Development and Application of a Blastocystis Subtype-Specific PCR Assay Reveals that Mixed-Subtype Infections Are Common in a Healthy Human Population

Pauline D. Scanlan, Christen Rune Stensvold, Paul D. Cotter

The human gut is host to a diversity of microorganisms including the single-celled microbial eukaryote Blastocystis. Research has shown that most carriers host a single Blastocystis subtype (ST), which is unusual given the considerable within-host species diversity observed for other microbial genera in this ecosystem. However, our limited knowledge of both the incidence and biological significance of Blastocystis diversity within hosts (i.e. so-called mixed infections) is likely due to problems with existing methodologies. Here, we developed and applied Blastocystis ST-specific PCRs for the investigation of the most common subtypes of Blastocystis, (ST1-ST4), to a healthy human cohort (N = 50). We detected mixed infections in 22% of the cases, all of which had been identified as single ST infections in an earlier study using state-of-the-art methods. Our results show that certain STs occur predominantly as either single (ST3 and 4) or mixed infections (ST1), which may reflect inter alia transient colonisation patterns and/or co-operative or competitive interactions between different STs. Comparative analyses with other primers that have been used extensively for ST-specific analysis found them unsuitable for detection of mixed and, in some cases, single ST infections. Collectively, our data shed new light on the diversity of Blastocystis within and between human hosts. Moreover, the development of these PCR assays will facilitate future work into the molecular epidemiology and significance of mixed infections in groups of interest, including health and disease cohorts, and also help identify sources of Blastocystis transmission to humans, including identifying potential animal and environmental reservoirs.