Albrechtsen, Hans-Jørgen

Krogfelt, Karen A.

Krøjgaard, Louise Hjelmar

Uldum, Søren A.

During the investigation of a small outbreak of legionnaires’ disease in a newly built block of flats, several risk factors were uncovered. The outbreak encompassed two men (age 44 and 65) of which the oldest one died. Two interventions (heat treatments of 70ºC for 12 and 24 hours respectively) and permanent changes of the flow and temperature were conducted to overcome the high concentrations of Legionella in the water. Water samples (104 in total, both cold and warm water) from taps and shower hoses were collected and cultivated. The samples consisted of: A samples: the first one litre, B samples: one litre collected after flushing until constant temperature and water from shower hoses at a temperature of 38ºC. The survey pointed at important risk factors 1) low temperature of the hot water in some of the most distant taps 2) low flow of the water circulating system because of too small pipe dimensions and too low pump capacity 3) high numbers of Legionella in the shower hoses 4) stagnancy of water before residents move in and in unoccupied apartments and 5) lack of or inappropriate control measures. Following cultivation, DNA from all water samples was extracted by a simple method with Chelex-100. The samples will be investigated by an in-house q-PCR (Quantitative Real Time PCR). The q-PCR is a Taq-Man based assay with 5S primers detecting Legionella spp and mip primers detecting Legionella pneumophila. The two Legionella q-PCR assays have been validated according to the Afnor standard (T90 471) with standards calibrated according to the international standard from Legionelles centre National de Référence (SRM_LEGDNA_01 and CQE_LEGDNA_01). The results from culture (CFU/L) and PCR (GU/L) will be compared, and on this background it will be evaluated if q-PCR could have been used for fast risk assessment in the initial phase of the investigation and to follow the effect of the interventions in the later phase of the investigation

Online Research Database In Technology

                       General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal   If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.     Downloaded from orbit.dtu.dk on: Dec 18, 2017Risk assessment and monitoring of Legionella by culture and q-PCR in a newly builtblock of flats associated with a small outbreak of legionnaires´ diseaseKrøjgaard, Louise Hjelmar; Krogfelt, Karen A.; Albrechtsen, Hans-Jørgen; Uldum, Søren A.Published in:Programme European Working Group For Legionella InfectionsPublication date:2010Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Krøjgaard, L. H., Krogfelt, K. A., Albrechtsen, H-J., & Uldum, S. A. (2010). Risk assessment and monitoring ofLegionella by culture and q-PCR in a newly built block of flats associated with a small outbreak of legionnaires´disease. In Programme European Working Group For Legionella Infections Copenhagen, Denmark: EWGLI.80 81P 2.07 Detection of Legionella-decontamination in water by real-time-PCR and pre-treatment with ethidium monoazide - Differentiation of viable and dead cellsCatharina Unger, Christin Eichhorn, Christian Lück University of Technology Dresden, Institute of Medical Microbiology and Hygiene, Dresden, GermanyThe contamination of drinking water supplies in hotels, hospitals and public buildings with Legionella is well known. Hospital acquired legionnaires disease is often associated with a high fatality rate. In order to suppress the multiplication of Legionella in these buildings technical measures and decontamina-tion by hypoclorination or super heating are applied. For the exact detection of the contamination the culture method is currently mostly used, but the method with the increasing impact is the real-time-PCR. Since PCR-based assays cannot discriminate between live and dead bacteria further studies are needed.Ethidium monoazide (EMA) is a photo activated stain, which penetrates only dead bacterial cells with compromised cell walls and cell membranes. It intercalated into DNA of those cells and also in free extracellular DNA. The covalent binding of EMA to DNA is induced by photo activation. The photo induced cross linking result in the insolubility of DNA. Following this DNA can’t be extracted.For the first experiment we prepared different artificial water samples including Legionella pneumophi-la strain Corby. To get an overview of the affectivity of EMA we tested different incubation-times, dif-ferent concentrations and different exposure times. In the most experiments we use 5 min EMA treat-ment with 50µmol and an exposure-time of 2 min. For the real-time-PCR we use a 529 bp-fragment, because statistically EMA intercalate into DNA only every 10-80bp. We treated the water samples with heat, sodium hypochlorite, silver, formaldehyde, Ampicilin and UV-light to get a reduction of the viable bacteria.With some of these methods a reduction of cultivable bacteria can be verified with the help of EMA, but in some others not. This effect is probably caused by the different antimicrobial mechanisms of the different decontamination-methods. P 2.08 Risk assessment and monitoring of Legionella by culture and q-PCR in a newly built block of flats associated with a small outbreak of legionnaires’ disease Louise Hjelmar Krøjgaard a, Karen A. Krogfelt a, Hans-Jørgen Albrechtsen b, Søren A. Uldum a a Statens Serum Institut, Copenhagen, Denmarkb Technical University of Denmark, Kgs.Lyngby, DenmarkDuring the investigation of a small outbreak of legionnaires’ disease in a newly built block of flats, several risk factors were uncovered. The outbreak encompassed two men (age 44 and 65) of which the oldest one died. Two interventions (heat treatments of 70ºC for 12 and 24 hours respectively) and permanent changes of the flow and temperature were conducted to overcome the high concentra-tions of Legionella in the water. Water samples (104 in total, both cold and warm water) from taps and shower hoses were collected and cultivated. The samples consisted of: A samples: the first one litre, B samples: one litre collected after flushing until constant temperature and water from shower hoses at a temperature of 38ºC. The survey pointed at important risk factors 1) low temperature of the hot water in some of the most distant taps 2) low flow of the water circulating system because of too small pipe dimensions and too low pump capacity 3) high numbers of Legionella in the shower hoses 4) stagnancy of water before residents move in and in unoccupied apartments and 5) lack of or inap-propriate control measures. Following cultivation, DNA from all water samples was extracted by a simple method with Chelex-100. The samples will be investigated by an in-house q-PCR (Quantitative Real Time PCR). The q-PCR is a Taq-Man based assay with 5S primers detecting Legionella spp and mip primers detecting Le-gionella pneumophila. The two Legionella q-PCR assays have been validated according to the Afnor standard (T90 471) with standards calibrated according to the international standard from Legionelles centre National de Référence (SRM_LEGDNA_01 and CQE_LEGDNA_01). The results from culture (CFU/L) and PCR (GU/L) will be compared, and on this background it will be evaluated if q-PCR could have been used for fast risk assessment in the initial phase of the investigation and to follow the effect of the interventions in the later phase of the investigation. POSTER SESSION II – ECOLOGY / RISK ASSESSMENT / DETECTION / PREVENTION AND CONTROLPOSTER SESSION II – ECOLOGY / RISK ASSESSMENT / DETECTION / PREVENTION AND CONTROL25th meeting of the European Working Group for Legionella Infections (EWGLI) in Copenhagen.  

Risk assessment and monitoring of Legionella by culture and q-PCR in a newly built block of flats associated with a small outbreak of legionnaires´ disease

http://orbit.dtu.dk/ws/files/5878376/ENV2010-226.pdf

Risk assessment and monitoring of Legionella by culture and q-PCR in a newly built block of flats associated with a small outbreak of legionnaires´ disease

Abstract

Similar works

Full text

Available Versions

Online Research Database In Technology