- Email: [email protected]
Microbiological diagnosis and molecular typing of Legionella strains during an outbreak of legionellosis in Southern Germany
Accepted Manuscript Title: Microbiological Diagnosis and Molecular Typing of Legionella strains during an Outbreak of Legionellosis in Southern Germany Author: Andreas Essig Heike von Baum Theodor Gonser Georg Haerter Christian L¨uck PII: DOI: Reference:
S1438-4221(16)30001-7 http://dx.doi.org/doi:10.1016/j.ijmm.2016.01.001 IJMM 51021
To appear in: Received date: Revised date: Accepted date:
10-12-2015 22-1-2016 25-1-2016
Please cite this article as: Essig, A., von Baum, H., Gonser, T., Haerter, G., L¨uck, C.,Microbiological Diagnosis and Molecular Typing of Legionella strains during an Outbreak of Legionellosis in Southern Germany, International Journal of Medical Microbiology (2016), http://dx.doi.org/10.1016/j.ijmm.2016.01.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Microbiological Diagnosis and Molecular Typing of Legionella strains during an Outbreak of Legionellosis in Southern Germany
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Andreas Essiga, Heike von Bauma, Theodor Gonserb, Georg Haerterc, Christian Lückd*
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Institute of Medical Microbiology and Hygiene, Ulm University Hospital, Germany
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Public Health Authority Ulm, Germany
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Department of Internal Medicine III, Section of Infectious Diseases, Ulm, University Hospital. Now: MVZ Endokrinologikum Ulm, Center for Endocrinology and Infectious Diseases, Ulm, Germany d
Institute of Medical Microbiology and Hygiene, German Consiliary laboratory for Legionella, Dresden University of Technology, Germany
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Key words: Legionnaire’s Disease, outbreak, cooling tower, Legionella pneumophila, molecular typing, pneumonia
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*Corresponding author: Christian Lück, Institute of Medical Microbiology and Hygiene, National Consiliary Laboratory for Legionella, Medical Faculty "Carl Gustav Carus", University of Technology Dresden, Fetscherstrasse 74, D-01307 Dresden
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Email: [email protected]
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Phone: 0049-351-458 16580 Fax: 0049-351-458 6311
International Journal of Medical Microbiology 2015, Category: Bacteriology
Presented in part at the annual meeting of the ICAAC, (abstract 187); Annual Meeting of the European Legionella Surveillance network ELDSNet, Copenhagen September 2010.
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26 Abstract
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An explosive outbreak of Legionnaires’ disease with 64 reported cases occurred in Ulm/ NeuUlm in the South of Germany in December 2009/January 2010 caused by Legionella (L.) pneumophila serogroup 1, monoclonal (mAb) subtype Knoxville, sequence type (ST) 62. Here we present the clinical microbiological results from 51 patients who were diagnosed at the University hospital of Ulm, the results of the environmental investigations and of molecular typing of patients and environmental strains. All 50 patients from whom urine specimens were available were positive for L. pneumophila antigen when an enzyme-linked immunosorbent assay (EIA) was used following concentration of those urine samples that tested initially negative. The sensitivity of the BinaxNow rapid immunographic assay (ICA), after 15 minutes reading and after 60 min reading were 70% and 84%, respectively. Direct typing confirmed the monoclonal subtype Knoxville in 5 out of 8 concentrated urine samples. Real time PCR testing of respiratory tract specimens for L. pneumophila was positive in 15 out of 25 (60%) patients. Direct nested sequence based typing (nSBT) in some of these samples allowed partial confirmation of ST62. L. pneumophila serogroup 1, monoclonal subtype Knoxville ST62, defined as the epidemic strain was isolated from 8 out of 31 outbreak patients (26%) and from one cooling tower confirming it as the most likely source of the outbreak. While rapid detection of Legionella antigenuria was crucial for the recognition and management of the outbreak, culture and molecular typing of the strains from patients and environmental specimens was the clue for the rapid identification of the source of infection.
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1. Introduction
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Legionellae are ubiquitous gram-negative bacteria, which occupy natural and manmade aquatic environments. Currently the genus Legionella comprises 58 species (Rizzardi et al., 2015) and more than 70 serogroups with Legionella ( L.) pneumophila serogroup (sg) 1 causing the majority of human infections (Helbig et al., 2002).
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Legionnaires’ disease (LD) occurs mostly as a sporadic pneumonia caused by inhalation of Legionella-containing aerosols generated from warm water supplies, cooling towers, or whirl pool spas. Approximately 10% of cases occur in outbreaks or clusters (Mercante and Winchell, 2015) . Legionella outbreaks may present as serious threats of public health and management of outbreaks can be extremely difficult, especially if the source of infection is unknown. In general, Legionella spp. are responsible for 2-15% of community acquired pneumonia (CAP) (Torres et al., 2014). 860 cases have been reported to the German Health Authorities in 2014 (Robert-Koch-Institut, 2015). Thus, significant underdiagnosing and/ or underreporting must be considered.
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Here we describe the results of the microbiological and epidemiological investigation during an outbreak of LD with 64 reported cases between December 2009 and January 2010 in Ulm and Neu-Ulm, twin cities with a total of 175 000 inhabitants in Southern Germany. A cooling tower in the city center of Ulm was finally identified as the most likely source of the outbreak (von Baum H. et al., 2010). The majority of patients was diagnosed and treated in our hospital thus offering a unique opportunity to study the microbiological and epidemiological methods applied.
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2. Materials and methods
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2.1. Case Detection and criteria for Legionella pneumonia
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During the outbreak investigation, the case definitions of the European Centre for Disease Prevention and Control (European Legionnaires’ Disease Surveillance Network (ELDSNet) were applied (von Baum H. et al., 2010). A case of Legionnaire’s disease was defined as a patient who had (1) confirmed pneumonia, (2) laboratory evidence of L. pneumophila sg 1 infection (positive urinary antigen test, isolation of Legionella from respiratory specimens), (3) onset of disease between December 22th 2009 and January 7th 2010 and (4) lived in or visited the cities of Ulm/Neu-Ulm in the 2 weeks preceding the development of CAP. 2.2. Detection of Legionella spp. in clinical specimens
Detection of urinary antigen. For the qualitative detection of L. pneumophila soluble antigen in urine, specimens were tested using a rapid immunochromatographic card assay (ICA), BinaxNow (Allere, Düsseldorf, Germany) as well as a microtiter based enzyme-linked immunosorbent assay (EIA) (Biotest Legionella Urine Antigen EIA; BioRad, Munich Germany) according to the manufacturer’s instructions. In case of negative results the incubation time of the Binax NOW assay was prolonged to 60 min. Furthermore, the urines were concentrated 25-fold using an ultrafiltration device (Minicon concentrator, 15 kDa exclusion size, Millipore) and retested with the Biotest EIA but not with the Binax Now assay. In eight urine samples a direct monoclonal typing approach was applied. From the other samples the amount left was not sufficient for this analysis. Briefly, the urine specimens were 3
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first concentrated five-fold by using the Vivaspin 6 columns (Vivascience, Fisher Scientific, Schwerte, Germany, exclusion size 5kDa) and added to wells of Binax Legionella urinary antigen EIA carrying immobilised anti-Legionella antibodies (Allere Düsseldorf, Germany). After washing, the bound antigens were detected by monoclonal antibodies (mAbs) following incubation with horseradish peroxidase labelled anti-mouse IgG (Sigma, Deisenhofen, Germany) and substrate reaction (Helbig et al., 2012).
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Detection of Legionella DNA from respiratory samples. Respiratory samples including sputum samples, tracheal and bronchial aspirates were processed for detection of Legionella specific DNA by nucleic acid amplification. DNA was isolated from respiratory samples using the Magna pure system (Roche, Germany) and a L. pneumophila specific real-time LightCycler PCR was run targeting the macrophage infectivity potentiator (mip) gene as described previously (Wellinghausen et al., 2001). To define the sequence type directly from clinical samples nested SBT was performed on PCR positive samples according to the ESGLI protocol (Mentasti et al., 2012) .
