This Month in Blastocystis Research (SEP 2015)

The month of September saw the publication of the first data on Blastocystis subtypes going out from Qatar. Abu-Madi and colleagues--who have already been quite prolific in terms of surveying intestinal parasitic infections in Qatar--studied the positive rate of Blastocystis in 608 apparently healthy subjects arriving in Qatar for the first time, identifying a prevalence of 71% as identified by PCR. Strikingly, the positive rate by microscopy of the corresponding samples was only 7%. Three subtypes were idenfied, with ST3 being the most common subtype, followed in prevalence by ST1 and ST2. The study is important for at least two reasons: It confirms the drawback of basing Blastocystis epidemiological research on data generated using microscopy alone, and it confirms the virtual absence of ST4 outside of Europe.

Increased sensitivity of PCR relative to microscopy was also confirmed in a study carried out in Malaysia (I presume) by Ragavan and colleagues. This group surveyed the Blastocystis positivity rate among IBS and non-IBS patients analyzing colonic aspirates, including a total of 109 individuals. Given the data available on Blastocystis prevalence, I was quite surprised to learn that this group failed to detect Blastocystis in any of the samples by microscopy and culture. Using PCR (the subtype-specific [STS] primers were used as diagnostic primers), the group identified Blastocystis in 6 IBS patients and 4 non-IBS patients. Also these figures appear quite low. However, there is very little information available on the non-IBS patients, and since all study individuals were subject to colonscopy, this group of individuals might be suffering chronic and potentially severe intestinal disease, including for instance colorectal cancer, inflammatory bowel disease, etc., which would explain the low prevalence of Blastocystis observed among these individuals. Indeed, evidence is accumulating that the more "gut healthy" you are, the larger the probability of being Blastocystis-positive. I noticed that the colonic aspirates were spun down using 3,000 rpm prior to culture and microscopy; this process might have had an impact on cell viability and morphology; still, DNA should be detectable following this process. Meanwhile, we recently showed (Scanlan et al., 2015) that the sensitivity of the STS primers is relatively low, which is why the use of real-time PCR is recommendable for PCR-based screening. To see an example of how the STS primers perform relative to barcoding primers, go here (Suppl Table 2).
Moreover, care should be taken when reading this paper, since I'm fairly convinced that the subtype terminology used in the study is different from the consensus terminology (Stensvold et al., 2007). It says that the subtypes detected included ST2, ST3, ST4, and ST5; if this reflects the terminology that went along with the original description of the STS primers, these subtypes correspond to ST7, ST3, ST6, and ST2, which to me would be a more likely subtype distribution, taking this particular region into consideration, and given the fact that ST5 appears to be extremely rare in humans. 

It's always interesting to expand on the natural host spectrum of Blastocystis. The parasite has been found in a perplexing array of hosts, but some host specificity has been observed. When it comes to animals held by humans as livestock or pets, we know that pigs and cattle are commonly, if not consistently, colonised by Blastocystis with some quite specific subtypes. With regard to pets, dogs and cats have been found positive, but there seems to be increasing evidence that these animals are not natural hosts (see also Wang et al., 2013). Osman and colleagues, recently published a survey on Cryptosporidium and Blastocystis in dogs using sensitive molecular methods, demonstrating a prevalence of Blastocystis of only about 3%. Moreover, the subtypes 2 and 10 were found, and ST10 is found mostly in cattle, and never before in dogs, as far as I know, which could suggest accidental colonisation - and possibly not a very long-lasting one. Similarly, when humans are found to be colonised with subtypes rarely found in humans, such as ST6, ST7, and ST8, it would be interesting to know for how long these subtypes are capable of "staying put" in the human intestine.

