Showing posts with label microscopy. Show all posts
Showing posts with label microscopy. Show all posts

Friday, April 1, 2016

This Month in Blastocystis Research (MAR 2016)

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

Please go here to see the case.

Creepy Dreadful Wonderful Parasites: Case of the Week 390.

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

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

Tuesday, March 31, 2015

This Month in Blastocystis Research - MAR 2015

"Show me your gut bacteria and I'll tell you if you're infected with Entamoeba"

One of my 'partners in crime', science reporter Jop de Vrieze, made me aware of a study just published now by Elise R Morton and colleagues. The study appeared in bioRxiv—The Preprint Server for Biology, operated by Cold Spring Harbor Laboratory. The study is totally in line with one of the research foci in our lab.

The paper is called 'Variation in rural African gut microbiomes is strongly shaped by parasitism and diet', and can be downloaded here. The backbone in this type of research is the recognition that studies revealing a large contrast between the microbiomes of populations in developing countries and those of populations in urban industrialised areas have shown that geography is an important factor associated with the gut microbiome, but that such studies yet have to disentangle the effects of factors such as climate, diet, host genetics, hygiene and parasitism.

It's very refreshing that for once, 'parasitism' is included in such considerations. As mentioned in one or more of my previous blog posts, we have metagenomics data stongly indicating that Blastocystis colonisation is associated with certain microbial communities. As of yet, we have no idea about cause and effet, but the idea alone is immensely intriguing.

A large and a small cyst of Entamoeba coli. Courtesy of Dr Marianne Lebbad.
Now, Morton et al. have produced data that suggest that the presence of Entamoeba—another gut-associated eukaryotic genus comprising multiple species of varying pathogencitiy—is strongly correlated with microbial composition and diversity. They showed that an individual's liability to being infected by Entamoeba could be predicted with 79% accuracy based on gut microbiome composition.

The authors used 16S PCR and Illumina-based sequencing of 16S amplicons, and I could have wished that molecular assays, e.g., the 18S PCR that we have developed in our lab + associated software, had also been used to test the faecal samples from the 64 individuals enrolled in the study in order to obtain more precise data, not only on Entamoeba but also on other human-associated gut protists, such as Blastocystis.

While alpha (intra-host) diversity of Entamoeba-positive individuals was significantly higher than that of Entamoeba-negative individuals, analysis of the beta (inter-host) diversity revealed that gut communities across Entamoeba-positive individuals were more similar than across Entamoeba-negative individuals, suggesting that, as alpha diversity increases, there are fewer potential stable states for individual gut communities, or that infection by Entamoeba drives changes in the microbiome that are dominant over other factors.

Right—this is Entamoeba, I know, but in principle, the type of analyses that were performed in the present study could be applicable to Blastocystis, Dientamoeba, and other gut parasites, which may help us understand their role in health and disease. Are these parasites able to influence gut microbiota? Can they be used for gut microbiota manipulation? Or do they only infect people with certain microbiota profiles? Time will show... maybe.

For those of you who would like to read more about what is shaping our microbiomes and how the gut microbiota may impact on our gastrointestinal health, I recently did a couple of blog posts for United European Gastroenterology (UEG) Education that might be of some interest:

Are we finally saluting the fungal kingdom as a co-ruler of GI health and disease?

The intestinal microbiome—Rosetta Stone or Tower of Babel?


Morton ER, Lynch J, Froment A, Lafosse S, Heyer E, Przeworski M, Blekham R, Segurel L.
Variation in rural African gut microbiomes is strongly shaped by parasitism and diet. bioRxiv doi

Monday, January 27, 2014

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 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.

Tuesday, March 12, 2013

Blastocystis video

Just saw this on YouTube and had to share it. This is Blastocystis (and other microorganisms) viewed through a microscopy (light microscopy). Note that this is Blastocystis from a chicken, but Blastocystis from humans looks the same; at least I don't know how to tell the difference. I wonder whether this is from a culture or a completely fresh egestion... looks more like a culture to me. Note how the Blastocystis looks almost like fat cells...

The video comes with some nice music as well!

Friday, February 22, 2013

Bubbly Blasto!

Yesterday, I was checking up on a fresh Blastocystis culture. I loaded 20 µL of the culture "sediment" on to a glass slide, placed the cover slip on top and examined it by light microscopy. While examining the slide, I observed a multitude of dividing cells, indicating vigorous growth and a thriving strain, and once again I was struck by the appearance of dividing Blastocystis. This is basically what they may look like:

Like soap bubbles really, only a lot smaller obviously (mikrons), and somewhat opaque! You'll see them in different sizes and the way they divide looks just like this. Apparently some sort of random budding or multiple fission. You'll see little more than this bubbly structure, which means that there are very few morphological hallmarks to describe. A few nuclei may be discernible along the cytoplasmatic rim, but that's about it when you use light microscopy. Ultrastructural and biochemical analysis is required if you hope to be able to describe some of the processes involved in reproduction.

We often say that Blastocystis organisms representing different subtypes are morphologically indistinguishable; what this actually means is that we do not have the tools to differentiate them morphologically. There may actually be great variation between strains in terms of for instance how they grow in vivo and in vitro and maybe also how they reproduce. Vacuolar forms are the most common form seen in xenic cultures, but other morphotypes are sometimes observed, for instance the granular stage, which, in my experience, is typically seen in cultures that are not “well looked after”, i.e. where medium is not being replaced about twice a week. Dunn and colleagues. (1989) observed that the granular stage could arise from vacuolar stages in cultures where the concentration of horse serum was increased.