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Culture of Legionella species from clinical samples. Respiratory samples from patients with antigenuria were cultured on non-selective buffered charcoal- yeast extract (BCYE) agar and a selective agar containing cefamandol, polymyxin B and anisomycin (BMPA (Stout et al., 2003) at 36°C in humified atmosphere supplemented with 5% CO2 following heat treatment at 50°C for 30 minutes. Culture plates were inspected daily up to day 10. Colonies suspected for Legionella were cultured on non-selective buffered charcoal yeast extract (BCYE) agar and Columbia blood agar. Isolated strains that did only grow on BCYE agar but not on blood agar were initially serotyped by using a Latex agglutination test (Oxoid, Wesel, Germany) and confirmed by using a panel of mAb as described elsewhere (Helbig et al., 2002). Genotyping was performed by using the European sequence based typing (SBT) schema (http://www.hpabioinformatics.org.uk/legionella/legionella_sbt/php/sbt_homepage.php).
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2.3 Environmental Investigation
The epidemiological data suggested a wet cooling tower (CT) as the source of the outbreak (Freudenmann et al., 2011;von Baum H. et al., 2010). Therefore the search for possible sources of the outbreak focussed on 30 wet cooling towers identified in the area of interest. CTs were inspected, and 1 to 4 water samples were collected and processed according to ISO 11731/1998 (ISO 11731, 1998). Samples were collected from the cooling tower ponds in all CT, and if accessible water samples were also taken from 1 to 3 additional sites like water incomes, the water system of the building and other sites. Environmental L. pneumophila isolates were typed by mAb typing and SBT as described for the clinical isolates. Nonpneumophila strains were typed by mip gene sequencing (Ratcliff et al., 1998).
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3. Results
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3.1. Case detection
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Altogether, 64 epidemiologically linked cases (66% male, 34% female) met the European case definition of a confirmed case and were reported to the local public health authorities during this outbreak. The median age of the patients was 68 (27-96) years. Sixty patients had to be hospitalized and 5 patients died from infection (four of whom were older than 80 years)(Freudenmann et al., 2011). Fifty-one patients (80%) were diagnosed at the University Hospital of Ulm. Prominent clinical and laboratory findings included radiological infiltrates (94%), fever (83%), non-productive cough (69%), central nervous system symptoms (58%), muscle pain or pain in the limbs (41%), abdominal pain/diarrhea (28%), CRP>100 mg/l (92%), LDH elevation (74%), Troponin I elevation (71%), hyponatremia (60%) and leukocytosis (50%). The interdisciplinary management of the outbreak has been published previously (Freudenmann et al., 2011).
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3.2 Detection of Legionella in clinical specimens
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Detection and typing of urinary antigen. Urine samples for detection of soluble L. pneumophila antigen were available from 50 of 51 patients diagnosed and treated at the University hospital of Ulm. Specimens were investigated by a rapid BinaxNow card assay and by the Biotest EIA. Using the BinaxNow visible bands were detected in 70% of specimens at 15 min. Prolongation of the reading time to 60 minutes increased the detection rate to 82% indicating that 18% of specimens remained negative in the BinaxNow assay even if the incubation period was extended to 60 minutes. Using nonconcentrated urine samples the Biotest EIA yielded positive results in 74% of cases. 25fold concentration of urine samples yielded another 13 positive tests resulting in a 100% detection rate. Seven out of 9 specimens that tested negative with the BinaxNow were also negative by EIA using nonconcentrated urines but became positive by EIA when urine samples were concentrated (Table 1). In 5/8 fivefold concentrated urine samples the direct monoclonal subtyping identified the monoclonal subtype Knoxville of the causative strain. In three concentrated urines no reactivity with any of the mAbs could be achieved (Table 2). From other patients the amounts of urine were not sufficient for this investigation.
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Culture of Legionella species from clinical samples. For isolation procedures respiratory tract specimens from 31 patients with positive antigenuria and/or positive nucleic acid detection of Legionella pneumophila were available. Eight out of 31 patients were culture positive (26%). Six isolates were cultured from sputum samples, and two isolates were grown from tracheal and bronchial aspirates, respectively. All clinical isolates were typed as Legionella pneumophila serogroup 1, mAb subtype Knoxville, ST62, which was defined as the epidemic strain.
Detection and direct typing of DNA from respiratory specimens. Nucleic acid based detection of L. pneumophila was performed by a real time PCR assay targeting the mip gene. PCR was positive in 15 out of 25 available respiratory tract specimens tested (60%). PCR-positive samples included 13 sputum specimens, 1 tracheal aspirate and 1 bronchial aspirate. All of the culture-positive specimens (8/23) yielded also positive PCR results. DNA sequences of selected alleles in concordance with alleles of ST62 were detected by direct nested SBT in 18 respiratory samples from 15 patients (Table 3). Due to the limited amount of DNA the 5
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alleles flaA, pilE, mip, proA, neuA were chosen because this combination of alleles is unique in ST62.
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3.3. Environmental investigations
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Thirty wet cooling towers were inspected by an expert team of the local public health authorities. Immediately after water samples were collected all cooling towers were shut down, cleaned and disinfected as a prophylactic measure. L. pneumophila was recovered from 9 water samples out of 30 cooling towers in the city of Ulm (Table 4). L. pneumophila sg 1 strains were isolated from 7 cooling towers. However strains of the mAb subtype Knoxville (mAb 3-1 were found in only one of them (CT 1A) making this the most likely source of the infection. Genotyping of these isolates revealed ST62, the epidemic strain. This cooling tower showed the highest degree of contamination, with a maximum of 92 500 cfu (colony forming units) per 100ml. Additionally, we identified other strains of L. pneumophila and L. rubrilucens in this cooling tower (Table 4), the epidemic strain, however, represented the majority (19 colonies out of 32 = 60%) of the Legionella population in this cooling tower. A second cooling tower (CT 1B) of the same design on the same building was contaminated only with the L. pneumophila sg 8 strain and L. rubrilucens strain. Altogether we serotyped 96 colonies from CT1B but were unable to detect the epidemic strain. However, the sg 8strains from both cooling towers (1A and 1B) had the same unique ST 1373 arguing for a common source of the contamination of both CT.
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The contamination of the other cooling towers was substantially lower (Table 4). Many of the environmental isolates were unique or only rarely reported in the European data base including a putative new species (Table 4).
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4. Discussion
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This paper presents the results of the microbiological investigation from the majority of patients hospitalized due to LD during an outbreak in the German twin cities of Ulm/NeuUlm. In general, only less than 10% of the reported cases of LD occur in outbreaks or clusters (Mercante and Winchell, 2015). However, each outbreak of LD must be regarded as a serious threat for public health since LD is a potentially life threatening disease with case fatality rates of up to 15% (Mercante and Winchell, 2015).We can show that the application of a broad spectrum of microbiological methods including rapid detection of antigenuria, monoclonal typing directly in urine specimens, PCR for Legionella including direct SBT and culture of respiratory specimens can quickly detect the epidemic strain. Subsequently, the collection of environmental specimens and the comparison of clinical and environmental strains or clinical specimens by using modern molecular typing seems mandatory for an efficient outbreak management (Mercante and Winchell, 2015) .
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The diagnosis of LD always requires laboratory confirmation since the clinical picture of LD is not specific (Torres et al., 2014). In this outbreak urinary antigen testing by using the EIA was positive in all cases. However 30% of cases would have been missed if the diagnosis had been based only on the reading of the rapid BinaxNow at 15 minutes as recommended by the manufacturer. Extension of the incubation time to 60 minutes increased the detection rate to 82%. Using the EIA we had a relatively low detection rate of 74% in nonconcentrated urines as well, but a detection rate of 100% when the urine specimens were concentrated. Thus, extension of the reading time or concentration of the urinary samples might be necessary for an optimized outcome. Monoclonal subtyping directly in highly positive urine samples confirmed that five patients were infected by a subtype Knoxville strain, the monoclonal subtype of the epidemic strain (Table 2). As shown here this is very useful in epidemiological investigations since MAb 3-1 positive strains cause the majority of community acquired infections but are seldom isolated from epidemiologically unrelated water sources (Harrison et al., 2009;Kozak-Muiznieks et al., 2014).
