Establishing the Performance of Next-Generation Amplicon Sequencing for Detection of Giardia duodenalis in Ready-to-Eat Packaged Leafy Greens

• Published in: Journal of food protection Vol. 87; no. 10; p. 100355
• Main Authors: Nichols, Holly, Santin, Monica, Maloney, Jenny G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-10-2024; Elsevier
• Subjects: Beta-giardin gene; Foodborne; Giardia duodenalis; NGS; Parasite; Vegetables; Parasite; Beta-giardin gene; Vegetables; Giardia duodenalis; NGS; Foodborne
• ISSN: 0362-028X; 1944-9097; 1944-9097
• DOI: 10.1016/j.jfp.2024.100355

•Utility of NGS to detect G. duodenalis in packaged leafy greens was established.•As few as 20 cysts could be detected in a 25 g sample of leafy greens.•50% of samples seeded with 20 cysts were positive using NGS.•100% of samples seeded with 100, 200, and 1,000 cysts were positive using NGS.•Combining BAM Chapter 19b and NGS can assist with Giardia identification in produce.
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 the 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 (n = 5), 20 (n = 10), 100 (n = 10), 200 (n = 10), or 1,000 (n = 10) G. duodenalis cysts of the assemblage B strain H3. Detection of G. duodenalis was successful in 100% of the samples seeded with 1,000, 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.