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

Is This A New Subtype?

To quote one of my colleagues attending the recent IWOP 2012 meeting in Tarrytown, NY, Blastocystis subtyping in humans and animals is becoming 'trendy', and so we keep trying to advocate for a standardisation of the metholodology of Blastocystis subtyping.

We recently changed the title of our page at www.pubmlst.org/blastocystis so that now it is called Blastocystis Subtype (18S) and Sequence Typing (MLST) Databases, and we added some text to front page:

In terms of genetic markers, the barcode region (Scicluna et al., 2006) is by far the best represented in publicly available sequence databases, and the correct subtype can be identified by BLAST analysis in the sequence database at the present site. Blasting against this database has the added advantages, compared to using GenBank, of automatically assigning allele types to the SSU-rDNA as well as using the consensus subtype nomenclature (unlike GenBank where the subtype is included only if one was part of the accession submission and no attempt to impose a standard nomenclature is made). In case the sequence does not match any of the ones in the database despite full coverage of the region, this indicates that the sequence represents a new allele or maybe even a new subtype depending on the amount of variation. If a new subtype is suspected, we suggest doing PCR and sequencing of the complete SSU rRNA gene with subsequent phylogenetic analysis using reference sequences.

Now, the last bit is extremely important. We have seen examples of researchers (including ourselves!) assigning sequences to a new a subtype in the absence of complete SSU rDNA data (in fact complete sequences for ST10-ST14 are not yet publicly available!). Doing so has a least two major limitations/drawbacks: Far from all SSU rDNA regions have been validated as being representative of the whole SSU rRNA gene in terms of phylogenetic analysis, and therefore phylogenetic inferences based on non-validated regions may have little or at least less support than anticipated. Moreover, if someone analyses e.g. position 600-1600, and phylogenetic analysis based on this region reveals a potentially new subtype, this makes it impossible for his/her colleague who has data covering positions 1-600 from a Blastocystis isolate that may also represent a new subtype to ascertain whether it might be same subtype (see example below)!

Obtaining complete SSU rDNA sequences directly from faecal DNA may be a cumbersome task but is sometimes possible by combining sequence-specific primers with low-specificity primers such as the RD5 and the RD3 primers (Clark, 1997). If a cultured isolate is available, obviously this makes complete SSU rDNA sequencing much easier.

While it appears that the number of subtypes occurring in humans stays around 9, our gut feeling is that we are yet to uncover quite a few subtypes colonising non-human mammals, and it's great to see an increasing number of teams exploring the genetic diversity of Blastocystis. For instance, Dr Ronald Fayer and his group recently published exciting data on a new Blastocystis subtype in cattle, which they named ST14 (Fayer et al., 2012).

Importantly, caution should be taken to avoid creating confusion in subtype terminology. Confusion can arise when independent researchers assign the same new subtype name (e.g. ST14, ST15, etc.) to novel sequences which in fact belong to different ribosomal lineages, or when incomplete SSU rDNA sequence data are used; this situation was seen recently, when Petrasova et al. (2011), assigned a Colobus sequence to ST5, although it was in fact a ST13 sequence (Clark et al., in press); the situation arose, since Petrasova et al. (2011) did not have data covering the region currently available for ST13 (Parkar et al., 2010), and therefore believed that their sequence was a unique ST5 variant. As for ST14, less than 500 bp are currently available, and these 500 bp are not in the barcode region, making it difficult for all teams using barcoding to compare their data. And so we would like to advocate for making complete SSU rDNA sequences publicly available (Genbank) for potentially new subtypes, for at least two reasons:

1. Phylogenetic inferences based on the complete SSU rDNA will be more robust than those obtained from analysing shorter sequence streches.

2. Complete seqeunces are needed for reference since subtype screening typically includes a single round PCR such as barcoding (Scicluna et al., 2006) amplifying about 550 bp; in the situation where complete SSU rDNAs are available for all known subtypes, it will be quick to analyse, whether a sequence may represent a new subtype, since this will be independent on the SSU rDNA region studied.We therefore hope that complete SSU rDNA sequences will soon be made publicly available for ST10-ST14.

So, when does a complete SSU rDNA sequence represent a new subtype? Well, we have a review paper in press in Advances in Parasitology on recent developments in Blastocystis research, which will be published in less than six months probably, and which also touches on this topic; once the paper is published, I will try and make a summary our thoughts on this...

Further reading:

Clark CG (1997). Extensive genetic diversity in Blastocystis hominis. Molecular and biochemical parasitology, 87 (1), 79-83 PMID: 9233675

Fayer R, Santin M, & Macarisin D (2012). Detection of concurrent infection of dairy cattle with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and microscopic methods. Parasitology research PMID: 22710524

Parkar U, Traub RJ, Vitali S, Elliot A, Levecke B, Robertson I, Geurden T, Steele J, Drake B, & Thompson RC (2010). Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers. Veterinary parasitology, 169 (1-2), 8-17 PMID: 20089360

Petrášová J, Uzlíková M, Kostka M, Petrželková KJ, Huffman MA, & Modrý D (2011). Diversity and host specificity of Blastocystis in syntopic primates on Rubondo Island, Tanzania. International journal for parasitology, 41 (11), 1113-20 PMID: 21854778
 
Scicluna SM, Tawari B, & Clark CG (2006). DNA barcoding of blastocystis. Protist, 157 (1), 77-85 PMID: 16431158

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.

Novel Blastocystis real-time PCR

Our new real-time PCR for Blastocystis has been published in JCM.

Check it out at

Development and evaluation of a genus-specific, probe-based, internal process controlled real-time PCR assay for sensitive and specific detection of Blastocystis.

or

http://www.ncbi.nlm.nih.gov/pubmed/22422846

and use it!