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The sensitivity of urinary antigen detection varied depending on the test used even for L. pneumophila sg 1 infection. In general, it was shown that infections caused by mAb 3-1 positive strains were detected with a high sensitivity (Helbig et al., 2003). Furthermore, a strict correlation between the severity of illness and the positivity rate has been reported (Blazquez et al., 2005;von Baum H. et al., 2008;Yzerman et al., 2002). Persons with only mild symptoms might not have sought medical care and thus were not identified during this outbreak. However, general practitioners and hospitals in the region were immediately informed about the outbreak and reminded of the German Guideline on community-acquired pneumonia recommending Legionella diagnostics by using urinary antigen detection in severe cases (Höffken et al., 2010). It must be emphasized that in all cases of severe pneumonia an empirical targeted therapy must be initiated on clinical grounds irrespective of the results of diagnostic tests (Torres et al., 2014) . Detection of Legionella DNA in clinical samples is the second method applied here as well as in other epidemiological investigations. The reported sensitivity is rather high (Mentasti et al., 2012;Murdoch et al., 2013) although we found only a detection rate of 60%. We do not know if this was due to the well-known limited purulence of sputa from patients with Legionnaires disease however we were able to detect the organism by PCR in one case where urine samples were not available for antigen detection. Beside the fact that DNA detection is quick this method has a broader spectrum of detection. Depending from the gene target selected all 7
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sgs of L. pneumophila, several or all Legionella species could be detected (Benitez and Winchell, 2013;Mentasti et al., 2012). Furthermore, direct typing from clinical specimens could confirm the genotype of the epidemic strain thus allowing a culture–independent confirmation of a suspected water supply system as the source of the infection (Mentasti et al., 2012). This approach was successfully used in this outbreak investigation. From 7 culture-negative patients the partial ST could be determined, for further 2 patients this was not successful (Table 3). Thus, it confirms that in addition to eight culture-confirmed cases additional patients were infected with the epidemic strain.
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In eight patients with LD L. pneumophila sg 1 could be cultivated from respiratory samples. The sensitivity of the culture method in our patients was 8/31 (26%) equivalent to a relatively low sensitivity. If compared to reports from other outbreak investigations, where culturing of Legionella ssp. was successful in 3/30 (Hugosson et al., 2007), 6/106 (Blazquez et al., 2005), 3/21(Brown et al., 1999) 6/21 (Coetzee et al., 2012) and 16/179 (Bennett et al., 2013) samples, respectively, our results seem promising. In general, clinicians should always be encouraged to obtain respiratory specimens for PCR and/or culture in addition to urine samples for antigen testing (Murdoch et al., 2013).
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Since we did not apply all diagnostic methods for all patients the exact sensitivity of the different tests cannot be finally estimated. PCR and urinary antigen detection are quick methods in establishing the diagnosis in patients suspected of LD which is the first and crucial step in detecting a possible outbreak.
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In this outbreak, as in previous instances the availability of cultures from both clinical and environmental isolates and their comparison using molecular typing techniques permitted a definite identification of the source of the outbreak (Bennett et al., 2013;Coetzee et al., 2012;Garcia-Fulgueiras et al., 2003;Levesque et al., 2014;McCormick et al., 2012;Nygard et al., 2008;Shivaji et al., 2014;Walser et al., 2014). For the environmental investigation of CTs in the area of interest water samples were processed applying the two step molecular strain typing approach, first line monoclonal antibody (mAb) subtyping and second line sequence based typing (SBT) (Helbig et al., 2002) (Table 4). The hypothesis that a contaminated CT was responsible for the outbreak was first suspected after mAb subtyping and later confirmed by SBT results. It seems noteworthy that the occurrence of this specific ST62 is worldwide nearly always associated with disease, defining this ST as one of the highly virulent clones (http://www.hpa-bioinformatics.org.uk/legionella/legionella_sbt/php/sbt_homepage.php). As of 07.10.2015 data from 10174 strains were deposited in the European Study Group on Legionella Infections (ESGLI) database 6587 (65,1%) were of clinical, 3503 (34,1%) of environmental and 84 (0,8%) of unknown origin. Among the ST62 strains 329 were isolated from patients but only 31 from water. Only 9 strains were not related to human infections (Borchardt et al., 2008;Harrison et al., 2009;Reimer et al., 2010;Tijet et al., 2010). It seems noteworthy that a large outbreak of Pontiac fever was caused by a strain with the same ST (Kaufmann et al., 1981). Further large outbreaks caused by ST62 were reported from the Dutch flower show (Bosch et al., 2015) and in Quebec (Canada) (Levesque et al., 2014).
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Recently, we applied whole genome sequencing of the epidemic strain. This study revealed the presence of an Lvh type IV secretion system containing all components of a functional CRISPR-Cas system that has been proposed to encode a functional anti alien –DNA system and that may be transferred horizontally to other L. pneumophila strains. CRISPR–Cas typing of the clinical and environmental strains further confirmed CT 1A as the source of the infection (Lück et al., 2015). Reporting a cluster of cases triggers a series of epidemiological 8
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and environmental investigations, which, theoretically, can limit the number of cases and allow earlier identification of the source of the infections. Our multidisciplinary microbiological, environmental and epidemiological investigation demonstrated that the source of this outbreak was in all likelihood a cooling tower in the city center of Ulm contaminated with a highly virulent L. pneumophila Sg 1, mAb subtype Knoxville, ST62 strain (Jepras et al., 1985;Lück et al., 2015).
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289 Acknowledgements:
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The authors acknowledge the contribution of all those individuals involved in the outbreak – local, regional and national Health Agency staff, the Local Authority, Environmental Health Officers, hospital staff and microbiologists – for their dedication and professionalism in the face of such a large outbreak. We are grateful to Monika Nolle-Volz, Sonja Rothenberger, Beathe Wirths, Carola Maier, Ulrike Simnacher (Ulm), Kerstin Lück, Susann Menzel and Ines Wolf Dresden) for expert technical assistance. Own studies on molecular typing were supported by the Robert-Koch-Institute (German Federal Ministry of Health) grant 1369-351.
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Table 1
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Clinical microbiological results from 51 legionnaires disease patients from the outbreak Patients tested (n)
Positive in % 70
ICA Urinary antigen, reading 50 after 60 min
82
EIA Urinary antigen nonconcentrated urine
50
74
EIA Urinary antigen concentrated urine
50
100
Culture respiratory samples
31
PCR respiratory samples
25
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ICA Urinary antigen, reading 50 after 15 min
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300
60
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Microbiological Method
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Table 2
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Results from direct monoclonal subtyping in concentrated urine samples
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8/5
3/1
3
8/4
10/6
20/1
Philadelphia (ATCC 33152)
+
+
o
+
o
Benidorm (ATCC 43108)
+
+
o
o
o
Knoxville (ATCC 33153)
+
+
+
o
o
OLDA (ATCC 43109)
+
o
o
+
o
Bellingham (ATCC 43111)
+
o
o
o
L10/23 (epidemic strain)
+
+
+
Urine samples A (n=5)2
+
+
Urine samples B (n=3) 3
o
o
12/2
o
+
+
o
o
o
o
o
o
+
+
+
+
+
+
o
o
o
o
o
+
o
o
o
o
o
o
o
o
o
o
o
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26/1
Positive/ negative: +/o : Cut-off values for the optical density were calculated as mean plus three-fold standard deviation for five-fold concentrated negative control urine samples (Helbig et al., 2012)
