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The Impact of Chlorine and Chloramine on the Detection and Quantification of Legionella Pneumophila and Mycobacterium Spp

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The Impact of Chlorine and Chloramine on the Detection and Quantification of Legionella Pneumophila and Mycobacterium Spp The impact of chlorine and chloramine on the detection and quantification of Legionella pneumophila and Mycobacterium spp. Maura J. Donohue Ph.D. Office of Research and Development Center of Environmental Response and Emergency Response (CESER): Water Infrastructure Division (WID) Small Systems Webinar January 28, 2020 Disclaimer: The views expressed in this presentation are those of the author and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. A Tale of Two Bacterium… Legionellaceae Mycobacteriaceae • Legionella (Genus) • Mycobacterium (Genus) • Gram negative bacteria • Nontuberculous Mycobacterium (NTM) (Gammaproteobacteria) • M. avium-intracellulare complex (MAC) • Flagella rod (2-20 µm) • Slow grower (3 to 10 days) • Gram positive bacteria • Majority of species will grow in free-living • Rod shape(1-10 µm) amoebae • Non-motile, spore-forming, aerobic • Aerobic, L-cysteine and iron salts are required • Rapid to Slow grower (1 week to 8 weeks) for in vitro growth, pH: 6.8 to 7, T: 25 to 43 °C • ~156 species • ~65 species • Some species capable of causing disease • Pathogenic or potentially pathogenic for human 3 NTM from Environmental Microorganism to Opportunistic Opponent Genus 156 Species Disease NTM =Nontuberculous Mycobacteria MAC = M. avium Complex Mycobacterium Mycobacterium duvalii Mycobacterium litorale Mycobacterium pulveris Clinically Relevant Species Mycobacterium abscessus Mycobacterium elephantis Mycobacterium llatzerense. Mycobacterium pyrenivorans, Mycobacterium africanum Mycobacterium europaeum Mycobacterium madagascariense Mycobacterium rhodesiae Mycobacterium agri Mycobacterium fallax Mycobacterium mageritense, Mycobacterium riyadhense Mycobacterium aichiense Mycobacterium farcinogenes Mycobacterium malmoense Mycobacterium rufum M. avium, M. intracellulare, Mycobacterium algericum Mycobacterium flavescens Mycobacterium mantenii Mycobacterium rutilum Mycobacterium alsense Mycobacterium florentinum. Mycobacterium marinum Mycobacterium salmoniphilum ( M. fortuitum, M. chelonae, Mycobacterium alvei. Mycobacterium fluoranthenivorans Mycobacterium massiliense Mycobacterium saopaulense Mycobacterium angelicum Mycobacterium fortuitum Mycobacterium microti Mycobacterium saskatchewanense M. kansasii, M. abscessus, Mycobacterium anyangense Mycobacterium franklinii Mycobacterium minnesotense Mycobacterium scrofulaceum Mycobacterium arabiense. Mycobacterium frederiksbergense Mycobacterium monacense Mycobacterium sediminis etc. Mycobacterium aromaticivorans Mycobacterium gadium Mycobacterium montefiorense Mycobacterium senegalense Mycobacterium arosiense Mycobacterium gastri Mycobacterium moriokaense, Mycobacterium senuense Mycobacterium arupense Mycobacterium genavense Mycobacterium mucogenicum Mycobacterium seoulense, Mycobacterium asiaticum Mycobacterium gilvum Mycobacterium murale Mycobacterium septicum • Pulmonary NTM lung Mycobacterium aurum Mycobacterium goodii Mycobacterium neoaurum Mycobacterium setense Mycobacterium austroafricanum Mycobacterium gordonae Mycobacterium nebraskense Mycobacterium sherrisii, disease Mycobacterium avium Mycobacterium haemophilum Mycobacterium neworleansense Mycobacterium shimoidei Mycobacterium bacteremicum Mycobacterium hassiacum Mycobacterium nonchromogenicum Mycobacterium shinjukuense Mycobacterium boenickei Mycobacterium heckeshornense. Mycobacterium noviomagense Mycobacterium shottsii, • Chronic bronchopulmonary Mycobacterium spp. Mycobacterium botniense Mycobacterium heidelbergense Mycobacterium novocastrense Mycobacterium simiae Mycobacterium bouchedurhonense Mycobacterium hiberniae Mycobacterium pallens Mycobacterium smegmatis disease Mycobacterium bourgelatii Mycobacterium hippocampi Mycobacterium palustre Mycobacterium sphagni Mycobacterium bovis Mycobacterium hodleri Mycobacterium paraense Mycobacterium stomatepiae • Cervical or other Mycobacterium brisbanense Mycobacterium holsaticum Mycobacterium paraffinicum Mycobacterium szulgai Mycobacterium brumae Mycobacterium houstonense Mycobacterium parafortuitum Mycobacterium terrae lymphadenitis Mycobacterium canariasense Mycobacterium immunogenum Mycobacterium paragordonae. Mycobacterium thermoresistibile Mycobacterium caprae Mycobacterium insubricum Mycobacterium paraintracellulare Mycobacterium timonense Mycobacterium celatum Mycobacterium interjectum Mycobacterium parakoreense. Mycobacterium tokaiense • Skin and soft tissue Mycobacterium celeriflavum Mycobacterium intermedium Mycobacterium parascrofulaceum Mycobacterium triplex Mycobacterium chelonae Mycobacterium intracellulare Mycobacterium paraseoulense Mycobacterium triviale diseases