Validación de un método de aislamiento para Legionella pneumophila por medio de técnicas de concentración

El aislamiento y detección de Legionella pneumophila en agua potable puede ser un proceso difícil, debido a que algunas de las técnicas existentes no concentran ni recuperan la cantidad estandarizada por entes internacionales, así mismo, es un microorganismo exigente, y lo tanto, requiere de medios...

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Autores:
Cufiño Castiblanco, Cristian Sebastián
Lara Sanabria, María Alejandra
Tipo de recurso:
Trabajo de grado de pregrado
Fecha de publicación:
2022
Institución:
Colegio Mayor de Cundinamarca
Repositorio:
Repositorio Colegio Mayor de Cundinamarca
Idioma:
spa
OAI Identifier:
oai:repositorio.unicolmayor.edu.co:unicolmayor/5684
Acceso en línea:
https://repositorio.unicolmayor.edu.co/handle/unicolmayor/5684
Palabra clave:
Dilución
Filtración por membrana
Validación
Límite de detección
Legionella pneumophila,
Medio BCYE
Celltrazone
Rights
closedAccess
License
Derechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2022
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oai_identifier_str oai:repositorio.unicolmayor.edu.co:unicolmayor/5684
network_acronym_str UCOLMAYOR2
network_name_str Repositorio Colegio Mayor de Cundinamarca
repository_id_str
dc.title.spa.fl_str_mv Validación de un método de aislamiento para Legionella pneumophila por medio de técnicas de concentración
title Validación de un método de aislamiento para Legionella pneumophila por medio de técnicas de concentración
spellingShingle Validación de un método de aislamiento para Legionella pneumophila por medio de técnicas de concentración
Dilución
Filtración por membrana
Validación
Límite de detección
Legionella pneumophila,
Medio BCYE
Celltrazone
title_short Validación de un método de aislamiento para Legionella pneumophila por medio de técnicas de concentración
title_full Validación de un método de aislamiento para Legionella pneumophila por medio de técnicas de concentración
title_fullStr Validación de un método de aislamiento para Legionella pneumophila por medio de técnicas de concentración
title_full_unstemmed Validación de un método de aislamiento para Legionella pneumophila por medio de técnicas de concentración
title_sort Validación de un método de aislamiento para Legionella pneumophila por medio de técnicas de concentración
dc.creator.fl_str_mv Cufiño Castiblanco, Cristian Sebastián
Lara Sanabria, María Alejandra
dc.contributor.advisor.none.fl_str_mv Gómez Garzón, Marcela
Rodríguez Aristizába, Mónica Alejandra
dc.contributor.author.none.fl_str_mv Cufiño Castiblanco, Cristian Sebastián
Lara Sanabria, María Alejandra
dc.subject.lemb.none.fl_str_mv Dilución
Filtración por membrana
Validación
Límite de detección
topic Dilución
Filtración por membrana
Validación
Límite de detección
Legionella pneumophila,
Medio BCYE
Celltrazone
dc.subject.proposal.spa.fl_str_mv Legionella pneumophila,
Medio BCYE
Celltrazone
description El aislamiento y detección de Legionella pneumophila en agua potable puede ser un proceso difícil, debido a que algunas de las técnicas existentes no concentran ni recuperan la cantidad estandarizada por entes internacionales, así mismo, es un microorganismo exigente, y lo tanto, requiere de medios especiales para su crecimiento. El objetivo del estudio fue validar un método de concentración por medio de la implementación del equipo Celltrazone para mejorar la recuperación de la bacteria en agua potable. Para lograr una mejor concentración de L. pneumophila y que pudiera reflejarse en el medio de cultivo, se ensayaron varias técnicas de tratamiento para el filtro obtenido por el equipo como el vortex, sonicado y agitación, sin embargo, el no aplicar ningún procedimiento y realizar una siembra directa desde la membrana obtuvo los mejores resultados. El análisis estadístico demostró que el método aplicado puede concentrar hasta 1x102 (100 UFC), se alcanzó una sensibilidad del 100% y una especificidad del 80% y un valor aceptable de exactitud (87,5%). El método de filtración de membrana utilizando el equipo Celltrazone permitió aumentar la concentración del L. pneumophila en agua potable, cumpliendo con los intervalos normalizados por ISO y la OMS, por lo tanto, el método es apto para su aplicación permitiendo obtener resultados confiables
publishDate 2022
dc.date.accessioned.none.fl_str_mv 2022-10-05T21:31:08Z
dc.date.available.none.fl_str_mv 2022-10-05T21:31:08Z
dc.date.issued.none.fl_str_mv 2022
dc.type.spa.fl_str_mv Trabajo de grado - Pregrado
dc.type.coar.spa.fl_str_mv http://purl.org/coar/resource_type/c_7a1f
dc.type.coarversion.spa.fl_str_mv http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.content.spa.fl_str_mv Text
dc.type.driver.spa.fl_str_mv info:eu-repo/semantics/bachelorThesis
dc.type.redcol.spa.fl_str_mv https://purl.org/redcol/resource_type/TP
dc.type.version.spa.fl_str_mv info:eu-repo/semantics/publishedVersion
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status_str publishedVersion
dc.identifier.uri.none.fl_str_mv https://repositorio.unicolmayor.edu.co/handle/unicolmayor/5684
url https://repositorio.unicolmayor.edu.co/handle/unicolmayor/5684
dc.language.iso.spa.fl_str_mv spa
language spa
dc.relation.references.spa.fl_str_mv Lucas RMO. Vigilancia epidemiológica, prevención y control de la legionella en el hospital. In: Revista de Salud Ambiental [Internet]. 2015 [cited 2020 Sep 25]. Available from: https://ojs.diffundit.com/index.php/rsa/article/view/738/681
Icontec. Guía Técnica Colombiana GTC 257 de 2015. Bogotá; 2015
Burillo A, Pedro-Botet ML, Bouza E. Microbiology and Epidemiology of Legionnaire’s Disease. Infectious Disease Clinics of North America [Internet]. 2017 Mar 1 [cited 2020 Sep 25];31(1):7–27. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0891552016300885?via%3Dihub
Abdel-Nour M, Duncan C, Low DE, Guyard C. Biofilms: The Stronghold of Legionella pneumophila. International Journal of Molecular Sciences [Internet]. 2013 Oct 31 [cited 2020 Sep 25];14(11):21660. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3856027/
Fields BS, Benson RF, Besser RE. Legionella and Legionnaires’ Disease: 25 Years of Investigation. Clinical Microbiology Reviews [Internet]. 2002 [cited 2020 Sep 27];15(3):506. Available from: /pmc/articles/PMC118082/
Cunha BA, Burillo A, Bouza E. Legionnaires’ disease. The Lancet [Internet]. 2016 Jan 23 [cited 2020 Sep 27];387(10016):376–85. Available from: http://www.thelancet.com/article/S0140673615600782/fulltext
