of patients with hematological malignancies. Journal of clinical microbiology, 49 (3), 975-83 PMID: 21177897

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

Scanlan PD, & Marchesi JR (2008). Micro-eukaryotic diversity of the human distal gut microbiota: qualitative assessment using culture-dependent and -independent analysis of faeces. The ISME journal, 2 (12), 1183-93 PMID: 18670396

Stensvold CR, Ahmed UN, Andersen LO, & Nielsen HV (2012). Development and Evaluation of a Genus-Specific, Probe-Based, Internal-Process-Controlled Real-Time PCR Assay for Sensitive and Specific Detection of Blastocystis spp. Journal of clinical microbiology, 50 (6), 1847-51 PMID: 22422846

Stensvold CR, Arendrup MC, Jespersgaard C, Mølbak K, & Nielsen HV (2007). Detecting Blastocystis using parasitologic and DNA-based methods: a comparative study. Diagnostic microbiology and infectious disease, 59 (3), 303-7 PMID: 17913433

Stensvold CR, & Nielsen HV (2012). Comparison of microscopy and PCR for detection of intestinal parasites in Danish patients supports an incentive for molecular screening platforms. Journal of clinical microbiology, 50 (2), 540-1 PMID: 22090410

Sunday, May 20, 2012

Brave New World

Using Blastocystis as an example, we have only recently realised the fact that conventional diagnostic methods in many cases fail to detect Blastocystis in faecal samples, which is why we have started using molecular diagnostics for Blastocystis. I was also surprised to realise that apparently no single drug can be used to treat Blastocystis, and that in fact we do not know which combo of drugs will actually consistently eradicate Blastocystis (Stensvold et al., 2010).

There will come a time - and it will be soon - where it will be common to use data from genome sequencing of pathogenic micro-organisms to identify unique signatures suitable for molecular diagnostic assays and to predict suitable targets (proteins) for chemotherapeutic intervention; in fact this is already happening (Hung et al., in press). However, despite already harvesting the fruits of recent technological advances, we will have to bear in mind that the genetic diversity seen within groups of micro-organisms infecting humans may be quite extensive. This of course will hugely impact our ablility to detect these organisms by nucleic acid-based techniques. For many of the micro-eukaryotic organisms which are common parasites of our guts, we still have only very little data available. For Blastocystis, data is building up in GenBank and at the Blastocystis Sequence Typing Databases, but for other parasites such as e.g. some Entamoeba species, Endolimax and Iodamoeba, we have very little data available. We only recently managed to sequence the small subunit ribosomal RNA gene of Iodamoeba, and we demonstrated tremendous genetic variation within the genus; it is now clear that Iodamoeba in humans comprises a species complex rather than "just" Iodamoeba bütschlii (Stensvold et al, 2012).

Cysts of Iodamoeba
Ribosomal RNA is present in all living cells and is the RNA component of the ribosome. We often use this gene for infering phylogenetic relationships, i.e. explaining how closely or distantly related one organism is to another. This again assists us in hypothesising on transmission patterns, pathogenicity, evolution, drug susceptibility and other things. Since ribosomal RNA gene data are available for most known parasites, we often base our molecular diagnostics on such data. However, the specificity and sensitivity of our molecular diagnostic assays such as real-time PCRs are of course always limited by the data available at a given point in time (Stensvold et al., 2011). Therefore substantial sampling from many parts of the world is warranted in order to increase the amount of data available for analysis. In terms of intestinal micro-eukaryotes, we have only seen the beginning. It's great to know data are currently builiding up for Blastocystis from many parts of the world, - recently also from South America (Malheiros et al., 2012) - but the genetic diversity and host specificity of many micro-eukaryotes are still to be explored. It may be somewhat tricky to obtain information, since conventional PCR and sequencing offer significant challenges in terms of obtaining sequence data; such challenges can potentially be solved by metagnomic approaches - today's high throughput take on cloning; however, although the current next generation sequencing technology hype makes us feel that we are almost there, it seems we still have a long way to go - extensive sampling is key!

Cited literature:

Hung, G., Nagamine, K., Li, B., & Lo, S. (2012). Identification of DNA Signatures Suitable for Developing into Real-Time PCR assays by Whole Genome Sequence Approaches: Using Streptococcus pyogenes as a pilot study Journal of Clinical Microbiology DOI: 10.1128/JCM.01155-12

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

Stensvold, C., Lebbad, M., & Clark, C. (2011). Last of the Human Protists: The Phylogeny and Genetic Diversity of Iodamoeba Molecular Biology and Evolution, 29 (1), 39-42 DOI: 10.1093/molbev/msr238  

Stensvold, C., Lebbad, M., & Verweij, J. (2011). The impact of genetic diversity in protozoa on molecular diagnostics Trends in Parasitology, 27 (2), 53-58 DOI: 10.1016/

Stensvold, C., Smith, H., Nagel, R., Olsen, K., & Traub, R. (2010). Eradication of Blastocystis Carriage With Antimicrobials: Reality or Delusion? Journal of Clinical Gastroenterology, 44 (2), 85-90 DOI: 10.1097/MCG.0b013e3181bb86ba