Mycobacterium chitae Mycobacterium iranicum Mycobacterium paratuberculosis. Mycobacterium tuberculosis Mycobacterium chlorophenolicum Mycobacterium kansasii Mycobacterium parmense Mycobacterium tusciae • Disseminated infections Mycobacterium chubuense Mycobacterium komossense Mycobacterium peregrinum Mycobacterium ulcerans Mycobacterium colombiense Mycobacterium koreense. Mycobacterium phlei Mycobacterium vaccae Mycobacterium conceptionense Mycobacterium kubicae. Mycobacterium phocaicum Mycobacterium vanbaalenii. • Catheter-related infections Mycobacterium confluentis Mycobacterium kumamotonense Mycobacterium pinnipedii Mycobacterium vulneris. Mycobacterium conspicuum , Mycobacterium kyorinense Mycobacterium porcinum Mycobacterium wolinskyi Mycobacterium cookii Mycobacterium lacus. Mycobacterium poriferae, Mycobacterium xenopi Mycobacterium cosmeticum Mycobacterium lentiflavum Mycobacterium pseudoshottsii Mycobacterium yongonense Mycobacterium crocinum Mycobacterium leprae Mycobacterium psychrotolerans Mycobacterium doricum Mycobacterium lepraemurium 4 Legionellosis: Respiratory Disease National Notifiable Disease Surveillance Disease System (NNDSS) Annual Cost of Legionellosis = pneumonia 2019 National Reportable Disease List: contained Treatment in the U.S. the names of over 110 Diseases/Microorganisms • Legionaries' Disease (severe) • Pontiac Fever (mild) Number of Cases Reported in 2019: 7,802 cases Number of Hospitalization/year Signs/Symptoms 8,000-18,000 cases Pneumonia avg(13,000) Marston, (1997) (Signs/Symptoms) High Fever Cough Headache Chills Total Hospitalization Cost: Muscle $433,758,000 Aches Diarrhea LEGIONELLOSIS. Incidence,* by year — United States, 2000–2019, Source NNDSS Collier, S.A. et al 2012: Direct healthcare costs of selected disease primarily or partially transmitted by water. Epidemiology Infection, 140, p2003-2013 5 Nontuberculous Mycobacteria Infection/Disease: Primarily Respiratory Diseases Infection/Disease State Health Departments Annual Cost of • Pulmonary NTM lung disease Treatment in the U.S. Laboratory Reports • Chronic bronchopulmonary *Report is defined as the presence of culturable NTM from a human disease specimen (Lavage, Sputum, Blood, and/or Tissue) • Cervical or other lymphadenitis Number of Cases Reported in 2010: Est 86,244 cases Number of Hospitalization/year • Skin and soft tissue diseases Nontuberculous Mycobacteria 16,386 cases: 2007 • Disseminated infections 14 Total Hospitalization Cost: • Catheter-related infections 12 10 $425,788,469 8 Signs/Symptoms Collier, S.A. et al 2012: 6 Direct healthcare costs of selected disease primarily or partially transmitted by water. Epidemiology Infection, 140, p2003-2013 Fever Chest Pain 4 2 Number of NTM Cases Headache Fatigue 0 Isolation rate per 100,000 persons 1970 1980 1990 2000 2010 2020 est 86,244 cases: 2010 Year NONTUBERCULOUS MYCOBACTERIA. Total Hospitalization Cost: Isolation Rate,* by year — United States, 1980–2013, Sources: Good et al. (1980), CDC, NTM 1993-1996 report (1999), Donohue et al. $815,098,690 (2016) Strollo S.E. et al 2015: The Burden of Pulmonary N0ntuberculous Mycobacterial Disease in the United States. AnnalsATS, 12, p1458-1464 6 Exposure Routes: Environmental Sources Legionella pneumophila and Mycobacterium avium are microorganisms of the natural environment found in soil and water. AIR Route * Treated WATER Route WATER Route SOIL Route * The following study examines exposure potentials from portable water 7 What Is the Purpose of this Study? Clear Well Houses/Building Source Water Waterworks Distribution Point of Use Lp = 25% (6/24) Lp = 4% (1/24) MA = 25% (6/24) MA = 25% (6/25) MI = 25% (6/24) MI = 25% (6/25) Y X King et al 2016: SOTEN Donohue et al 2019: JAM & AEM 8 Study Design Structure/ Point of Use 46 States and Territories 5 qPCR Assays Water Quality Testing 358 Samples . Legionella pneumophila Donohue et al. 2014 • Total Chlorine . L. pneumophila Sg1 Merault et al 2011l DPD Total Chlorine Test . Mycobacterium avium • Monochloramine Houses Chen et al. 2015 Monochlor F Test . Mycobacterium intracellulare Chen et al. 2015 • Temperature . Mycobacterium abscessus Cold water line Steindor M. et al. 2015 Hot water line Buildings • Heterotrophic Plate . Detection Frequency (FD) Counts (HPC) . Persistence Standard Method 9215 . Concentration Sampling Time Frame : 2011 – 2017 9 Scope and Sample Design • Chlorine (Cl) - Chloramine (CLM) • Residual (Totatl Chlorine Test) – Temperature (Cold vs Hot) • L. pneumophila (Lp) – Mycobacterium spp.(Myco) Molecular Testing: qPCR Water Membrane Filtration DNA Extraction qPCR Method Polycarbonate 0.4 µm Bead Beating 40 cycles DNA precipitation 1hr 45 min Assays 1. Genus: Gen-L (16S) 96 plate well: Well Format • Assay/Mastermix 2. Species: Lp-16S (16S) • Primer/Probes • Template(DNA/ddH2O) 10 µL reaction ~ 200mL 1 Well = 1 Reaction • Internal Control 3. Species: Lp-Mip (Mip) Overview
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