Garcia-Nuñez M, Sopena N, Ragull S, Pedro-Botet ML, Morera J, Sabria M. Persistence of Legionella in hospital water supplies and nosocomial Legionnaires’ disease. FEMS immunology and medical microbiology [Internet]. 2008 Mar [cited 2020 Sep 27];52(2):202–6. Available from: https://pubmed.ncbi.nlm.nih.gov/18093139/
. Patiño-Barbosa AM, Gil-Restrepo AF, Restrepo-Montoya V, Villamil-Gomez WE, CardonaOspina JA, Rodriguez-Morales AJ. Is Legionellosis Present and Important in Colombia? An Analyses of Cases from 2009 to 2013. Cureus [Internet]. 2017 Mar 28 [cited 2022 Sep 28];9(3). Available from: /pmc/articles/PMC5409816/
Fraser DW, Tsai TR, Orenstein W, Parkin WE, Beecham HJ, Sharrar RG, et al. Legionnaires’ Disease. http://dx.doi.org/101056/NEJM197712012972201 [Internet]. 2010 Jan 13 [cited 2020 Sep 30];297(22):1189–97. Available from: https://www.nejm.org/doi/10.1056/NEJM197712012972201
Clayton AJ. Legionnaires' disease: an illness whose time has come. Can Med Assoc J [Internet]. 1979 [cited 2020 sep 30];120(12):1482-1483. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1704222/
. Steele TW. Legionnaires' disease in South Australia, 1979-1988. Med J Aust [Internet]. 1989 [cited 2020 sep 30];151(6):322-328. Available from: https://pubmed.ncbi.nlm.nih.gov/2687665/
Morris GK, Patton CM, Feeley JC, Johnson SE, Gorman G, Martin WT, et al. Isolation of the Legionnaires’ disease bacterium from environmental samples. Annals of Internal Medicine [Internet]. 1979 [cited 2020 Oct 7];90(4):664–6. Available from: https://www.acpjournals.org/doi/10.7326/0003-4819-90-4-664?url_ver=Z39.88- 2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed
Fliermans, CB, Cherry WB, Orrison LH, Thacker AL. Isolation of Legionella pneumophila from Nonepidemic-Related Aquatic Habitats. Applied and environmental microbiology [Internet]. 1979 [cited 2022 Feb 9];37(6):1239–42. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC243385/pdf/aem00210-0201.pdf
Shands KM, Ho JL, Meyer RD, Gorman GW, Edelstein PH, Mallison GF, et al. Potable Water as a Source of Legionnaires’ Disease. JAMA [Internet]. 1985 Mar 8 [cited 2020 Oct 8];253(10):1412–6. Available from: https://jamanetwork.com/journals/jama/fullarticle/397245
Glavin FL, Winn WC, Craighead JE. Ultrastructure of lung in Legionnaires’ disease. Observations of three biopsies done during the Vermont epidemic. Annals of internal medicine [Internet]. 1979 [cited 2020 Oct 2];90(4):555–9. Available from: https://pubmed.ncbi.nlm.nih.gov/434634/
Tison DL, Pope DH, Cherry WB, Fliermans CB. Growth of Legionella pneumophila in Association with Blue-Green Algae (Cyanobacteria). Applied and environmental microbiology [Internet]. 1980 [cited 2020 Oct 7];39(2):456–9. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC291353/pdf/aem00232-0184.pdf
Rowbotham TJ. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. Journal of Clinical Pathology [Internet]. 1980 [cited 2020 Oct 7];33(12):1179. Available from: /pmc/articles/PMC1146371/?report=abstract
Feeley JC, Gorman GW, Weaver RE, Mackel DC, Smith HW. Primary isolation media for Legionnaires disease bacterium. Journal of Clinical Microbiology [Internet]. 1978 [cited 2020 Sep 30];8(3):320. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC275239/
Tilton RC. Legionnaires’ disease antigen detected by enzyme-linked immunosorbent assay. Annals of Internal Medicine [Internet]. 1979 [cited 2020 Oct 9];90(4):697–8. Available from: https://www.acpjournals.org/doi/10.7326/0003-4819-90-4-697?url_ver=Z39.88- 2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed
Berdal BP, Farshy CE, Feeley JC. Detection of Legionella pneumophila Antigen in Urine by Enzyme-Linked Immunospecific Assay. Journal of clinical microbiology. 1979;575–8.
.Kohler RB, Zimmerman SE, Wilson E, Allen SD, Edelstein PH, Wheat LJ, White A. Rapid radioimmunoassay diagnosis of Legionnaires' disease: detection and partial characterization of urinary antigen. Ann Intern Med. 1981 May;94(5):601-605. Available from: https://pubmed.ncbi.nlm.nih.gov/7235392/
Kohler RB, Winn WC, Wheat1 LJ. Onset and Duration of Urinary Antigen Excretion in Legionnaires Disease. Journal of clinical microbiology [Internet]. 1984 [cited 2020 Oct 11];20(4):605–7. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC271393/pdf/jcm00123-0035.pdf
Horwitz MA. Formation of a novel phagosome by the legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. [cited 2020 Oct 4]; Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2187375/pdf/je15841319.pdf
Doebbeling BN, Bale MJ, Koontz FP, Helms CM, Wenzel RP, Pfaller MA. Prospective evaluation of the Gen-Probe assay for detection of legionella in respiratory specimens. Eur J Clin Microbiol Infect Dis. 1988 Dec;7(6):748-52
Cianciotto NP, Eisenstein BI, Mody CH, Toews GB, Cary Englebergl N. A Legionella pneumophila Gene Encoding a Species-Specific Surface Protein Potentiates Initiation of Intracellular Infection. INFECTION AND IMMUNITY [Internet]. 1989 [cited 2020 Oct 14];57(4):1255–62. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC313258/pdf/iai00064-0257.pdf
Mercante JW, Winchell JM. Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations. Clinical Microbiology Reviews [Internet]. 2015 Jan 1 [cited 2022 Oct 21];28(1):95. Available from: /pmc/articles/PMC4284297/
Cianciotto NP, Fieldst BS. Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages (intracellular parastism/evolution/Hwimannela/Tebwhymena/FK506-bnidng proteins). 1992 [cited 2020Oct 14];89:5188–91. Available from
. Keserue HA, Baumgartner A, Felleisen R, Egli T. Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry. Microbial biotechnology [Internet]. 2012 Nov [cited 2020 Oct 24];5(6):753. Available from: /pmc/articles/PMC3815896/
Schoch CL, Ciufo S, Domrachev M, Hotton CL, Kannan S, Khovanskaya R, et al. NCBI Taxonomy: A comprehensive update on curation, resources and tools. Database [Internet]. 2020 [cited 2020 Nov 8];2020. Available from: https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=446&lvl=3&lin =f&keep=1&srchmode=1&unlock
Manske C, Hilbi H. Metabolism of the vacuolar pathogen Legionella and implications for virulence. Frontiers in Cellular and Infection Microbiology [Internet]. 2014 [cited 2020 Nov 14];4(SEP). Available from: /pmc/articles/PMC4158876/
García-Núñez M. Colonización, citopatogenicidad y persistencia de Legionella spp. en agua sanitaria hospitalaria. Tesis Doctoral [Internet]. 2009 [cited 2020 Dec 9]. Available from: https://ddd.uab.cat/pub/tesis/2009/tdx-0217110-115805/mgn1de1.pdf
