Eva Nohýnková

We developed a simple 24-hour viability assay to evaluate the tolerance of the parasite Giardia intestinalis to metronidazole (MTZ) in vitro . Our findings emphasize the importance of test conditions and evaluation parameters. Using this assay with minimum lethal concentration (MLC) as an evaluation parameter, we identified two isolates that are naturally tolerant to MTZ. Natural MTZ tolerance was detected only under microaerophilic conditions, as both isolates were susceptible to MTZ under anaerobic conditions. These isolates were derived from stool samples of patients with MTZ-refractory giardiasis, suggesting that the parasite tolerance was responsible for the failure of MTZ treatment. However, two other isolates from patients with MTZ-refractory giardiasis were susceptible under both test conditions, indicating that MTZ-refractory giardiasis likely has a multifactorial cause. HIGHLIGHTS: In vitro test conditions are significant to assess metronidazole tolerance in Giardia In vitro test should be done under both anaerobic and microaerophilic conditions MLC, but not IC ₅₀ , is a valuable parameter for evaluating MTZ tolerance Failure to treat giardiasis with metronidazole probably has a multifactorial cause

Advanced imaging of microorganisms, including protists, is challenging due to their small size. Specimen expansion prior to imaging is thus beneficial to increase resolution and cellular details. Here, we present a sample preparation workflow for improved observations of the single-celled eukaryotic pathogen Giardia intestinalis (Excavata, Metamonada). The binucleated trophozoites colonize the small intestine of humans and animals and cause a diarrhoeal disease. Their remarkable morphology includes two nuclei and a pronounced microtubular cytoskeleton enabling cell motility, attachment and proliferation. By use of expansion and confocal microscopy, we resolved in a great detail subcellular structures and organelles of the parasite cell. The acquired spatial resolution of 43 nm enabled novel observations of centrin localisation at Giardia basal bodies. Interestingly, non-luminal centrin localization between the Giardia basal bodies was observed, which is an atypical eukaryotic arrangement. Our protocol includes antibody staining and can be used for the localisation of epitope-tagged proteins, as well as for differential organelle labelling by amino reactive esters. This fast and simple protocol is suitable for routine use without a superresolution microscopy equipment.

Background: The flagellated parasite Giardia duodenalis is a major and global cause of diarrhoeal disease. Eight genetically very distinct groups, known as assemblages A to H, have been recognized in the G. duodenalis species complex, two of which (assemblages A and B) infect humans and other mammalian hosts. Informative typing schemes are essential to understand transmission pathways, characterize outbreaks and trace zoonotic transmission. In this study, we evaluated a published multi-locus sequence typing (MLST) scheme for G. duodenalis assemblage A, which is based on six polymorphic markers. Methods: We genotyped 60 human-derived and 11 animal-derived G. duodenalis isolates collected in Europe and on other continents based on the published protocol. After retrieving previously published genotyping data and excluding isolates whose sequences showed allelic sequence heterozygosity, we analysed a dataset comprising 146 isolates. Results: We identified novel variants at five of the six markers and identified 78 distinct MLST types in the overall dataset. Phylogenetic interpretation of typing data confirmed that sub-assemblage AII only comprises human-derived isolates, whereas sub-assemblage AI comprises all animal-derived isolates and a few human-derived isolates, suggesting limited zoonotic transmission. Within sub-assemblage AII, isolates from two outbreaks, which occurred in Sweden and Italy, respectively, had unique and distinct MLST types. Population genetic analysis showed a lack of clustering by geographical origin of the isolates. Conclusion: The MLST scheme evaluated provides sufficient discriminatory power for epidemiological studies of G. duodenalis assemblage A. Graphical

Water suspensions of cysts of a pathogenic clinical isolate of Acanthamoeba sp. were prepared, and the cysts were inactivated either in suspension or placed on the surface of contact lenses by the non-thermal plasma produced by the DC corona transient spark discharge. The efficacy of this treatment was determined by cultivation and the presence of vegetative trophozoites indicating non-inactivated cysts. The negative discharge appeared to be more effective than the positive one. The complete inactivation occurred in water suspension after 40 min and on contaminated lenses after 50 min of plasma exposure. The properties of lenses seem to not be affected by plasma exposure; that is, their optical power, diameter, curvature, water content and infrared and Raman spectra remain unchanged.