Next generation amplicon sequencing improves detection of Blastocystis mixed subtype infections

Blastocystis is a highly prevalent enteric protist parasite of humans and animals. Transmission occurs via the fecal-oral route through ingestion of contaminated food or water. Genetic diversity studies have identified numerous subtypes (STs) within the genus Blastocystis based on polymorphism at the SSU rRNA gene. Although there is evidence of frequent mixed subtype infections, the extent of within-host subtype diversity remains largely unexplored. Accurate assessment of Blastocystis ST diversity is crucial to understand epidemiology and sources of Blastocystis transmission to humans. Here, we report the application of next generation sequencing (NGS) for detection and characterization of Blastocystis subtypes to investigate intra-host Blastocystis diversity. A total of 75 specimens obtained from cattle feces, previously identified as Blastocystis positive, were examined using next generation amplicon sequencing. A fragment of the SSU rRNA gene was amplified using Blastocystis-specific primers and resulting amplicons were used for NGS. Comparison of Sanger and NGS results suggest greater sensitivity using the NGS approach. Using Sanger sequencing, mixed infections were suspected in 18 specimens but only confirmed through cloning in three, while NGS identified 49 mixed infections (16 times more). In addition, NGS revealed greater diversity of subtypes with 14 detected compared to 11 by Sanger. Nine more infections with potentially zoonotic STs were detected by NGS than Sanger. Indeed, subtype 3, the most common subtype found in humans, was found in 37% (28) of specimens tested by NGS but in only four specimens using Sanger. Our findings indicate that mixed Blastocystis infections may be far more common than previously thought due to the limitations of current detection methods. This next generation amplicon sequencing strategy improves detection of mixed subtype infections and low abundance subtypes and represents a valuable resource for future Blastocystis studies to improve our understanding of its epidemiology.