Van der Kooij D, Brouwer-Hanzens AJ, Veenendaal HR, Wullings BA. Multiplication of Legionella pneumophila Sequence Types 1, 47, and 62 in Buffered Yeast Extract Broth and Biofilms Exposed to Flowing Tap Water at Temperatures of 38°C to 42°C. Applied and Environmental Microbiology [Internet]. 2016 [cited 2022 Dec 9];82(22):6691. Available from: /pmc/articles/PMC5086563/
Stewart CR, Muthye V, Cianciotto NP. Legionella pneumophila Persists within Biofilms Formed by Klebsiella pneumoniae, Flavobacterium sp., and Pseudomonas fluorescens under Dynamic Flow Conditions. PLoS ONE [Internet]. 2012 Nov 21 [cited 2022 Dec 17];7(11). Available from: /pmc/articles/PMC3503961/
Yu VL, Plouffe JF, Pastoris MC, Stout JE, Schousboe M, Widmer A, et al. Distribution of Legionella species and serogroups isolated by culture in patients with sporadic communityacquired legionellosis: an international collaborative survey. The Journal of infectious diseases [Internet]. 2002 Jul 1 [cited 2022 Dec 17];186(1):127–8. Available from: https://pubmed.ncbi.nlm.nih.gov/12089674/
Mudali G, Kilgore PE, Salim A, McElmurry SP, Zervos M. Trends in Legionnaires’ DiseaseAssociated Hospitalizations, United States, 2006–2010. Open Forum Infectious Diseases [Internet]. 2020 Aug 1 [cited 2022 Dec 17];7(8). Available from: https://academic.oup.com/ofid/article/7/8/ofaa296/5872667
Arias J. Neumonía atípica, importancia de la detección oportuna de legionella pneumophila en Colombia. a propósito de un caso importado detectado en Bogotá en el año 2015 [Internet]. AREANDINA. Fundación Universitaria del Área Andina; 2016 [cited 2020 Dec 18]. Available from: https://digitk.areandina.edu.co/handle/areandina/634
Harrison TG, Taylor AG. A rapid microagglutination test for the diagnosis of Legionella pneumophila (serogroup 1) infection. Journal of Clinical Pathology [Internet]. 1982 [cited 2020 Dec 19];35(9):1028. Available from: /pmc/articles/PMC497857/?report=abstract
Ta AC, Stout JE, Yu VL, Wagener MM. Comparison of Culture Methods for Monitoring Legionella Species in Hospital Potable Water Systems and Recommendations for Standardization of Such Methods. JOURNAL OF CLINICAL MICROBIOLOGY [Internet]. 1995 [cited 2021 Jan 18];33(8):2118–23. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC228346/pdf/332118.pdf
Centers for Disease Control and Prevention. Legionnaires Disease: Laboratory Guidance for Processing Samples | CDC [Internet]. 1992 [cited 2021 Jan 18]. Available from: https://www.cdc.gov/legionella/labs/procedures-manual.htm
De Giglio O, Diella G, Trerotoli P, Consonni M, Palermo R, Tesauro M, et al. Legionella Detection in Water Networks as per ISO 11731:2017: Can Different Filter Pore Sizes and Direct Placement on Culture Media Influence Laboratory Results? International Journal of Environmental Research and Public Health Article [Internet]. 2020 [cited 2021 Jan 18]; Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142469/pdf/ijerph-17- 02077.pdf
Sepúlveda M, Rodríguez M, Arboleda C, Arismendy L, Betancur J. Verificación del método de filtración por membrana para la detección y cuantificación de legionella spp. en agua potable [Internet]. 2016 [cited 2021 Jan 18]. Available from: https://revistas.elpoli.edu.co/index.php/pol/article/view/881/1480
Lösch LS, Merino LA. Presencia de Legionella spp. en depósitos domiciliarios de agua potable en Resistencia, Chaco, Argentina. Informe preliminar. Revista Argentina de Microbiología [Internet]. 2016 Oct 1 [cited 2021 Jan 18];48(4):329–32. Available from: https://www.sciencedirect.com/science/article/pii/S0325754116300554
Dimitriadi D, Velonakis E. Detection of Legionella spp. from Domestic Water in the Prefecture of Arta, Greece . Journal of Pathogens [Internet]. 2014 [cited 2021 Jan 18];2014:1–5. Available from: https://www.hindawi.com/journals/jpath/2014/407385/
Díaz-Flores Á, Montero JC, Castro FJ, Alejandres EM, Bayón C, Solís I, et al. Comparing methods of determining Legionella spp. in complex water matrices. BMC Microbiology [Internet]. 2015 Dec 12 [cited 2021 Jan 19];15(1). Available from: /pmc/articles/PMC4436101/
Párraga-Niño N, Quero S, Ventós-Alfonso A, Uria N, Castillo-Fernandez O, Ezenarro JJ, et al. New system for the detection of Legionella pneumophila in water samples. Talanta [Internet]. 2018 Nov 1 [cited 2021 Jan 19];189:324–31. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0039914018307057?via%3Dihub
Reinthaler FF, Sattler J, Schaffler-Dullnig K, Weinmayr B, Marth E. Comparative study of procedures for isolation and cultivation of Legionella pneumophila from tap water in hospitals. Journal of Clinical Microbiology [Internet]. 1993 [cited 2021 Jan 20];31(5):1213. Available from: /pmc/articles/PMC262906/?report=abstrac
Van Kenhove E, Dinne K, Janssens A, Laverge J. Overview and comparison of Legionella regulations worldwide. American Journal of Infection Control [Internet]. 2019 Aug 1 [cited 2021 Jan 20];47(8):968–78. Available from: http://www.ajicjournal.org/article/S0196655318309957/fulltext
European Society of Clinical Microbiology and Infectious Diseases. ESGLI European Technical Guidelines for the Prevention, Control and Investigation, of Infections Caused by Legionella species. 2017 [cited 2021 Jan 20]; Available from: https://www.escmid.org/fileadmin/src/media/PDFs/3Research_Projects/ESGLI/ESGLI_Europ ean_Technical_Guidelines_for_the_Prevention_Control_and_Investigation_of_Infections_Ca used_by_Legionella_species_June_2017.pdf
AENOR. UNE-EN ISO 11731:2017 Calidad del agua. Recuento de Legionella [Internet]. Madrid; 2017. Available from: https://tienda.aenor.com/norma-une-en-iso-11731-2017- n0059300
Ministerio de Trabajo y Asuntos Sociales. NTP 538: Legionelosis: medidas de prevención y control en instalaciones de suministro de agua [Internet]. Madrid; 1999. Available from:https://www.insst.es/documents/94886/327064/ntp_538.pdf/4f52d14a-744b-46ee-ac49- ea8619dd00e9
Ministerio de Sanidad y Consumo. BOLETÍN OFICIAL DEL ESTADO LEGISLACIÓN CONSOLIDADA Real Decreto 865/2003 [Internet]. Jul 4, 2003. Available from: https://www.boe.es/buscar/pdf/2003/BOE-A-2003-14408-consolidado.pdf
Serrano Suárez A. Relación de Legionella spp. con parametros microbiológicos y fisicoquímicos en aguas [Internet]. TDX (Tesis Doctorals en Xarxa). Universitat de Barcelona; 2009 [cited 2021 Jan 22]. Available from: http://www.tdx.cat/handle/10803/284890