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Reactivity with monoclonal antibodies mAbs (+/o)1
ip t
L. pneumophila sg 1 type strains /urine samples from patients
2
OD values of the nonconcentrated urine :>0,9
3
OD values of the nonconcentrated urine :<0,9
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Table 3
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Results from direct nested SBT obtained with 5 genes in 18 respiratory samples from 15 patients with Legionnaires’ disease in the outbreak
Epidemic strain
nSBT typing directly from respiratory sample SBT allele sequence designation
ip t
Results from PCR in respiratory samples
flaA
pilE
8
cr
Results from Culture
proA
neuA
15
1
6
10
15
1
6
0
15
1
6
0
10
15
0
6
8
10
15
1
6
10
us
Number of respiratory samples
mip
positive
positive
8
1
positive
positive
8
3
positive
positive
1
positive
positive
4
negative
positive
8
10
15
1
6
1
negative
positive
0
0
15
0
6
2
negative
0
0
0
0
0
1 311 312 313 314
M
d positive
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1
an
4
negative
positive
0
10
0
0
0
negative
positive
0
10
15
0
6
Explanations: 0 no amplification/ readable sequences, loci asd and momp were not tested because not sufficient amount of DNA
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Table 4
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Characterization of L. pneumophila strains isolated from humans and cooling towers during the outbreak of Ulm, December 2009 /January 2010
CT = Cooling tower
Maximum colony forming units cfu/100ml
Sequence type ST flaA
pilE
asd
mip
momp
proA
neuA
Occurrence of this strain in other regions according to the EWGLI/ESGLI Database (as by 7.10.2015 Related to clinical cases, not isolated from environmental sources not related to disease
(N = colonies tested)
(N = colonies tested)
1 Knoxville1
62
8
10
3
15
18
1
6
ce
pt
8 patients from the outbreak
Serogroup / mAb subtype
ed
Source
M
315
92500
1 Knoxville1 (n=19)
62 (n=4)
8
10
3
15
18
1
6
1. CT 1A
92500
1 OLDA2 (n=1)
1 (n=1)
1
4
3
1
1
1
1
Commonly found among clinical and environmental isolates worldwide
92500
10 (n=1)
908 (n=1)
1
4
3
5
1
1
6
Unique
1. CT 1A
92500
8 (n=3)
1373 (n=2)
5
2
3
10
6
25
203
Unique
1. CT 1A
92500
3 (n=8)
984 (n=1)
12
29
2
20
50
20
15
Unique
1. CT 1A
92500
L. rubrilucens3 (n=1)
NA5
2. CT 1B
70 000
8 (n=94)
1373 (n=2)
5
2
3
10
6
25
203
Unique
2. CT 1B
70 000
L. rubrilucens3 (n=2)
NA5
1. CT 1A
Ac
1. CT 1A
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cr us 300
1 Bellingham2 (n=1)
4. CT
3000
4 (n=1)
4. CT
3000
5. CT
1
6. CT
1
7. CT
130
an
3. CT
2
6
17
6
13
11
11
Isolated from clinical and environmental sources in Germany and the Netherlands
681 (n=1)
11
14
16
25
7
13
6
Isolated once in France
6 (n=1)
956 (n=1)
6
10
3
3
9
1
9
Isolated once in Poland
1 OLDA2 (n=1)
1 (n=1)
1
4
3
1
1
1
1
Commonly found among clinical and environmental isolates worldwide
pt
ed
M
334 (n=1)
NA5
1 Bellingham2 (n=1)
334 (n=1)
2
6
17
6
13
11
11
Isolated from clinical and environmental sources in Germany and the Netherlands
15 000
1 OLDA2 (n=2)
595 (n=1)
2
14
16
16
15
13
2
Isolated once in Japan
8. CT
6
4 (n=1)
1369 (n=1)
8
14
16
25
7
13
207
Unique
9. CT
5
1 OLDA2 (n=1)
1 (n=1)
1
4
3
1
1
1
1
Commonly found among clinical and environmental isolates worldwide
10. CT
1
1 OLDA2 (n=1)
104 (n=1)
3
10
1
1
14
9
1
Isolated 9x from clinical and environmental Sources in the UK
Ac
8. CT
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Putative new Legionella species (n=1)3, 4
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mAb 3-1 positive
318
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mAb 3-1 negative
319
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based on mip (macrophage infectivity potentiator) sequence (Ratcliff et al., 1998)
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mip sequence Accession number HE613854; 80% related to L. brunensis , Acc Nr U92227; 99% identity to Legionella brunensis like strain Tver-868.1 isolated in Russia, NCBI Acc Nr. FJ357014.1;
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5
ed
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317
pt
NA, non applicable for non-pneumophila strains
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Reference List
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Benitez, A.J. and Winchell, J.M., 2013. Clinical Application of a Multiplex Real-Time PCR Assay for Simultaneous Detection of Legionella Species, Legionella pneumophila, and Legionella pneumophila Serogroup 1. J. Clin. Microbiol. 51, 348-351.
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Bennett, E., Ashton, M., Calvert, N., Chaloner, J., Cheesbrough, J., Egan, J., Farrell, I., Hall, I., Harrison, T.G., Naik, F.C., Partridge, S., Syed, Q., and Gent, R.N., 2013. Barrow-in-Furness: a large community legionellosis outbreak in the UK. Epidemiol. Infect. 1-15.
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Blazquez, R.M., Espinosa, F.J., Martnez-Toldos, C.M., Alemany, L., Garca-Orenes, M.C., and Segovia, M., 2005. Sensitivity of urinary antigen test in relation to clinical severity in a large outbreak of Legionella pneumonia in Spain. Eur J Clin Microbiol Infect Dis 24, 488-491.
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Borchardt, J., Helbig, J.H., and Lück, P.C., 2008. Occurrence and distribution of sequence types among Legionella pneumophila strains isolated from patients in Germany: common features and differences to other regions of the world. Eur J Clin Microbiol Infect Dis 27, 29-36.
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Bosch, T., Euser, S.M., Landman, F., Bruin, J.P., Ijzerman, E.P., Den Boer, J.W., and Schouls, L.M., 2015. Whole-Genome Mapping as a Novel High-Resolution Typing Tool for Legionella pneumophila. J Clin Microbiol 53, 3234-3238.
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Brown, C.M., Nuorti, P.J., Breiman, R.F., Hathcock, A.L., Fields, B.S., Lipman, H.B., Llewellyn, G.C., Hofmann, J., and Cetron, M., 1999. A community outbreak of Legionnaires' disease linked to hospital cooling towers: an epidemiological method to calculate dose of exposure. Int J Epidemiol 28, 353-359.
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Coetzee, N., Duggal, H., Hawker, J., Ibbotson, S., Harrison, T.G., Phin, N., Laza-Stanca, V., Johnston, R., Iqbal, Z., Rehman, Y., Knapper, E., Robinson, S., and Aigbogun, N., 2012. An outbreak of Legionnaires' disease associated with a display spa pool in retail premises, Stoke-onTrent, United Kingdom, July 2012. Eurosurveillance 17, 6-8.
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Freudenmann, M., Kurz, S., von, B.H., Reick, D., Schreff, A.M., Essig, A., Lück, C., Gonser, T., Brockmann, S.O., Härter, G., Eberhardt, B., Embacher, A., and Höller, C., 2011. [Interdisciplinary management of a large Legionella outbreak in Germany]. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 54, 1161-1169.
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Garcia-Fulgueiras, A., Navarro, C., Fenoll, D., Garica, J., Gonzles-Diego, P., Jimnez-Buuelas, T., Rodriguez, M., Lopez, R., Pacheco, F., Ruiz, J., Segovia, M., Balandrn, B., and Pelaz, C., 2003. Legionnaires disease outbreak in Murcia, Spain. Emerg Infect Dis 9, 915-921.
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Harrison, T.G., Afshar, B., Doshi, N., Fry, N.K., and Lee, J.V., 2009. Distribution of Legionella pneumophila serogroups, monoclonal antibody subgroups and DNA sequence types in recent
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clinical and environmental isolates from England and Wales (2000-2008). Eur J Clin Microbiol Infect Dis 28, 781-791.
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Helbig, J.H., Bernander, S., Castellani, P.M., Etienne, J., Gaia, V., Lauwers, S., Lindsay, D., Lück, P.C., Marques, T., Mentula, S., Peeters, M.F., Pelaz, C., Struelens, M., Uldum, S.A., Wewalka, G., and Harrison, T.G., 2002. Pan-European study on culture-proven Legionnaires disease: distribution of Legionella pneumophila serogroups and monoclonal subgroups. Eur J Clin Microbiol Infect Dis 21, 710-716.
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Helbig, J.H., Jacobs, E., and Lück, C., 2012. Legionella pneumophila urinary antigen subtyping using monoclonal antibodies as a tool for epidemiological investigations. Eur J Clin Microbiol Infect Dis 31, 1673-1677.
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Helbig, J.H., Uldum, S.A., Bernander, S., Lück, P.C., Wewalka, G., Abraham, B., Gaia, V., and Harrison, T.G., 2003. Clinical utility of urinary antigen detection for diagnosis of communityacquired, travel-associated, and nosocomial legionnaires disease. J Clin Microbiol 41, 838-840.
