Assessment of next generation amplicon sequencing of the beta-giardin gene for the detection of Giardia duodenalis assemblages and mixed infections

Authors: Maloney, Jenny; Molokin, Aleksey; Santin-Duran, Monica

Submitted to: Food and Waterborne Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/12/2020
Publication Date: 11/17/2020
Citation: Maloney, J.G., Molokin, A., Santin, M. 2020. Assessment of next generation amplicon sequencing of the beta-giardin gene for the detection of Giardia duodenalis assemblages and mixed infections. Food and Waterborne Parasitology. 21:e00098. https://doi.org/10.1016/j.fawpar.2020.e00098.
DOI: https://doi.org/10.1016/j.fawpar.2020.e00098

Interpretive Summary: Giardia duodenalis is one of the most common intestinal parasites of humans and is an important cause of intestinal illness including diarrhea and abdominal pain. The parasite is divided into genetic groups called assemblages with different assemblages infecting different animal and human hosts. Mixtures of assemblages have been documented in both human and animal hosts, but the importance of these mixtures in causing disease remains unclear in part because better technology is needed to quickly and easily detect mixed infections. To address this problem, we have developed a new tool for mixed assemblage detection using next generation amplicon sequencing (NGS) of the beta-giardin gene. We demonstrate NGS works just as well as traditional Sanger sequencing for detecting Giardia species and assemblages. Furthermore, we show that NGS does a superior job of detecting mixed assemblage infections, low abundance assemblages, and intra-assemblage variation which would have been missed using traditional Sanger sequencing. NGS represents a powerful new tool which can be used to fill important knowledge gaps in our understanding of Giardia epidemiology. This information should be useful to other scientists and public health agencies working on detection of Giardia.

Technical Abstract: Giardia duodenalis is an enteric protozoan parasite commonly found in humans and many other animals around the world. The parasite is grouped into genetically related strains called assemblages which display differing degrees of host specificity. Although mixed assemblage infections have been documented, the full extent of the occurrence and importance of mixed infections remains to be characterized as current sequencing technologies lack the sensitivity to readily detect mixed infections. Here we have developed a next generation amplicon sequencing (NGS) protocol and analysis pipeline for detecting Giardia assemblages using the beta-giardin gene. NGS was validated using 37 isolates that included Giardia muris and six assemblages (A-F) of Giardia duodenalis obtained from seven different hosts. NGS was compared to traditional PCR and direct Sanger sequencing for its ability to detect Giardia species, assemblages, and mixed assemblage infections. We demonstrate that NGS works as well as PCR and Sanger sequencing for assemblage detection as the same assemblage was observed in all samples by both methods . NGS has the further benefit of detecting mixed assemblage infections, low abundance assemblages, and intra-assemblage variation in samples which would have been missed using direct Sanger sequencing alone. NGS represents a powerful new tool for exploring Giardia infections not only in infected hosts but also in environmental specimens which may aide in understanding Giardia epidemiology.