Institute of Environmental Epidemiology Ministry of the Environment. Code for Practice for the Control of Legionella Bacteria in Cooling Towers [Internet]. Singapore; Available from: https://www.nea.gov.sg/docs/default-source/resource/practices-/code-of-practice-for-controlof-legionella-bacteria-in-cooling-towers.pdf
Ministry of Health. The Prevention of Legionellosis in New Zealand: Guidelines for the control of legionella bacteria | Ministry of Health NZ [Internet]. New Zealand.; 2011 [cited 2021 Jan 24]. Available from: https://www.health.govt.nz/publication/prevention-legionellosis-newzealand-guidelines-control-legionella-bacteria
Health and Human Services. A guide to developing risk management plans for cooling tower systems [Internet]. [cited 2021 Jan 27]. Available from: https://silo.tips/download/a-guide-todeveloping-risk-management-plans-for-cooling-tower-systems
Asociación Española de Abastecimientos de Agua y Saneamiento. OPERATING GUIDE: WATER ANALYSIS LABORATORIES PART II CRITERIA TO VALIDATE PHYSICOCHEMICAL AND MICROBIOLOGICAL ANALYSIS METHODS DRAFTING TEAM Experts in microbiological analysis. [cited 2021 Jan 31]; Available from: https://www.aeas.es/images/publicaciones/manuales/AEAS_Operating_Guide_Water_Analy sis_Laboratories_Part_II.pdf
World Health Organization. Who expert committee on specifications for pharmaceutical preparation. [Internet]. 1992 [cited 2021 Feb 1]. Available from: http://apps.who.int/iris/bitstream/handle/10665/39645/WHO_TRS_823.pdf;jsessionid=3AF81 4E252888AB5A77CAB77EBD7672F?sequence=1
Asociación Española de Normalización. UNE-EN ISO 8199:2019 Calidad del agua. Requisitos y orientaciones generales para el recuento de microorganismos mediante cultivo [Internet]. [cited 2021 Feb 1]. Available from: https://www.une.org/encuentra-tu-norma/buscatu-norma/norma?c=N0061957
Celltrazone. HURO PATH S Slide Sample Processor User manual [Internet]. Celltrazone. Geumcheon-Gu, Seoul, Korea; [cited 2021 Feb 3]. Available from: https://dmec.moh.gov.vn/documents/10182/8891204/upload_00086670_1542882291262.pdf ?version=1.0&fileId=8898267
Liofilchem. Legionella CYE Agar Base [Internet]. 2015 [cited 2021 Feb 4]. Available from: http://www.liofilchem.net/login/pd/ts/620125_TS.pdf
Liofilchem. Legionella BCYE Growth Supplement [Internet]. [cited 2021 Feb 4]. Available from: http://www.liofilchem.net/login/pd/pi/81056_PI.pdf
Invima. VALIDACION DE TECNICAS ANALITICAS [Internet]. 2015 Mar [cited 2021 Feb 20]. Available from: https://www.invima.gov.co/documents/20143/1433420/Validaci%C3%B3n+cualitativa+Micro biolog%C3%ADa.pdf
Giménez DF. Staining Rickettsiae in Yolk-Sac Cultures. http://dx.doi.org/103109/10520296409061219 [Internet]. 2009 [cited 2022 Jan 4];39(3):135– 40. Available from: https://www.tandfonline.com/doi/abs/10.3109/10520296409061219
Greer PW, Chandler FW, Hicklin MD. Rapid Demonstration of Legionella pneumophila in Unembedded Tissue: An Adaptation of the Giménez Stain. American Journal of Clinical Pathology [Internet]. 1980 Jun 1 [cited 2022 Jan 4];73(6):788–90. Available from: https://academic.oup.com/ajcp/article/73/6/788/1777877
Murcia L CAMM. Estandarización de la técnica de coloración de Gimenez para la tinción de cepas de Legionella pneumophila. [Bogota DC]; 2021.
Merck. Merck Microbiology Manual 12th Edition [Internet]. Merck . [cited 2022 Jan 23]. Available from: http://www.laboquimia.es/index.php
Rodríguez-Martínez S, Blanky M, Friedler E, Halpern M. Legionella spp. isolation and quantification from greywater. MethodsX [Internet]. 2015 Jan 1 [cited 2022 Mar 1];2:458–62. Available from: https://www.sciencedirect.com/science/article/pii/S221501611500059X
Neogen. Agar BCYE | Medio de aislamiento de Legionella | NEOGEN [Internet]. [cited 2022 Feb 3]. Available from: https://www.neogen.com/es/categories/microbiology/bcye-agarlegionella-isolation-medium/
Bartie C, Venter SN, Nel LH. Evaluation of detection methods for Legionella species using seeded water samples. Water SA [Internet]. 2001 Apr 1 [cited 2022 Feb 3];27(4):523–8. Available from: https://www.ajol.info/index.php/wsa/article/view/4966
Schulze-Röbbecke, R., Hartemann, P., Fimmers, R., & Hagenau, C. Comparison of membrane filtration methods for the recovery of legionellae from naturally contaminated domestic drinking water supplies. [Internet].1999 [cited 2022 Feb 3] Zentralblatt Für Hygiene Und Umweltmedizin, 202(1), 51–59. Available from: https://doi.org/10.1016/S0934- 8859(99)80053-4
Matuszewska, R., & Krogulska, B. [Detection and isolation of bacteria of Legionella species from the water environment]. [Internet] 2000 [cited 2022 Feb 3] Roczniki Panstwowego Available from: Zakladu Higieny, 51(2), 183–190. https://europepmc.org/article/med/10959198
Ezenarro JJ, Párraga-Niño N, Sabrià M, del Campo FJ, Muñoz-Pascual FX, Mas J, et al. Rapid Detection of Legionella pneumophila in Drinking Water, Based on Filter Immunoassay and Chronoamperometric Measurement. Biosensors [Internet]. 2020 Sep 1 [cited 2022 Feb 25];10(9). Available from: /pmc/articles/PMC7558583/
Cercenado E, Cantón R. Procedimientos en Microbiología Clínica Recomendaciones de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. [cited 2022 Feb 7]; Available from: https://www.seimc.org/contenidos/documentoscientificos/procedimientosmicrobiologia/seimc -procedimientomicrobiologia48.pdf
Cercenado E, Cantón R. Procedimientos en Microbiología Clínica Recomendaciones de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. [cited 2022 Feb 7]; Available from: https://www.seimc.org/contenidos/documentoscientificos/procedimientosmicrobiologia/seimc -procedimientomicrobiologia48.pdf
Gruas C, Álvarez I, Lara C, García CB, Savva D, Arruga MV. Identification of Legionella spp. in Environmental Water Samples by ScanVIT-LegionellaTM Method in Spain. Indian Journal of Microbiology [Internet]. 2013 Jun 1 [cited 2022 Feb 7];53(2):142. Available from: /pmc/articles/PMC3626958/
Boulanger, C. A., & Edelstein, P. H. Precision and accuracy of recovery of Legionella pneumophila from seeded tap water by filtration and centrifugation.[Internet] 1995 Applied and Environmental Microbiology, [cited 2022 Feb 7] 61(5), 1805–1809. Available from: /pmc/articles/PMC3626958/ https://doi.org/10.1128/AEM.61.5.1805-1809.1995
Sandle T. Approaching Microbiological Method Validation. 2015 [cited 2022 Feb 7]; Available from: https://www.researchgate.net/publication/287992312_Approaching_Microbiological_Method_ Validation