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Höffken, G., Lorenz, J., Kern, W., Welte, T., Bauer, T., Dalhoff, K., Dietrich, E., Ewig, S., Gastmeier, P., Grabein, B., Halle, E., Kolditz, M., Marre, R., and Sitter, H., 2010. Guidelines of the Paul-Ehrlich-Society of Chemotherapy, the German Respiratory Diseases Society, the German Infectious Diseases Society and of the Competence Network CAPNETZ for the Management of Lower Respiratory Tract Infections and Community-acquired Pneumonia. Pneumologie 64, 149-154.
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Hugosson, A., Hjorth, M., Bernander, S., Claesson, B.E., Johansson, A., Larsson, H., Nolskog, P., Pap, J., Svensson, N., and Ulleryd, P., 2007. A community outbreak of Legionnaires disease from an industrial cooling tower: assessment of clinical features and diagnostic procedures. Scand J Infect Dis 39, 217-224.
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ISO 11731, 1998. Water Quality - Detection and enumeration of Legionella. International Standard.
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Jepras, R.I., Fitzgeorge, R.B., and Baskerville, A., 1985. A comparison of virulence of two strains of Legionella pneumophila based on experimental aerosol infection of guinea-pigs. J Hyg (Lond) 95, 29-38.
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Kaufmann, A.F., McDade, J.E., Patton, C.M., Bennett, J.V., Skaliy, P., Feeley, J.C., Anderson, D.C., Potter, M.E., Newhouse, V.F., Gregg, M.B., and Brachman, P.S., 1981. Pontiac fever: isolation of the etiologic agent (Legionella pneumophila) and demonstration of its mode of transmission. Am. J. Epidemiol. 114, 337-347.
390 391
Kozak-Muiznieks, N.A., Lucas, C.E., Brown, E., Pondo, T., Taylor, T.H., Jr., Frace, M., Miskowski, D., and Winchell, J.M., 2014. Prevalence of sequence types among clinical and
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environmental isolates of Legionella pneumophila serogroup 1 in the United States from 1982 to 2012. J. Clin. Microbiol. 52, 201-211.
394 395 396 397
Levesque, S., Plante, P.L., Mendis, N., Cantin, P., Marchand, G., Charest, H., Raymond, F., Huot, C., Goupil-Sormany, I., Desbiens, F., Faucher, S.P., Corbeil, J., and Tremblay, C., 2014. Genomic characterization of a large outbreak of Legionella pneumophila serogroup 1 strains in Quebec City, 2012. PLoS. One. 9, e103852.
398 399 400
Lück, C., Brzuszkiewicz, E., Rydzewski, K., Koshkolda, T., Sarnow, K., Essig, A., and Heuner, K., 2015. Subtyping of the Legionella pneumophila "Ulm" outbreak strain using the CRISPRCas system. Int. J Med. Microbiol 305, 828-837.
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McCormick, D., Thorn, S., Milne, D., Evans, C., Stevenson, J., Llano, M., and Donaghy, M., 2012. Public health response to an outbreak of Legionnaires' disease in Edinburgh, United Kingdom, June 2012. Eurosurveillance 17, 6-9.
404 405 406 407 408
Mentasti, M., Fry, N., Afshar, B., Palepou-Foxley, C., Naik, F., and Harrison, T., 2012. Application of Legionella pneumophila-specific quantitative real-time PCR combined with direct amplification and sequence-based typing in the diagnosis and epidemiological investigation of Legionnaires' disease. European Journal of Clinical Microbiology & Infectious Diseases 31, 2017-2028.
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Mercante, J.W. and Winchell, J.M., 2015. Current and emerging Legionella diagnostics for laboratory and outbreak investigations. Clin Microbiol Rev. 28, 95-133.
411 412 413 414
Murdoch, D.R., Podmore, R.G., Anderson, T.P., Barratt, K., Maze, M.J., French, K.E., Young, S.A., Chambers, S.T., and Werno, A.M., 2013. Impact of routine systematic polymerase chain reaction testing on case finding for Legionnaires' disease: a pre-post comparison study. Clin. Infect. Dis. 57, 1275-1281.
415 416 417 418 419
Nygard, K., Werner-Johansen, O., Ronsen, S., Caugant, D.A., Simonsen, O., Kanestrom, A., Ask, E., Ringstad, J., Odegard, R., Jensen, T., Krogh, T., Hoiby, E.A., Ragnhildstveit, E., Aaberge, I.S., and Aavitsland, P., 2008. An Outbreak of Legionnaires Disease Caused by LongDistance Spread from an Industrial Air Scrubber in Sarpsborg, Norway. Clinical Infectious Diseases 46, 61-69.
420 421 422
Ratcliff, R.M., Lanser, J.A., Manning, P.A., and Heuzenroeder, M.W., 1998. Sequence-based classification scheme for the genus Legionella targeting the mip gene. J Clin Microbiol 36, 15601567.
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Reimer, A.R., Au, S., Schindle, S., and Bernard, K.A., 2010. Legionella pneumophila monoclonal antibody subgroups and DNA sequence types isolated in Canada between 1981 and 2009: Laboratory Component of National Surveillance. Eur J Clin Microbiol Infect Dis 29, 191205.
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Rizzardi, K., Winiecka-Krusnell, J., Ramliden, M., Alm, E., Andersson, S., and Byfors, S., 2015. Legionella norrlandica sp. nov., isolated from the biopurification systems of wood processing plants. Int. J Syst. Evol. Microbiol 65, 598-603.
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Robert-Koch-Institut, 2015. Infektionsepidemiologisches Jahrbuch meldepflichtiger Krankheiten für 2014.
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Shivaji, T., Sousa, P.C., San-Bento, A., Oliveira Serra, L.A., Valente, J., Machado, J., Marques, T., Carvalho, L., Nogueira, P.J., Nunes, B., and Vasconcelos, P., 2014. A large community outbreak of Legionnaires disease in Vila Franca de Xira, Portugal, October to November 2014. Euro Surveill 19, 20991.
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Stout, J.E., Rihs, J.D., and Yu, V.L., Legionella. Manual of Clinical Microbiology, ASM Press, 2003, pp. 809-823.
438 439 440
Tijet, N., Tang, P., Romilowych, M., Duncan, C., Ng, V., Fisman, D.N., Jamieson, F., Low, D.E., and Guyard, C., 2010. New endemic Legionella pneumophila serogroup I clones, Ontario, Canada. Emerg Infect Dis 16, 447-454.
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Torres, A., Blasi, F., Peetermans, W.E., Viegi, G., and Welte, T., 2014. The aetiology and antibiotic management of community-acquired pneumonia in adults in Europe: a literature review. Eur. J. Clin. Microbiol. Infect. Dis. 33, 1065-1079.
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von Baum H., Ewig, S., Marre, R., Suttorp, N., Gonschior, S., Welte, T., and Lück, C., 2008. Community-acquired Legionella pneumonia: new insights from the German competence network for community acquired pneumonia. Clin Infect Dis 46, 1356-1364.
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von Baum H., Härter, G., Essig, A., Lück, C., Gonser, T., Embacher, A., and Brockmann, S., 2010. Preliminary report: outbreak of Legionnaires disease in the cities of Ulm and Neu-Ulm in Germany, December 2009 - January 2010. Euro Surveill 15, 19472.
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Walser, S.M., Gerstner, D.G., Brenner, B., Holler, C., Liebl, B., and Herr, C.E., 2014. Assessing the environmental health relevance of cooling towers--a systematic review of legionellosis outbreaks. Int. J. Hyg. Environ. Health 217, 145-154.
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Wellinghausen, N., Frost, C., and Marre, R., 2001. Detection of legionellae in hospital water samples by quantitative real-time LightCycler PCR. Appl Environ Microbiol 67, 3985-3993.
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Yzerman, E.P., Den, B., Lettinga, K.D., Schellekens, J., Dankert, J., and Peeters, M., 2002. Sensitivity of three urinary antigen tests associated with clinical severity in a large outbreak of Legionnaires' disease in The Netherlands. J Clin Microbiol 40, 3232-3236.
