Beatriz Rosa Marques Zeferino¹, Letícia Bassani Bonato¹, Marcel Ivan Ramirez¹

Introduction
Giardia intestinalis is a flagellated intestinal protozoan responsible for giardiasis, a globally
prevalent diarrhoeal disease. Chronic infections, particularly in children and
immunocompromised individuals, often show reduced responsiveness to albendazole,
suggesting parasite adaptation. Among emerging virulence mechanisms, the release of
extracellular vesicles (EVs) has gained attention due to their role in host modulation and
persistence. This study aimed to identify Giardia clones with altered EV secretion, focusing
on a hypersecretory variant derived from a patient with chronic, drug-refractory giardiasis.

Materials and Methods
Clones were generated by limiting dilution from a WB strain passaged in a chronically
infected patient. After 72 h of culture, large EVs were isolated by differential centrifugation
and analysed by nanoparticle tracking analysis (NTA). Clone 05 (LR051), showing markedly
elevated EV output, was selected for characterisation. EV release was tested under basal
conditions and after stimulation with CaCl2 (2 mM) and albendazole (0.3 μM). EV biogenesis pathways were probed using inhibitors: bafilomycin A1, EGTA, Thapsigargin , and methyl-β-
cyclodextrin. Viability was verified by trypan blue exclusion.

Results
Clone 05 secreted 4–6 times more large EVs than the wild-type WB strain. This
hypersecretion was further enhanced by Ca2+ and albendazole exposure and was significantly
reduced (by 50–60 %) by all tested inhibitors, indicating involvement of defined vesicle
biogenesis mechanisms. The wild-type strain displayed lower secretion and reduced
inducibility.

Conclusion and Perspectives
Clone 05 represents a stable, inducible hypersecretory phenotype selected under host and drug
pressure. Future studies will compare this clone to wild-type G. intestinalis in a 3D Caco-2
intestinal model to explore its effects on TEER, claudin expression, and paracellular
permeability, providing insight into EV-driven mechanisms underlying chronic giardiasis.

Agradecimentos: Acknowledgments We thank the Graduate Program in Biosciences and Biotechnology at the Carlos Chagas Institute – Fiocruz Paraná for institutional support. We also acknowledge the EVAHPI research group for their technical and scientific guidance throughout the development of this work. Finally, we thank the Oswaldo Cruz Foundation for funding through the research scholarship grant.