Bedrina, B., Macián, S., Solís, I., Fernández-Lafuente, R., Baldrich, E., & Rodríguez, G. . Fast immunosensing technique to detect Legionella pneumophila in different natural and anthropogenic environments: comparative and collaborative trials. (2013) [cited 2022 Feb 7]BMC Microbiology, 13(1), 88. Available from: https://doi.org/10.1186/1471-2180-13-88
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spelling Gómez Garzón, Marcelad7301d7cefc1f029227a4dea7bdf6b5eRodríguez Aristizába, Mónica Alejandra92d982014baa025cff1a931c7f0dde2e600Cufiño Castiblanco, Cristian Sebastiánedb4273a23a8907c1d0251e29369ac17Lara Sanabria, María Alejandra53b900cec4c2ad9fa68dd6829550a6a32022-10-05T21:31:08Z2022-10-05T21:31:08Z2022https://repositorio.unicolmayor.edu.co/handle/unicolmayor/5684El aislamiento y detección de Legionella pneumophila en agua potable puede ser un proceso difícil, debido a que algunas de las técnicas existentes no concentran ni recuperan la cantidad estandarizada por entes internacionales, así mismo, es un microorganismo exigente, y lo tanto, requiere de medios especiales para su crecimiento. El objetivo del estudio fue validar un método de concentración por medio de la implementación del equipo Celltrazone para mejorar la recuperación de la bacteria en agua potable. Para lograr una mejor concentración de L. pneumophila y que pudiera reflejarse en el medio de cultivo, se ensayaron varias técnicas de tratamiento para el filtro obtenido por el equipo como el vortex, sonicado y agitación, sin embargo, el no aplicar ningún procedimiento y realizar una siembra directa desde la membrana obtuvo los mejores resultados. El análisis estadístico demostró que el método aplicado puede concentrar hasta 1x102 (100 UFC), se alcanzó una sensibilidad del 100% y una especificidad del 80% y un valor aceptable de exactitud (87,5%). El método de filtración de membrana utilizando el equipo Celltrazone permitió aumentar la concentración del L. pneumophila en agua potable, cumpliendo con los intervalos normalizados por ISO y la OMS, por lo tanto, el método es apto para su aplicación permitiendo obtener resultados confiablesThe isolation and detection of Legionella pneumophila in drinking water can be a difficult process, because some of the existing techniques do not concentrate or recover the amount standardized by international entities, likewise, it is a demanding microorganism, and therefore, requires special media for its growth. The objective of the study was to validate a concentration method through the implementation of Celltrazone equipment to improve the recovery of the bacterium in drinking water. To achieve a better concentration of L. pneumophila that could be reflected in the culture medium, several treatment techniques were tested for the filter obtained by the equipment, such as vortexing, sonication and agitation; however, not applying any procedure and performing a direct seeding from the membrane obtained the best results. Statistical analysis showed that the applied method can concentrate up to 1x102 (100 CFU), a sensitivity of 100% and a specificity of 80% and an acceptable accuracy value (87.5%) were achieved. The membrane filtration method using the Celltrazone equipment allowed to increase the concentration of L. pneumophila in drinking water, complying with the intervals standardized by ISO and WHO, therefore, the method is suitable for its application allowing to obtain reliable resultsRESUMEN 1. PLANTEAMIENTO DEL PROBLEMA….10 2. HIPÓTESIS11 3. OBJETIVOS..….12 3.1. Objetivo general13 3.2. Objetivos específicos13 4. JUSTIFICACIÓN15 5. ANTECEDENTES… 17 6. MARCO TEÓRICO...21 7. DISEÑO METODOLÓGICO34 7.1. Tipo de investigación...34 7.2. Nivel, alcance o enfoque de la investigación34 7.3. Población objeto de estudio....35 7.4. Muestra35 7.5. Definición de las variables...35 7.6. Procedimientos, técnicas y herramientas para la investigación36 7.7. Validación de los ensayos microbiológicos. 39 7.8. Confirmación en lámina mediante la coloración de Gimenez41 8. RESULTADOS.43 9. DISCUSIÓN..49 10.CONCLUSIONES.....57 11.PERSPECTIVAS PARA EL FUTURO.…58 12.ANEXOS.59 REFERENCIAS BIBLIOGRÁFICASPregradoAbogado(a)67p.application/pdfspaDerechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2022https://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/closedAccessAtribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0)http://purl.org/coar/access_right/c_14cbValidación de un método de aislamiento para Legionella pneumophila por medio de técnicas de concentraciónTrabajo de grado - Pregradohttp://purl.org/coar/resource_type/c_7a1fhttp://purl.org/coar/version/c_970fb48d4fbd8a85Textinfo:eu-repo/semantics/bachelorThesishttps://purl.org/redcol/resource_type/TPinfo:eu-repo/semantics/publishedVersionFacultad de Ciencias de la SaludBogotaBacteriología y Laboratorio ClínicoLucas RMO. Vigilancia epidemiológica, prevención y control de la legionella en el hospital. In: Revista de Salud Ambiental [Internet]. 2015 [cited 2020 Sep 25]. Available from: https://ojs.diffundit.com/index.php/rsa/article/view/738/681Icontec. Guía Técnica Colombiana GTC 257 de 2015. Bogotá; 2015Burillo A, Pedro-Botet ML, Bouza E. Microbiology and Epidemiology of Legionnaire’s Disease. Infectious Disease Clinics of North America [Internet]. 2017 Mar 1 [cited 2020 Sep 25];31(1):7–27. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0891552016300885?via%3DihubAbdel-Nour M, Duncan C, Low DE, Guyard C. Biofilms: The Stronghold of Legionella pneumophila. International Journal of Molecular Sciences [Internet]. 2013 Oct 31 [cited 2020 Sep 25];14(11):21660. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3856027/Fields BS, Benson RF, Besser RE. Legionella and Legionnaires’ Disease: 25 Years of Investigation. Clinical Microbiology Reviews [Internet]. 2002 [cited 2020 Sep 27];15(3):506. Available from: /pmc/articles/PMC118082/Cunha BA, Burillo A, Bouza E. Legionnaires’ disease. The Lancet [Internet]. 2016 Jan 23 [cited 2020 Sep 27];387(10016):376–85. Available from: http://www.thelancet.com/article/S0140673615600782/fulltextGarcia-Nuñez M, Sopena N, Ragull S, Pedro-Botet ML, Morera J, Sabria M. Persistence of Legionella in hospital water supplies and nosocomial Legionnaires’ disease. FEMS immunology and medical microbiology [Internet]. 2008 Mar [cited 2020 Sep 27];52(2):202–6. Available from: https://pubmed.ncbi.nlm.nih.gov/18093139/. Patiño-Barbosa AM, Gil-Restrepo AF, Restrepo-Montoya V, Villamil-Gomez WE, CardonaOspina JA, Rodriguez-Morales AJ. Is Legionellosis Present and Important in Colombia? An Analyses of Cases from 2009 to 2013. Cureus [Internet]. 