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S1438-4221(16)30001-7 http://dx.doi.org/doi:10.1016/j.ijmm.2016.01.001 IJMM 51021
To appear in: Received date: Revised date: Accepted date:
10-12-2015 22-1-2016 25-1-2016
Please cite this article as: Essig, A., von Baum, H., Gonser, T., Haerter, G., L¨uck, C.,Microbiological Diagnosis and Molecular Typing of Legionella strains during an Outbreak of Legionellosis in Southern Germany, International Journal of Medical Microbiology (2016), http://dx.doi.org/10.1016/j.ijmm.2016.01.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Microbiological Diagnosis and Molecular Typing of Legionella strains during an Outbreak of Legionellosis in Southern Germany
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Andreas Essiga, Heike von Bauma, Theodor Gonserb, Georg Haerterc, Christian Lückd*
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Institute of Medical Microbiology and Hygiene, Ulm University Hospital, Germany
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Public Health Authority Ulm, Germany
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Department of Internal Medicine III, Section of Infectious Diseases, Ulm, University Hospital. Now: MVZ Endokrinologikum Ulm, Center for Endocrinology and Infectious Diseases, Ulm, Germany d
Institute of Medical Microbiology and Hygiene, German Consiliary laboratory for Legionella, Dresden University of Technology, Germany
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Key words: Legionnaire’s Disease, outbreak, cooling tower, Legionella pneumophila, molecular typing, pneumonia
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*Corresponding author: Christian Lück, Institute of Medical Microbiology and Hygiene, National Consiliary Laboratory for Legionella, Medical Faculty "Carl Gustav Carus", University of Technology Dresden, Fetscherstrasse 74, D-01307 Dresden
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Email: [email protected]
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Phone: 0049-351-458 16580 Fax: 0049-351-458 6311
International Journal of Medical Microbiology 2015, Category: Bacteriology
Presented in part at the annual meeting of the ICAAC, (abstract 187); Annual Meeting of the European Legionella Surveillance network ELDSNet, Copenhagen September 2010.
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26 Abstract
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An explosive outbreak of Legionnaires’ disease with 64 reported cases occurred in Ulm/ NeuUlm in the South of Germany in December 2009/January 2010 caused by Legionella (L.) pneumophila serogroup 1, monoclonal (mAb) subtype Knoxville, sequence type (ST) 62. Here we present the clinical microbiological results from 51 patients who were diagnosed at the University hospital of Ulm, the results of the environmental investigations and of molecular typing of patients and environmental strains. All 50 patients from whom urine specimens were available were positive for L. pneumophila antigen when an enzyme-linked immunosorbent assay (EIA) was used following concentration of those urine samples that tested initially negative. The sensitivity of the BinaxNow rapid immunographic assay (ICA), after 15 minutes reading and after 60 min reading were 70% and 84%, respectively. Direct typing confirmed the monoclonal subtype Knoxville in 5 out of 8 concentrated urine samples. Real time PCR testing of respiratory tract specimens for L. pneumophila was positive in 15 out of 25 (60%) patients. Direct nested sequence based typing (nSBT) in some of these samples allowed partial confirmation of ST62. L. pneumophila serogroup 1, monoclonal subtype Knoxville ST62, defined as the epidemic strain was isolated from 8 out of 31 outbreak patients (26%) and from one cooling tower confirming it as the most likely source of the outbreak. While rapid detection of Legionella antigenuria was crucial for the recognition and management of the outbreak, culture and molecular typing of the strains from patients and environmental specimens was the clue for the rapid identification of the source of infection.
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1. Introduction
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Legionellae are ubiquitous gram-negative bacteria, which occupy natural and manmade aquatic environments. Currently the genus Legionella comprises 58 species (Rizzardi et al., 2015) and more than 70 serogroups with Legionella ( L.) pneumophila serogroup (sg) 1 causing the majority of human infections (Helbig et al., 2002).
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Legionnaires’ disease (LD) occurs mostly as a sporadic pneumonia caused by inhalation of Legionella-containing aerosols generated from warm water supplies, cooling towers, or whirl pool spas. Approximately 10% of cases occur in outbreaks or clusters (Mercante and Winchell, 2015) . Legionella outbreaks may present as serious threats of public health and management of outbreaks can be extremely difficult, especially if the source of infection is unknown. In general, Legionella spp. are responsible for 2-15% of community acquired pneumonia (CAP) (Torres et al., 2014). 860 cases have been reported to the German Health Authorities in 2014 (Robert-Koch-Institut, 2015). Thus, significant underdiagnosing and/ or underreporting must be considered.
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Here we describe the results of the microbiological and epidemiological investigation during an outbreak of LD with 64 reported cases between December 2009 and January 2010 in Ulm and Neu-Ulm, twin cities with a total of 175 000 inhabitants in Southern Germany. A cooling tower in the city center of Ulm was finally identified as the most likely source of the outbreak (von Baum H. et al., 2010). The majority of patients was diagnosed and treated in our hospital thus offering a unique opportunity to study the microbiological and epidemiological methods applied.
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2. Materials and methods
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2.1. Case Detection and criteria for Legionella pneumonia
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During the outbreak investigation, the case definitions of the European Centre for Disease Prevention and Control (European Legionnaires’ Disease Surveillance Network (ELDSNet) were applied (von Baum H. et al., 2010). A case of Legionnaire’s disease was defined as a patient who had (1) confirmed pneumonia, (2) laboratory evidence of L. pneumophila sg 1 infection (positive urinary antigen test, isolation of Legionella from respiratory specimens), (3) onset of disease between December 22th 2009 and January 7th 2010 and (4) lived in or visited the cities of Ulm/Neu-Ulm in the 2 weeks preceding the development of CAP. 2.2. Detection of Legionella spp. in clinical specimens
Detection of urinary antigen. For the qualitative detection of L. pneumophila soluble antigen in urine, specimens were tested using a rapid immunochromatographic card assay (ICA), BinaxNow (Allere, Düsseldorf, Germany) as well as a microtiter based enzyme-linked immunosorbent assay (EIA) (Biotest Legionella Urine Antigen EIA; BioRad, Munich Germany) according to the manufacturer’s instructions. In case of negative results the incubation time of the Binax NOW assay was prolonged to 60 min. Furthermore, the urines were concentrated 25-fold using an ultrafiltration device (Minicon concentrator, 15 kDa exclusion size, Millipore) and retested with the Biotest EIA but not with the Binax Now assay. In eight urine samples a direct monoclonal typing approach was applied. From the other samples the amount left was not sufficient for this analysis. Briefly, the urine specimens were 3
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first concentrated five-fold by using the Vivaspin 6 columns (Vivascience, Fisher Scientific, Schwerte, Germany, exclusion size 5kDa) and added to wells of Binax Legionella urinary antigen EIA carrying immobilised anti-Legionella antibodies (Allere Düsseldorf, Germany). After washing, the bound antigens were detected by monoclonal antibodies (mAbs) following incubation with horseradish peroxidase labelled anti-mouse IgG (Sigma, Deisenhofen, Germany) and substrate reaction (Helbig et al., 2012).
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Detection of Legionella DNA from respiratory samples. Respiratory samples including sputum samples, tracheal and bronchial aspirates were processed for detection of Legionella specific DNA by nucleic acid amplification. DNA was isolated from respiratory samples using the Magna pure system (Roche, Germany) and a L. pneumophila specific real-time LightCycler PCR was run targeting the macrophage infectivity potentiator (mip) gene as described previously (Wellinghausen et al., 2001). To define the sequence type directly from clinical samples nested SBT was performed on PCR positive samples according to the ESGLI protocol (Mentasti et al., 2012) .
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Culture of Legionella species from clinical samples. Respiratory samples from patients with antigenuria were cultured on non-selective buffered charcoal- yeast extract (BCYE) agar and a selective agar containing cefamandol, polymyxin B and anisomycin (BMPA (Stout et al., 2003) at 36°C in humified atmosphere supplemented with 5% CO2 following heat treatment at 50°C for 30 minutes. Culture plates were inspected daily up to day 10. Colonies suspected for Legionella were cultured on non-selective buffered charcoal yeast extract (BCYE) agar and Columbia blood agar. Isolated strains that did only grow on BCYE agar but not on blood agar were initially serotyped by using a Latex agglutination test (Oxoid, Wesel, Germany) and confirmed by using a panel of mAb as described elsewhere (Helbig et al., 2002). Genotyping was performed by using the European sequence based typing (SBT) schema (http://www.hpabioinformatics.org.uk/legionella/legionella_sbt/php/sbt_homepage.php).
