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Discrimination of Viable Acanthamoeba castellani Trophozoites and Cysts by Propidium Monoazide Real-Time Polymerase Chain Reaction

Authors

  • MARIANA FITTIPALDI,

    1. 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
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  • NANCY J. PINO RODRIGUEZ,

    1. 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
    2. Grupo de Diagnóstico y Control de la Contaminación (GDCON), Sede de Investigación Universitaria, Universidad de Antioquia, Calle 62 # 52-59, Medellín, Colombia
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  • BÁRBARA ADRADOS,

    1. 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
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  • GEMMA AGUSTÍ,

    1. 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
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  • GUSTAVO PEÑUELA,

    1. Grupo de Diagnóstico y Control de la Contaminación (GDCON), Sede de Investigación Universitaria, Universidad de Antioquia, Calle 62 # 52-59, Medellín, Colombia
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  • JORDI MORATÓ,

    1. 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
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  • FRANCESC CODONY

    1. 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
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Corresponding Author: F.Codony, Laboratori de Microbiologia Sanitària i Mediambiental (MSM-Lab)—Aquasost, UNESCO Chair in Sustainability, Universitat Politècnica de Catalunya, C/Sant Nebridi S/N, Edifici GAIA, TR14, Terrassa, 08222 Barcelona, Spain—Telephone number: +34 93 7398 566; FAX number: +34 93 7398 370; e-mail: codony@oo.upc.edu

Abstract

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.

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