2017 Mar 28 [cited 2022 Sep 28];9(3). Available from: /pmc/articles/PMC5409816/Fraser DW, Tsai TR, Orenstein W, Parkin WE, Beecham HJ, Sharrar RG, et al. Legionnaires’ Disease. http://dx.doi.org/101056/NEJM197712012972201 [Internet]. 2010 Jan 13 [cited 2020 Sep 30];297(22):1189–97. Available from: https://www.nejm.org/doi/10.1056/NEJM197712012972201Clayton AJ. Legionnaires' disease: an illness whose time has come. Can Med Assoc J [Internet]. 1979 [cited 2020 sep 30];120(12):1482-1483. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1704222/. Steele TW. Legionnaires' disease in South Australia, 1979-1988. Med J Aust [Internet]. 1989 [cited 2020 sep 30];151(6):322-328. Available from: https://pubmed.ncbi.nlm.nih.gov/2687665/Morris GK, Patton CM, Feeley JC, Johnson SE, Gorman G, Martin WT, et al. Isolation of the Legionnaires’ disease bacterium from environmental samples. Annals of Internal Medicine [Internet]. 1979 [cited 2020 Oct 7];90(4):664–6. Available from: https://www.acpjournals.org/doi/10.7326/0003-4819-90-4-664?url_ver=Z39.88- 2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmedFliermans, CB, Cherry WB, Orrison LH, Thacker AL. Isolation of Legionella pneumophila from Nonepidemic-Related Aquatic Habitats. Applied and environmental microbiology [Internet]. 1979 [cited 2022 Feb 9];37(6):1239–42. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC243385/pdf/aem00210-0201.pdfShands KM, Ho JL, Meyer RD, Gorman GW, Edelstein PH, Mallison GF, et al. Potable Water as a Source of Legionnaires’ Disease. JAMA [Internet]. 1985 Mar 8 [cited 2020 Oct 8];253(10):1412–6. Available from: https://jamanetwork.com/journals/jama/fullarticle/397245Glavin FL, Winn WC, Craighead JE. Ultrastructure of lung in Legionnaires’ disease. Observations of three biopsies done during the Vermont epidemic. Annals of internal medicine [Internet]. 1979 [cited 2020 Oct 2];90(4):555–9. Available from: https://pubmed.ncbi.nlm.nih.gov/434634/Tison DL, Pope DH, Cherry WB, Fliermans CB. Growth of Legionella pneumophila in Association with Blue-Green Algae (Cyanobacteria). Applied and environmental microbiology [Internet]. 1980 [cited 2020 Oct 7];39(2):456–9. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC291353/pdf/aem00232-0184.pdfRowbotham TJ. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. Journal of Clinical Pathology [Internet]. 1980 [cited 2020 Oct 7];33(12):1179. Available from: /pmc/articles/PMC1146371/?report=abstractFeeley JC, Gorman GW, Weaver RE, Mackel DC, Smith HW. Primary isolation media for Legionnaires disease bacterium. Journal of Clinical Microbiology [Internet]. 1978 [cited 2020 Sep 30];8(3):320. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC275239/Tilton RC. Legionnaires’ disease antigen detected by enzyme-linked immunosorbent assay. Annals of Internal Medicine [Internet]. 1979 [cited 2020 Oct 9];90(4):697–8. Available from: https://www.acpjournals.org/doi/10.7326/0003-4819-90-4-697?url_ver=Z39.88- 2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmedBerdal BP, Farshy CE, Feeley JC. Detection of Legionella pneumophila Antigen in Urine by Enzyme-Linked Immunospecific Assay. Journal of clinical microbiology. 1979;575–8..Kohler RB, Zimmerman SE, Wilson E, Allen SD, Edelstein PH, Wheat LJ, White A. Rapid radioimmunoassay diagnosis of Legionnaires' disease: detection and partial characterization of urinary antigen. Ann Intern Med. 1981 May;94(5):601-605. Available from: https://pubmed.ncbi.nlm.nih.gov/7235392/Kohler RB, Winn WC, Wheat1 LJ. Onset and Duration of Urinary Antigen Excretion in Legionnaires Disease. Journal of clinical microbiology [Internet]. 1984 [cited 2020 Oct 11];20(4):605–7. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC271393/pdf/jcm00123-0035.pdfHorwitz MA. Formation of a novel phagosome by the legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. [cited 2020 Oct 4]; Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2187375/pdf/je15841319.pdfDoebbeling BN, Bale MJ, Koontz FP, Helms CM, Wenzel RP, Pfaller MA. Prospective evaluation of the Gen-Probe assay for detection of legionella in respiratory specimens. Eur J Clin Microbiol Infect Dis. 1988 Dec;7(6):748-52Cianciotto NP, Eisenstein BI, Mody CH, Toews GB, Cary Englebergl N. A Legionella pneumophila Gene Encoding a Species-Specific Surface Protein Potentiates Initiation of Intracellular Infection. INFECTION AND IMMUNITY [Internet]. 1989 [cited 2020 Oct 14];57(4):1255–62. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC313258/pdf/iai00064-0257.pdfMercante JW, Winchell JM. Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations. Clinical Microbiology Reviews [Internet]. 2015 Jan 1 [cited 2022 Oct 21];28(1):95. Available from: /pmc/articles/PMC4284297/Cianciotto NP, Fieldst BS. Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages (intracellular parastism/evolution/Hwimannela/Tebwhymena/FK506-bnidng proteins). 1992 [cited 2020Oct 14];89:5188–91. Available from. Keserue HA, Baumgartner A, Felleisen R, Egli T. Rapid detection of total and viable Legionella pneumophila in tap water by immunomagnetic separation, double fluorescent staining and flow cytometry. Microbial biotechnology [Internet]. 2012 Nov [cited 2020 Oct 24];5(6):753. Available from: /pmc/articles/PMC3815896/Schoch CL, Ciufo S, Domrachev M, Hotton CL, Kannan S, Khovanskaya R, et al. NCBI Taxonomy: A comprehensive update on curation, resources and tools. Database [Internet]. 2020 [cited 2020 Nov 8];2020. Available from: https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Tree&id=446&lvl=3&lin =f&keep=1&srchmode=1&unlockManske C, Hilbi H. Metabolism of the vacuolar pathogen Legionella and implications for virulence. Frontiers in Cellular and Infection Microbiology [Internet]. 2014 [cited 2020 Nov 14];4(SEP). Available from: /pmc/articles/PMC4158876/García-Núñez M. Colonización, citopatogenicidad y persistencia de Legionella spp. en agua sanitaria hospitalaria. Tesis Doctoral [Internet]. 2009 [cited 2020 Dec 9]. Available from: https://ddd.uab.cat/pub/tesis/2009/tdx-0217110-115805/mgn1de1.pdfVan der Kooij D, Brouwer-Hanzens AJ, Veenendaal HR, Wullings BA. Multiplication of Legionella pneumophila Sequence Types 1, 47, and 62 in Buffered Yeast Extract Broth and Biofilms Exposed to Flowing Tap Water at Temperatures of 38°C to 42°C. Applied and Environmental Microbiology [Internet]. 