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.

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.

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

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: www.dpd.cdc.gov)

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

Vegegtative stages of Blastocystis (Trichrome stain) (source: www.dpd.cdc.gov)

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.