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Evaluation of Giardia duodenalis viability after metronidazole treatment by flow cytometry.

Barbosa J, Rodrigues AG, Pérez MJ, Pina-Vaz C - Mem. Inst. Oswaldo Cruz (2014)

Bottom Line: The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells.An excellent correlation was found between FC and the classical method.A novel, accurate and reliable method is now available to evaluate G. duodenalis viability.

View Article: PubMed Central - PubMed

Affiliation: Serviço de Microbiologia, Centro de Investigação em Tecnologias e Sistemas de Informação em Saúde, Faculdade de Medicina, Universidade do Porto, Porto, Portugal.

ABSTRACT
Giardia duodenalis (syn. lamblia; syn. intestinalis) susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC) assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ). Different concentrations of MTZ were added to cultures of trophozoites (10 5 /mL) and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability.

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Evaluation of of Giardia duodenalis trophozoite suspensionsafter treatment with metronidazole (MTZ). A: number of recovered trophozoitesevaluated by in vitro classic method; B: percentage of unstained trophozoites withpropidium iodide (PI-) evaluated on a FACSCalibur flow cytometer.
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f01: Evaluation of of Giardia duodenalis trophozoite suspensionsafter treatment with metronidazole (MTZ). A: number of recovered trophozoitesevaluated by in vitro classic method; B: percentage of unstained trophozoites withpropidium iodide (PI-) evaluated on a FACSCalibur flow cytometer.

Mentions: Regarding the susceptibility studies of G. duodenalis trophozoites to MTZ,a statistically significant correlation (p < 0.05) between the in vitro culture classicmethod and FC was observed at most of the incubation times (Table). Increasing drug concentrations and incubation times resulted indecreased numbers of adherent trophozoites and increased PI+ cells (Figure). At the longest incubation time, only 10% of the cysts wereable to excyst, as previously described (Sousa &Poiares-da-Silva 1999, Cruz et al. 2003,Sandhu et al. 2004). After 18 h, at MTZconcentrations > 5.0 µg/mL, more than 90% of cells were dead and, therefore, lost thecapacity to grow; these cells were classified as PI+ (Figure). Additionally, we determined that the IC50 value was 50.0µg/mL for a 9 h incubation and 5.0 µg/mL for an 18 h incubation. Through the obtainedresults, we showed an excellent correlation between the number of trophozoites counted insuspension and the percentage of PI- cells (Table).


Evaluation of Giardia duodenalis viability after metronidazole treatment by flow cytometry.

Barbosa J, Rodrigues AG, Pérez MJ, Pina-Vaz C - Mem. Inst. Oswaldo Cruz (2014)

Evaluation of of Giardia duodenalis trophozoite suspensionsafter treatment with metronidazole (MTZ). A: number of recovered trophozoitesevaluated by in vitro classic method; B: percentage of unstained trophozoites withpropidium iodide (PI-) evaluated on a FACSCalibur flow cytometer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4325610&req=5

f01: Evaluation of of Giardia duodenalis trophozoite suspensionsafter treatment with metronidazole (MTZ). A: number of recovered trophozoitesevaluated by in vitro classic method; B: percentage of unstained trophozoites withpropidium iodide (PI-) evaluated on a FACSCalibur flow cytometer.
Mentions: Regarding the susceptibility studies of G. duodenalis trophozoites to MTZ,a statistically significant correlation (p < 0.05) between the in vitro culture classicmethod and FC was observed at most of the incubation times (Table). Increasing drug concentrations and incubation times resulted indecreased numbers of adherent trophozoites and increased PI+ cells (Figure). At the longest incubation time, only 10% of the cysts wereable to excyst, as previously described (Sousa &Poiares-da-Silva 1999, Cruz et al. 2003,Sandhu et al. 2004). After 18 h, at MTZconcentrations > 5.0 µg/mL, more than 90% of cells were dead and, therefore, lost thecapacity to grow; these cells were classified as PI+ (Figure). Additionally, we determined that the IC50 value was 50.0µg/mL for a 9 h incubation and 5.0 µg/mL for an 18 h incubation. Through the obtainedresults, we showed an excellent correlation between the number of trophozoites counted insuspension and the percentage of PI- cells (Table).

Bottom Line: The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells.An excellent correlation was found between FC and the classical method.A novel, accurate and reliable method is now available to evaluate G. duodenalis viability.

View Article: PubMed Central - PubMed

Affiliation: Serviço de Microbiologia, Centro de Investigação em Tecnologias e Sistemas de Informação em Saúde, Faculdade de Medicina, Universidade do Porto, Porto, Portugal.

ABSTRACT
Giardia duodenalis (syn. lamblia; syn. intestinalis) susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC) assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ). Different concentrations of MTZ were added to cultures of trophozoites (10 5 /mL) and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability.

Show MeSH
Related in: MedlinePlus