2016 [cited 2022 Dec 9];82(22):6691. Available from: /pmc/articles/PMC5086563/Stewart CR, Muthye V, Cianciotto NP. Legionella pneumophila Persists within Biofilms Formed by Klebsiella pneumoniae, Flavobacterium sp., and Pseudomonas fluorescens under Dynamic Flow Conditions. PLoS ONE [Internet]. 2012 Nov 21 [cited 2022 Dec 17];7(11). Available from: /pmc/articles/PMC3503961/Yu VL, Plouffe JF, Pastoris MC, Stout JE, Schousboe M, Widmer A, et al. Distribution of Legionella species and serogroups isolated by culture in patients with sporadic communityacquired legionellosis: an international collaborative survey. The Journal of infectious diseases [Internet]. 2002 Jul 1 [cited 2022 Dec 17];186(1):127–8. Available from: https://pubmed.ncbi.nlm.nih.gov/12089674/Mudali G, Kilgore PE, Salim A, McElmurry SP, Zervos M. Trends in Legionnaires’ DiseaseAssociated Hospitalizations, United States, 2006–2010. Open Forum Infectious Diseases [Internet]. 2020 Aug 1 [cited 2022 Dec 17];7(8). Available from: https://academic.oup.com/ofid/article/7/8/ofaa296/5872667Arias J. Neumonía atípica, importancia de la detección oportuna de legionella pneumophila en Colombia. a propósito de un caso importado detectado en Bogotá en el año 2015 [Internet]. AREANDINA. Fundación Universitaria del Área Andina; 2016 [cited 2020 Dec 18]. Available from: https://digitk.areandina.edu.co/handle/areandina/634Harrison TG, Taylor AG. A rapid microagglutination test for the diagnosis of Legionella pneumophila (serogroup 1) infection. Journal of Clinical Pathology [Internet]. 1982 [cited 2020 Dec 19];35(9):1028. Available from: /pmc/articles/PMC497857/?report=abstractTa AC, Stout JE, Yu VL, Wagener MM. Comparison of Culture Methods for Monitoring Legionella Species in Hospital Potable Water Systems and Recommendations for Standardization of Such Methods. JOURNAL OF CLINICAL MICROBIOLOGY [Internet]. 1995 [cited 2021 Jan 18];33(8):2118–23. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC228346/pdf/332118.pdfCenters for Disease Control and Prevention. Legionnaires Disease: Laboratory Guidance for Processing Samples | CDC [Internet]. 1992 [cited 2021 Jan 18]. Available from: https://www.cdc.gov/legionella/labs/procedures-manual.htmDe Giglio O, Diella G, Trerotoli P, Consonni M, Palermo R, Tesauro M, et al. Legionella Detection in Water Networks as per ISO 11731:2017: Can Different Filter Pore Sizes and Direct Placement on Culture Media Influence Laboratory Results? International Journal of Environmental Research and Public Health Article [Internet]. 2020 [cited 2021 Jan 18]; Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142469/pdf/ijerph-17- 02077.pdfSepúlveda M, Rodríguez M, Arboleda C, Arismendy L, Betancur J. Verificación del método de filtración por membrana para la detección y cuantificación de legionella spp. en agua potable [Internet]. 2016 [cited 2021 Jan 18]. Available from: https://revistas.elpoli.edu.co/index.php/pol/article/view/881/1480Lösch LS, Merino LA. Presencia de Legionella spp. en depósitos domiciliarios de agua potable en Resistencia, Chaco, Argentina. Informe preliminar. Revista Argentina de Microbiología [Internet]. 2016 Oct 1 [cited 2021 Jan 18];48(4):329–32. Available from: https://www.sciencedirect.com/science/article/pii/S0325754116300554Dimitriadi D, Velonakis E. Detection of Legionella spp. from Domestic Water in the Prefecture of Arta, Greece . Journal of Pathogens [Internet]. 2014 [cited 2021 Jan 18];2014:1–5. Available from: https://www.hindawi.com/journals/jpath/2014/407385/Díaz-Flores Á, Montero JC, Castro FJ, Alejandres EM, Bayón C, Solís I, et al. Comparing methods of determining Legionella spp. in complex water matrices. BMC Microbiology [Internet]. 2015 Dec 12 [cited 2021 Jan 19];15(1). Available from: /pmc/articles/PMC4436101/Párraga-Niño N, Quero S, Ventós-Alfonso A, Uria N, Castillo-Fernandez O, Ezenarro JJ, et al. New system for the detection of Legionella pneumophila in water samples. Talanta [Internet]. 2018 Nov 1 [cited 2021 Jan 19];189:324–31. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0039914018307057?via%3DihubReinthaler FF, Sattler J, Schaffler-Dullnig K, Weinmayr B, Marth E. Comparative study of procedures for isolation and cultivation of Legionella pneumophila from tap water in hospitals. Journal of Clinical Microbiology [Internet]. 1993 [cited 2021 Jan 20];31(5):1213. Available from: /pmc/articles/PMC262906/?report=abstracVan Kenhove E, Dinne K, Janssens A, Laverge J. Overview and comparison of Legionella regulations worldwide. American Journal of Infection Control [Internet]. 2019 Aug 1 [cited 2021 Jan 20];47(8):968–78. Available from: http://www.ajicjournal.org/article/S0196655318309957/fulltextEuropean Society of Clinical Microbiology and Infectious Diseases. ESGLI European Technical Guidelines for the Prevention, Control and Investigation, of Infections Caused by Legionella species. 2017 [cited 2021 Jan 20]; Available from: https://www.escmid.org/fileadmin/src/media/PDFs/3Research_Projects/ESGLI/ESGLI_Europ ean_Technical_Guidelines_for_the_Prevention_Control_and_Investigation_of_Infections_Ca used_by_Legionella_species_June_2017.pdfAENOR. UNE-EN ISO 11731:2017 Calidad del agua. Recuento de Legionella [Internet]. Madrid; 2017. Available from: https://tienda.aenor.com/norma-une-en-iso-11731-2017- n0059300Ministerio de Trabajo y Asuntos Sociales. NTP 538: Legionelosis: medidas de prevención y control en instalaciones de suministro de agua [Internet]. Madrid; 1999. Available from:https://www.insst.es/documents/94886/327064/ntp_538.pdf/4f52d14a-744b-46ee-ac49- ea8619dd00e9Ministerio de Sanidad y Consumo. BOLETÍN OFICIAL DEL ESTADO LEGISLACIÓN CONSOLIDADA Real Decreto 865/2003 [Internet]. Jul 4, 2003. Available from: https://www.boe.es/buscar/pdf/2003/BOE-A-2003-14408-consolidado.pdfSerrano Suárez A. Relación de Legionella spp. con parametros microbiológicos y fisicoquímicos en aguas [Internet]. TDX (Tesis Doctorals en Xarxa). Universitat de Barcelona; 2009 [cited 2021 Jan 22]. Available from: http://www.tdx.cat/handle/10803/284890Institute of Environmental Epidemiology Ministry of the Environment. Code for Practice for the Control of Legionella Bacteria in Cooling Towers [Internet]. Singapore; Available from: https://www.nea.gov.sg/docs/default-source/resource/practices-/code-of-practice-for-controlof-legionella-bacteria-in-cooling-towers.pdfMinistry of Health. The Prevention of Legionellosis in New Zealand: Guidelines