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2.3 Environmental Investigation
The epidemiological data suggested a wet cooling tower (CT) as the source of the outbreak (Freudenmann et al., 2011;von Baum H. et al., 2010). Therefore the search for possible sources of the outbreak focussed on 30 wet cooling towers identified in the area of interest. CTs were inspected, and 1 to 4 water samples were collected and processed according to ISO 11731/1998 (ISO 11731, 1998). Samples were collected from the cooling tower ponds in all CT, and if accessible water samples were also taken from 1 to 3 additional sites like water incomes, the water system of the building and other sites. Environmental L. pneumophila isolates were typed by mAb typing and SBT as described for the clinical isolates. Nonpneumophila strains were typed by mip gene sequencing (Ratcliff et al., 1998).
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3. Results
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3.1. Case detection
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Altogether, 64 epidemiologically linked cases (66% male, 34% female) met the European case definition of a confirmed case and were reported to the local public health authorities during this outbreak. The median age of the patients was 68 (27-96) years. Sixty patients had to be hospitalized and 5 patients died from infection (four of whom were older than 80 years)(Freudenmann et al., 2011). Fifty-one patients (80%) were diagnosed at the University Hospital of Ulm. Prominent clinical and laboratory findings included radiological infiltrates (94%), fever (83%), non-productive cough (69%), central nervous system symptoms (58%), muscle pain or pain in the limbs (41%), abdominal pain/diarrhea (28%), CRP>100 mg/l (92%), LDH elevation (74%), Troponin I elevation (71%), hyponatremia (60%) and leukocytosis (50%). The interdisciplinary management of the outbreak has been published previously (Freudenmann et al., 2011).
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3.2 Detection of Legionella in clinical specimens
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Detection and typing of urinary antigen. Urine samples for detection of soluble L. pneumophila antigen were available from 50 of 51 patients diagnosed and treated at the University hospital of Ulm. Specimens were investigated by a rapid BinaxNow card assay and by the Biotest EIA. Using the BinaxNow visible bands were detected in 70% of specimens at 15 min. Prolongation of the reading time to 60 minutes increased the detection rate to 82% indicating that 18% of specimens remained negative in the BinaxNow assay even if the incubation period was extended to 60 minutes. Using nonconcentrated urine samples the Biotest EIA yielded positive results in 74% of cases. 25fold concentration of urine samples yielded another 13 positive tests resulting in a 100% detection rate. Seven out of 9 specimens that tested negative with the BinaxNow were also negative by EIA using nonconcentrated urines but became positive by EIA when urine samples were concentrated (Table 1). In 5/8 fivefold concentrated urine samples the direct monoclonal subtyping identified the monoclonal subtype Knoxville of the causative strain. In three concentrated urines no reactivity with any of the mAbs could be achieved (Table 2). From other patients the amounts of urine were not sufficient for this investigation.
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Culture of Legionella species from clinical samples. For isolation procedures respiratory tract specimens from 31 patients with positive antigenuria and/or positive nucleic acid detection of Legionella pneumophila were available. Eight out of 31 patients were culture positive (26%). Six isolates were cultured from sputum samples, and two isolates were grown from tracheal and bronchial aspirates, respectively. All clinical isolates were typed as Legionella pneumophila serogroup 1, mAb subtype Knoxville, ST62, which was defined as the epidemic strain.
Detection and direct typing of DNA from respiratory specimens. Nucleic acid based detection of L. pneumophila was performed by a real time PCR assay targeting the mip gene. PCR was positive in 15 out of 25 available respiratory tract specimens tested (60%). PCR-positive samples included 13 sputum specimens, 1 tracheal aspirate and 1 bronchial aspirate. All of the culture-positive specimens (8/23) yielded also positive PCR results. DNA sequences of selected alleles in concordance with alleles of ST62 were detected by direct nested SBT in 18 respiratory samples from 15 patients (Table 3). Due to the limited amount of DNA the 5
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alleles flaA, pilE, mip, proA, neuA were chosen because this combination of alleles is unique in ST62.
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3.3. Environmental investigations
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Thirty wet cooling towers were inspected by an expert team of the local public health authorities. Immediately after water samples were collected all cooling towers were shut down, cleaned and disinfected as a prophylactic measure. L. pneumophila was recovered from 9 water samples out of 30 cooling towers in the city of Ulm (Table 4). L. pneumophila sg 1 strains were isolated from 7 cooling towers. However strains of the mAb subtype Knoxville (mAb 3-1 were found in only one of them (CT 1A) making this the most likely source of the infection. Genotyping of these isolates revealed ST62, the epidemic strain. This cooling tower showed the highest degree of contamination, with a maximum of 92 500 cfu (colony forming units) per 100ml. Additionally, we identified other strains of L. pneumophila and L. rubrilucens in this cooling tower (Table 4), the epidemic strain, however, represented the majority (19 colonies out of 32 = 60%) of the Legionella population in this cooling tower. A second cooling tower (CT 1B) of the same design on the same building was contaminated only with the L. pneumophila sg 8 strain and L. rubrilucens strain. Altogether we serotyped 96 colonies from CT1B but were unable to detect the epidemic strain. However, the sg 8strains from both cooling towers (1A and 1B) had the same unique ST 1373 arguing for a common source of the contamination of both CT.
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The contamination of the other cooling towers was substantially lower (Table 4). Many of the environmental isolates were unique or only rarely reported in the European data base including a putative new species (Table 4).
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4. Discussion
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This paper presents the results of the microbiological investigation from the majority of patients hospitalized due to LD during an outbreak in the German twin cities of Ulm/NeuUlm. In general, only less than 10% of the reported cases of LD occur in outbreaks or clusters (Mercante and Winchell, 2015). However, each outbreak of LD must be regarded as a serious threat for public health since LD is a potentially life threatening disease with case fatality rates of up to 15% (Mercante and Winchell, 2015).We can show that the application of a broad spectrum of microbiological methods including rapid detection of antigenuria, monoclonal typing directly in urine specimens, PCR for Legionella including direct SBT and culture of respiratory specimens can quickly detect the epidemic strain. Subsequently, the collection of environmental specimens and the comparison of clinical and environmental strains or clinical specimens by using modern molecular typing seems mandatory for an efficient outbreak management (Mercante and Winchell, 2015) .
204 205 206 207 208 209 210 211 212 213 214 215 216 217
The diagnosis of LD always requires laboratory confirmation since the clinical picture of LD is not specific (Torres et al., 2014). In this outbreak urinary antigen testing by using the EIA was positive in all cases. However 30% of cases would have been missed if the diagnosis had been based only on the reading of the rapid BinaxNow at 15 minutes as recommended by the manufacturer. Extension of the incubation time to 60 minutes increased the detection rate to 82%. Using the EIA we had a relatively low detection rate of 74% in nonconcentrated urines as well, but a detection rate of 100% when the urine specimens were concentrated. Thus, extension of the reading time or concentration of the urinary samples might be necessary for an optimized outcome. Monoclonal subtyping directly in highly positive urine samples confirmed that five patients were infected by a subtype Knoxville strain, the monoclonal subtype of the epidemic strain (Table 2). As shown here this is very useful in epidemiological investigations since MAb 3-1 positive strains cause the majority of community acquired infections but are seldom isolated from epidemiologically unrelated water sources (Harrison et al., 2009;Kozak-Muiznieks et al., 2014).
230 231 232 233 234 235 236
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218 219 220 221 222 223 224 225 226 227 228 229
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191
The sensitivity of urinary antigen detection varied depending on the test used even for L. pneumophila sg 1 infection. In general, it was shown that infections caused by mAb 3-1 positive strains were detected with a high sensitivity (Helbig et al., 2003). Furthermore, a strict correlation between the severity of illness and the positivity rate has been reported (Blazquez et al., 2005;von Baum H. et al., 2008;Yzerman et al., 2002). Persons with only mild symptoms might not have sought medical care and thus were not identified during this outbreak. However, general practitioners and hospitals in the region were immediately informed about the outbreak and reminded of the German Guideline on community-acquired pneumonia recommending Legionella diagnostics by using urinary antigen detection in severe cases (Höffken et al., 2010). It must be emphasized that in all cases of severe pneumonia an empirical targeted therapy must be initiated on clinical grounds irrespective of the results of diagnostic tests (Torres et al., 2014) . Detection of Legionella DNA in clinical samples is the second method applied here as well as in other epidemiological investigations. The reported sensitivity is rather high (Mentasti et al., 2012;Murdoch et al., 2013) although we found only a detection rate of 60%. We do not know if this was due to the well-known limited purulence of sputa from patients with Legionnaires disease however we were able to detect the organism by PCR in one case where urine samples were not available for antigen detection. Beside the fact that DNA detection is quick this method has a broader spectrum of detection. Depending from the gene target selected all 7
Page 7 of 20
sgs of L. pneumophila, several or all Legionella species could be detected (Benitez and Winchell, 2013;Mentasti et al., 2012). Furthermore, direct typing from clinical specimens could confirm the genotype of the epidemic strain thus allowing a culture–independent confirmation of a suspected water supply system as the source of the infection (Mentasti et al., 2012). This approach was successfully used in this outbreak investigation. From 7 culture-negative patients the partial ST could be determined, for further 2 patients this was not successful (Table 3). Thus, it confirms that in addition to eight culture-confirmed cases additional patients were infected with the epidemic strain.