References

Abu-Madi M, Aly M, Behnke JM, Clark CG, & Balkhy H (2015). The distribution of Blastocystis subtypes in isolates from Qatar. Parasites & Vectors, 8 PMID: 26384209

Osman M, Bories J, El Safadi D, Poirel MT, Gantois N, Benamrouz-Vanneste S, Delhaes L, Hugonnard M, Certad G, Zenner L, & Viscogliosi E (2015). Prevalence and genetic diversity of the intestinal parasites Blastocystis sp. and Cryptosporidium spp. in household dogs in France and evaluation of zoonotic transmission risk. Veterinary Parasitology PMID: 26395822   

Ragavan, N., Kumar, S., Chye, T., Mahadeva, S., & Shiaw-Hooi, H. (2015). Blastocystis sp. in Irritable Bowel Syndrome (IBS) - Detection in Stool Aspirates during Colonoscopy PLOS ONE, 10 (9) DOI: 10.1371/journal.pone.0121173  

Scanlan PD, Stensvold CR, & Cotter PD (2015). Development and Application of a Blastocystis Subtype-Specific PCR Assay Reveals that Mixed-Subtype Infections Are Common in a Healthy Human Population. Applied and Environmental Microbiology, 81 (12), 4071-6 PMID: 25841010   

Stensvold CR, Suresh GK, Tan KS, Thompson RC, Traub RJ, Viscogliosi E, Yoshikawa H, & Clark CG (2007). Terminology for Blastocystis subtypes--a consensus. Trends in Parasitology, 23 (3), 93-6 PMID: 17241816

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

Where Are We On Blastocystis Subtypes?

As mentioned, Blastocystis exhibits remarkable intrageneric diversity, which is continuously being explored by us and our colleagues. We are convinced that the genus of Blastocystis comprises multiple species, but for now we call them "ribosomal lineages" or "subtypes" and allocate numbers to each subtype, hence ST1, ST2, etc. While the number of subtypes that can be found in humans remains stable, we and our colleagues are still expanding the subtype universe in non-human hosts (I will be blogging on this shortly).

Barcoding currently represents state-of-the-art in Blastocystis subtyping, and luckily this method appears to gain a foothold in labs across the world.

Nine subtypes have been found in humans, but some of them only on rare occasions. A recent study going out from London School of Hygiene and Tropical Medicine and led by Dr Alfellani and published just now in Acta Tropica looked at 356 Blastocystis sequences from samples from the UK and Libya, but also from sub-Saharan Africa, namely Liberia and Nigeria.

Blastocystis Subtyping in Routine Microbiology Labs

When I speak to colleagues in and outside Europe and visit research portals and social media, including Facebook groups, I get the impression that Blastocystis subtyping is something that is still very rarely done, despite the fact that most clinical microbiologists and biologists acknowledge that subtypes may differ in terms of clinical significance and in other respects. We get new data on Blastocystis subtypes in various cohorts from time to time from research groups around the world, but all reports are characterised by relatively small sample sizes and subtyping methodology has not yet been standardised. This type of research is moreover challenged by the fact that Blastocystis is common in healthy individuals (i.e. people not seeing their GPs for gastrointestinal problems), and this makes it extremely difficult to identify the subtype distribution in the "background" population.

Although we recommend barcoding (see one of my previous posts) as the subtyping method of choice, there is no "official report" identifying the Blastocystis subtyping gold standard. Therefore, I'm currently setting up a lab project that is going to help us compare the most common methods used for subtyping in order to identify the one most suitable. I emphasise that the best method used for subtyping is not the PCR that should be used for diagnostic purposes, mostly due to the fact that PCRs for subtyping amplify 300-600 bp, which are much longer amplicons than the one we go for in diagnostic PCRs (typically 80-100 bp). We therefore recommend our novel TaqMan-based real-time PCR for initial diagnosis, or culture, which is inexpensive and relatively easy and provides you with a good source of cells for DNA extraction.
I hope that we will be able to come up with some robust data soon that will allow us to recommend the most suitable approach and hope to publish our results in a clinical microbiology journal of high impact, and I hope that this will prompt Blastocystis subtyping in many labs. Once this report has been published, I intend to upload a protocol here at the site where lab procedures for diagnosis and subtyping will be described in detail. Stay tuned!