for the control of legionella bacteria | Ministry of Health NZ [Internet]. New Zealand.; 2011 [cited 2021 Jan 24]. Available from: https://www.health.govt.nz/publication/prevention-legionellosis-newzealand-guidelines-control-legionella-bacteriaHealth and Human Services. A guide to developing risk management plans for cooling tower systems [Internet]. [cited 2021 Jan 27]. Available from: https://silo.tips/download/a-guide-todeveloping-risk-management-plans-for-cooling-tower-systemsAsociación Española de Abastecimientos de Agua y Saneamiento. OPERATING GUIDE: WATER ANALYSIS LABORATORIES PART II CRITERIA TO VALIDATE PHYSICOCHEMICAL AND MICROBIOLOGICAL ANALYSIS METHODS DRAFTING TEAM Experts in microbiological analysis. [cited 2021 Jan 31]; Available from: https://www.aeas.es/images/publicaciones/manuales/AEAS_Operating_Guide_Water_Analy sis_Laboratories_Part_II.pdfWorld Health Organization. Who expert committee on specifications for pharmaceutical preparation. [Internet]. 1992 [cited 2021 Feb 1]. Available from: http://apps.who.int/iris/bitstream/handle/10665/39645/WHO_TRS_823.pdf;jsessionid=3AF81 4E252888AB5A77CAB77EBD7672F?sequence=1Asociación Española de Normalización. UNE-EN ISO 8199:2019 Calidad del agua. Requisitos y orientaciones generales para el recuento de microorganismos mediante cultivo [Internet]. [cited 2021 Feb 1]. Available from: https://www.une.org/encuentra-tu-norma/buscatu-norma/norma?c=N0061957Celltrazone. HURO PATH S Slide Sample Processor User manual [Internet]. Celltrazone. Geumcheon-Gu, Seoul, Korea; [cited 2021 Feb 3]. Available from: https://dmec.moh.gov.vn/documents/10182/8891204/upload_00086670_1542882291262.pdf ?version=1.0&fileId=8898267Liofilchem. Legionella CYE Agar Base [Internet]. 2015 [cited 2021 Feb 4]. Available from: http://www.liofilchem.net/login/pd/ts/620125_TS.pdfLiofilchem. Legionella BCYE Growth Supplement [Internet]. [cited 2021 Feb 4]. Available from: http://www.liofilchem.net/login/pd/pi/81056_PI.pdfInvima. VALIDACION DE TECNICAS ANALITICAS [Internet]. 2015 Mar [cited 2021 Feb 20]. Available from: https://www.invima.gov.co/documents/20143/1433420/Validaci%C3%B3n+cualitativa+Micro biolog%C3%ADa.pdfGiménez DF. Staining Rickettsiae in Yolk-Sac Cultures. http://dx.doi.org/103109/10520296409061219 [Internet]. 2009 [cited 2022 Jan 4];39(3):135– 40. Available from: https://www.tandfonline.com/doi/abs/10.3109/10520296409061219Greer PW, Chandler FW, Hicklin MD. Rapid Demonstration of Legionella pneumophila in Unembedded Tissue: An Adaptation of the Giménez Stain. American Journal of Clinical Pathology [Internet]. 1980 Jun 1 [cited 2022 Jan 4];73(6):788–90. Available from: https://academic.oup.com/ajcp/article/73/6/788/1777877Murcia L CAMM. Estandarización de la técnica de coloración de Gimenez para la tinción de cepas de Legionella pneumophila. [Bogota DC]; 2021.Merck. Merck Microbiology Manual 12th Edition [Internet]. Merck . [cited 2022 Jan 23]. Available from: http://www.laboquimia.es/index.phpRodríguez-Martínez S, Blanky M, Friedler E, Halpern M. Legionella spp. isolation and quantification from greywater. MethodsX [Internet]. 2015 Jan 1 [cited 2022 Mar 1];2:458–62. Available from: https://www.sciencedirect.com/science/article/pii/S221501611500059XNeogen. Agar BCYE | Medio de aislamiento de Legionella | NEOGEN [Internet]. [cited 2022 Feb 3]. Available from: https://www.neogen.com/es/categories/microbiology/bcye-agarlegionella-isolation-medium/Bartie C, Venter SN, Nel LH. Evaluation of detection methods for Legionella species using seeded water samples. Water SA [Internet]. 2001 Apr 1 [cited 2022 Feb 3];27(4):523–8. Available from: https://www.ajol.info/index.php/wsa/article/view/4966Schulze-Röbbecke, R., Hartemann, P., Fimmers, R., & Hagenau, C. Comparison of membrane filtration methods for the recovery of legionellae from naturally contaminated domestic drinking water supplies. [Internet].1999 [cited 2022 Feb 3] Zentralblatt Für Hygiene Und Umweltmedizin, 202(1), 51–59. Available from: https://doi.org/10.1016/S0934- 8859(99)80053-4Matuszewska, R., & Krogulska, B. [Detection and isolation of bacteria of Legionella species from the water environment]. [Internet] 2000 [cited 2022 Feb 3] Roczniki Panstwowego Available from: Zakladu Higieny, 51(2), 183–190. https://europepmc.org/article/med/10959198Ezenarro JJ, Párraga-Niño N, Sabrià M, del Campo FJ, Muñoz-Pascual FX, Mas J, et al. Rapid Detection of Legionella pneumophila in Drinking Water, Based on Filter Immunoassay and Chronoamperometric Measurement. Biosensors [Internet]. 2020 Sep 1 [cited 2022 Feb 25];10(9). Available from: /pmc/articles/PMC7558583/Cercenado E, Cantón R. Procedimientos en Microbiología Clínica Recomendaciones de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. [cited 2022 Feb 7]; Available from: https://www.seimc.org/contenidos/documentoscientificos/procedimientosmicrobiologia/seimc -procedimientomicrobiologia48.pdfCercenado E, Cantón R. Procedimientos en Microbiología Clínica Recomendaciones de la Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. [cited 2022 Feb 7]; Available from: https://www.seimc.org/contenidos/documentoscientificos/procedimientosmicrobiologia/seimc -procedimientomicrobiologia48.pdfGruas C, Álvarez I, Lara C, García CB, Savva D, Arruga MV. Identification of Legionella spp. in Environmental Water Samples by ScanVIT-LegionellaTM Method in Spain. Indian Journal of Microbiology [Internet]. 2013 Jun 1 [cited 2022 Feb 7];53(2):142. Available from: /pmc/articles/PMC3626958/Boulanger, C. A., & Edelstein, P. H. Precision and accuracy of recovery of Legionella pneumophila from seeded tap water by filtration and centrifugation.[Internet] 1995 Applied and Environmental Microbiology, [cited 2022 Feb 7] 61(5), 1805–1809. Available from: /pmc/articles/PMC3626958/ https://doi.org/10.1128/AEM.61.5.1805-1809.1995Sandle T. Approaching Microbiological Method Validation. 2015 [cited 2022 Feb 7]; Available from: https://www.researchgate.net/publication/287992312_Approaching_Microbiological_Method_ ValidationBedrina, B., Macián, S., Solís, I., Fernández-Lafuente, R., Baldrich, E., & Rodríguez, G. . Fast immunosensing technique to detect Legionella pneumophila in different natural and anthropogenic environments: comparative and collaborative trials. (2013) [cited 2022 Feb 7]BMC Microbiology, 13(1), 88. 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Unicolmayorrepositorio@unicolmayor.edu.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