title: Discrimination of viable Acanthamoeba castellani trophozoites and cysts by propidium monoazide real-time polymerase chain reaction.
abstract: Even though the advent of quantitative polymerase chain reaction (PCR) has improved the detection of pathogen microorganisms in most of areas of microbiology, a serious limitation of this method may arise from the inability to discriminate between viable and nonviable pathogens. To overcome it, the use of real-time PCR and selective nucleic acid intercalating dyes like propidium monoazide (PMA) have been effectively evaluated for different microorganisms. To assess whether PMA pretreatment can inhibit PCR amplification of nonviable amoeba DNA, Acanthamoeba castellani survival was measured using cell culture and real-time PCR with and without PMA pretreatment. Autoclave and contact lens disinfecting solutions were used to inactivate amoebae. After these inactivation treatments, the results indicated that the PMA pretreatment approach is appropriate for differentiating viable A. castellani, both trophozoites and cysts. Therefore, the PMA-PCR approach could be useful as a rapid and sensitive analytical tool for monitoring treatment and disease control, assessing effective disinfection treatments, and for a more reliable understanding of the factors that contribute to the interaction amoeba-pathogenic bacteria.
authors.affiliation: Laboratori de Microbiologia Sanitària i Mediambiental (MSM-Lab)-Aquasost, UNESCO Chair in Sustainability, Universitat Politècnica de Catalunya, C/Sant Nebridi S/N, Terrassa, 08222, Barcelona, Spain.
authors.forename: Mariana
authors.initials: M
authors.lastname: Fittipaldi
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Pino Rodriguez
authors.forename: Bárbara
authors.initials: B
authors.lastname: Adrados
authors.forename: Gemma
authors.initials: G
authors.lastname: Agustí
authors.forename: Gustavo
authors.initials: G
authors.lastname: Peñuela
authors.forename: Jordi
authors.initials: J
authors.lastname: Morató
authors.forename: Francesc
authors.initials: F
authors.lastname: Codony
mesh_terms: D048248:Acanthamoeba castellanii / Q000166:cytology* / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D001386:Azides; D018639:Contact Lens Solutions; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives*; D013194:Staining and Labeling; D053058:Trophozoites / Q000166:cytology* / Q000502:physiology
chemical_list: D001386:Azides; D018639:Contact Lens Solutions; D016054:DNA, Protozoan; C533957:propidium monoazide; D011419:Propidium