Establishing the performance of next generation amplicon sequencing for detection of Giardia duodenalis in ready-to-eat packaged leafy greens

Authors: NICHOLS, HOLLY - Orise Fellow; Santin-Duran, Monica; Maloney, Jenny

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/27/2024
Publication Date: 8/30/2024
Citation: Nichols, H., Santin, M., Maloney, J.G. 2024. Establishing the performance of next generation amplicon sequencing for detection of Giardia duodenalis in ready-to-eat packaged leafy greens. Journal of Food Protection. 87(10): P100355. https://doi.org/10.1016/j.jfp.2024.100355.
DOI: https://doi.org/10.1016/j.jfp.2024.100355

Interpretive Summary: Giardia duodenalis is an intestinal parasite and an important human pathogen. It is spread through food and water, and fooborne transmission is currently considered an underreported transmission route in part due methodological limitations of detection methods for food. In the present study a next generation amplicon sequencing method to improve our ability to detect and differentiate Giardia in fresh leafy greens was validated. This method can now be used by researchers, regulatory agencies, or producers for surveillance of leafy greens, identification of genetic variants of Giardia that most frequently contaminate food sources, and investigations of foodborne illness outbreaks.

Technical Abstract: Giardia duodenalis is a globally distributed intestinal parasite that commonly infects both humans and animals. G. duodenalis is a species complex, which includes eight assemblages that vary both in genetic structure and host specificity. The prevalence of mixed-assemblage G. duodenalis cysts on food, an understudied infection route for G. duodenalis, remains unknown. In the present study, a method able to detect G. duodenalis mixed-assemblage infections using next generation amplicon sequencing (NGS) of the beta-giardin gene was applied in combination with the US-FDA’s BAM Chapter 19b protocol for detection of G. duodenalis from fresh produce to ascertain the limit of detection of G. duodenalis on leafy greens. Ready-to-eat baby romaine lettuce was inoculated with 5, 20, 100, 200, or 1000 G. duodenalis cysts. Detection of G. duodenalis was successful in 100% of the samples seeded with 1000, 200, and 100 cysts, in 50% of the samples seeded with 20 cysts, and in none of the samples seeded with 5 cysts. We thus demonstrate robust detection of G. duodenalis on packaged leafy greens using the BAM Chapter 19B method coupled with assemblage-sensitive NGS. This protocol provides a new diagnostic tool useful for both prevalence studies and outbreak investigations involving fresh produce that may assist in better describing the role of G. duodenalis in foodborne illness and in protecting consumers from contaminated fresh produce.