I have previously stated that there is no evidence for phagocytosis in Blastocystis. Actually, Dunn et al. (1989) captured what they thought to be bacterial engulfment by ultra-structural analysis, and they also observed bacteria-engulfing pseudopodia in amoeboid stages, in which degraded bacteria were observed. I don't think that I've ever come across this amoeboid stage, but it has been described by quite a few researchers.

Anyway, let's hope for another kind of bubbles this Friday night!

Suggested reading:

Dunn LA, Boreham PF, & Stenzel DJ (1989). Ultrastructural variation of Blastocystis hominis stocks in culture. International Journal for Parasitology, 19 (1), 43-56 PMID: 2707962

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

Monday, May 7, 2012

Blastocystis: To Treat or Not to Treat...

This year, Coyle et al. published a Clinical Practice paper in Clinical Infectious Diseases, a journal with a 5-year impact factor of almost 8. It is still difficult to get papers on Blastocystis published in clinical, peer-reviewed journals of major impact, probably due to the fact that evidence of Blastocystis' pathogenicity is so far only indicative, so it is great to see that the authors have managed to get their manuscript past those iron doors!

A few issues have come to my attention. When reading the abstract the reader will get the impression that subtypes are synonymous with genotypes, which is not the case. In the case of Blastocystis, a subtype is equivalent to a species; one of the reasons why we haven't allocated species names to Blastocystis from humans, other mammals and birds yet, is that we do not have sufficient data on genetic diversity and host specificity to come up with relevant names.

It says in the first page (pdf) that Blastocystis subtype (ST) 3 is found only in humans, which is not true. This subtype is common in non-human primates and can be seen in other, larger animals, including dogs, and also birds, if I remember correctly. However, so far, we only have multilocus sequence typing data from human and non-human primates, and these data indicate that ST3 found in non-human primates is often different from ST3 found in humans.

The authors recommend that asymptomatic individuals with few cysts should not be treated. Then what about asymptomatic individuals with many cysts? Also, with the diagnostic short-comings of microscopy of faecal concentrates, the suggested cut-off at 5 organisms per visual field appears arbitrary and, in best case, fortuitous.

In the abstract, the authors state that metronidazole is the drug of choice, although they appear to be quite aware that this drug has limited effect in terms of eradicating Blastocystis. So, why is metronidazole the drug of choice? Blastocystis is a parasite lodged primarily in the large intestine, and therefore we must anticipate that metronidazole often fails to reach the the parasite in sufficient concentrations due to absorption proximally in the gut. Luminal agents, such as paromomycin, are probably more likely to work, maybe in combination with metronidazole, although we have had a case, where even this combination was not effective.

When reviewing studies of treatment, it is important to acknowledge that insensitive methods have been used to evaluate drug efficacy. Culture combined with PCR is in my opinion the best method available in this respect. I prefer adding culture to the test, since culture detects viable Blastocystis (as opposed to PCR which will detect both viable and non-viable cells). Future randomised controlled treatment studies should therefore use culture and PCR to identify carriers both pre- and post-treatment. Whether Blastocystis-positive stool post-treatment is due to recrudescence, resistance or reinfection is not easily evaluated, but some useful information can be achieved by multi-locus sequence typing of isolates pre- and post-treatment.

Literature cited:

Coyle CM, Varughese J, Weiss LM, & Tanowitz HB (2012). Blastocystis: to treat or not to treat... Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 54 (1), 105-10 PMID: 22075794  

Stensvold CR, Alfellani M, & Clark CG (2012). Levels of genetic diversity vary dramatically between Blastocystis subtypes. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 12 (2), 263-73 PMID: 22116021  

Stensvold CR, Smith HV, Nagel R, Olsen KE, & Traub RJ (2010). Eradication of Blastocystis carriage with antimicrobials: reality or delusion? Journal of clinical gastroenterology, 44 (2), 85-90 PMID: 19834337

Sunday, April 8, 2012

A Few Words On Blastocystis Morphology and Diagnosis

Blastocystis is a sinlge-celled parasite. The parasite produces cysts (probably the transmissible form) and vegetative stages (including the stage commonly referred to as the vacuolar stage). Vegetative stages are commonly seen in fresh faecal samples and in culture. This is what they look like under light microscopy:

Vegetative stages of Blastocystis (unstained) (source:

Using permanent staining of fixed faecal material, the eccentrically located nuclei become more apparent:

Vegegtative stages of Blastocystis (Trichrome stain) (source:

Although sensitive, permanent staining techniques (e.g. Trichrome, Giemsa and Iron Haematoxylin) are relatively time-consuming, impractical and expensive. Since also conventional concentration of unfixed stool using e.g. the Formol Ethyl-Acetate Concentration Technique is not appropriate for diagnosis (Blastocystis cysts are very difficult to pick up, and vacuolar stages become distorted or disintegrate), we recommend short-term in-vitro culture (using Jones' or Robinson's medium) and/or Real-Time-PCR on genomic DNAs extracted directly from faeces using QIAGEN Stool Mini Kit (QIAGEN, Hilden, Germany) or - in modern laboratories - by automated DNA extraction robots. Once genomic DNAs have been extracted and screened by PCR, positive samples can be submitted to subtyping using the barcoding method, and DNAs can be screened for other parasites by PCR as well. In fact the use of insensitive methods to distinguish carriers from non-carriers is one of our greatest obstacles to obtaining valid prevalence data on Blastocystis.

Having an isolate in culture adds the benefit of having a continuous source of DNA for further genetic characterisation (for instance complete SSU-rDNA sequencing) in case a particular isolate turns out to be genetically different from those already present in GenBank or the isolate database at Blastocystis Sequence Typing Home Page. And chances are that there are quite a few "novel" subtypes out there... especially in animals. However, Blastocystis from animals may not always be successfully established in culture.