245 246 247 248 249 250 251 252
In eight patients with LD L. pneumophila sg 1 could be cultivated from respiratory samples. The sensitivity of the culture method in our patients was 8/31 (26%) equivalent to a relatively low sensitivity. If compared to reports from other outbreak investigations, where culturing of Legionella ssp. was successful in 3/30 (Hugosson et al., 2007), 6/106 (Blazquez et al., 2005), 3/21(Brown et al., 1999) 6/21 (Coetzee et al., 2012) and 16/179 (Bennett et al., 2013) samples, respectively, our results seem promising. In general, clinicians should always be encouraged to obtain respiratory specimens for PCR and/or culture in addition to urine samples for antigen testing (Murdoch et al., 2013).
253 254 255 256
Since we did not apply all diagnostic methods for all patients the exact sensitivity of the different tests cannot be finally estimated. PCR and urinary antigen detection are quick methods in establishing the diagnosis in patients suspected of LD which is the first and crucial step in detecting a possible outbreak.
257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276
In this outbreak, as in previous instances the availability of cultures from both clinical and environmental isolates and their comparison using molecular typing techniques permitted a definite identification of the source of the outbreak (Bennett et al., 2013;Coetzee et al., 2012;Garcia-Fulgueiras et al., 2003;Levesque et al., 2014;McCormick et al., 2012;Nygard et al., 2008;Shivaji et al., 2014;Walser et al., 2014). For the environmental investigation of CTs in the area of interest water samples were processed applying the two step molecular strain typing approach, first line monoclonal antibody (mAb) subtyping and second line sequence based typing (SBT) (Helbig et al., 2002) (Table 4). The hypothesis that a contaminated CT was responsible for the outbreak was first suspected after mAb subtyping and later confirmed by SBT results. It seems noteworthy that the occurrence of this specific ST62 is worldwide nearly always associated with disease, defining this ST as one of the highly virulent clones (http://www.hpa-bioinformatics.org.uk/legionella/legionella_sbt/php/sbt_homepage.php). As of 07.10.2015 data from 10174 strains were deposited in the European Study Group on Legionella Infections (ESGLI) database 6587 (65,1%) were of clinical, 3503 (34,1%) of environmental and 84 (0,8%) of unknown origin. Among the ST62 strains 329 were isolated from patients but only 31 from water. Only 9 strains were not related to human infections (Borchardt et al., 2008;Harrison et al., 2009;Reimer et al., 2010;Tijet et al., 2010). It seems noteworthy that a large outbreak of Pontiac fever was caused by a strain with the same ST (Kaufmann et al., 1981). Further large outbreaks caused by ST62 were reported from the Dutch flower show (Bosch et al., 2015) and in Quebec (Canada) (Levesque et al., 2014).
277 278 279 280 281 282
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Recently, we applied whole genome sequencing of the epidemic strain. This study revealed the presence of an Lvh type IV secretion system containing all components of a functional CRISPR-Cas system that has been proposed to encode a functional anti alien –DNA system and that may be transferred horizontally to other L. pneumophila strains. CRISPR–Cas typing of the clinical and environmental strains further confirmed CT 1A as the source of the infection (Lück et al., 2015). Reporting a cluster of cases triggers a series of epidemiological 8
Page 8 of 20
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and environmental investigations, which, theoretically, can limit the number of cases and allow earlier identification of the source of the infections. Our multidisciplinary microbiological, environmental and epidemiological investigation demonstrated that the source of this outbreak was in all likelihood a cooling tower in the city center of Ulm contaminated with a highly virulent L. pneumophila Sg 1, mAb subtype Knoxville, ST62 strain (Jepras et al., 1985;Lück et al., 2015).
Ac ce pt e
283 284 285 286 287 288
9
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289 Acknowledgements:
291 292 293 294 295 296 297
The authors acknowledge the contribution of all those individuals involved in the outbreak – local, regional and national Health Agency staff, the Local Authority, Environmental Health Officers, hospital staff and microbiologists – for their dedication and professionalism in the face of such a large outbreak. We are grateful to Monika Nolle-Volz, Sonja Rothenberger, Beathe Wirths, Carola Maier, Ulrike Simnacher (Ulm), Kerstin Lück, Susann Menzel and Ines Wolf Dresden) for expert technical assistance. Own studies on molecular typing were supported by the Robert-Koch-Institute (German Federal Ministry of Health) grant 1369-351.
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10
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298
Table 1
299
Clinical microbiological results from 51 legionnaires disease patients from the outbreak Patients tested (n)
Positive in % 70
ICA Urinary antigen, reading 50 after 60 min
82
EIA Urinary antigen nonconcentrated urine
50
74
EIA Urinary antigen concentrated urine
50
100
Culture respiratory samples
31
PCR respiratory samples
25
us
cr
ICA Urinary antigen, reading 50 after 15 min
an
26
300
60
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301
ip t
Microbiological Method
11
Page 11 of 20
301
Table 2
302
Results from direct monoclonal subtyping in concentrated urine samples
306 307 308
8/5
3/1
3
8/4
10/6
20/1
Philadelphia (ATCC 33152)
+
+
o
+
o
Benidorm (ATCC 43108)
+
+
o
o
o
Knoxville (ATCC 33153)
+
+
+
o
o
OLDA (ATCC 43109)
+
o
o
+
o
Bellingham (ATCC 43111)
+
o
o
o
L10/23 (epidemic strain)
+
+
+
Urine samples A (n=5)2
+
+
Urine samples B (n=3) 3
o
o
12/2
o
+
+
o
o
o
o
o
o
+
+
+
+
+
+
o
o
o
o
o
+
o
o
o
o
o
o
o
o
o
o
o
us
an
M
cr
o
d
1
26/1
Positive/ negative: +/o : Cut-off values for the optical density were calculated as mean plus three-fold standard deviation for five-fold concentrated negative control urine samples (Helbig et al., 2012)
Ac ce pt e
303 304 305
Reactivity with monoclonal antibodies mAbs (+/o)1
ip t
L. pneumophila sg 1 type strains /urine samples from patients
2
OD values of the nonconcentrated urine :>0,9
3
OD values of the nonconcentrated urine :<0,9
12
Page 12 of 20
308
Table 3
309 310
Results from direct nested SBT obtained with 5 genes in 18 respiratory samples from 15 patients with Legionnaires’ disease in the outbreak
Epidemic strain
nSBT typing directly from respiratory sample SBT allele sequence designation
ip t
Results from PCR in respiratory samples
flaA
pilE
8
cr
Results from Culture
proA
neuA
15
1
6
10
15
1
6
0
15
1
6
0
10
15
0
6
8
10
15
1
6
10
us
Number of respiratory samples
mip
positive
positive
8
1
positive
positive
8
3
positive
positive
1
positive
positive
4
negative
positive
8
10
15
1
6
1
negative
positive
0
0
15
0
6
2
negative
0
0
0
0
0
1 311 312 313 314
M
d positive
Ac ce pt e
1
an
4
negative
positive
0
10
0
0
0
negative
positive
0
10
15
0
6
Explanations: 0 no amplification/ readable sequences, loci asd and momp were not tested because not sufficient amount of DNA
13
Page 13 of 20
cr us an
Table 4
316
Characterization of L. pneumophila strains isolated from humans and cooling towers during the outbreak of Ulm, December 2009 /January 2010
CT = Cooling tower
Maximum colony forming units cfu/100ml
Sequence type ST flaA
pilE
asd
mip
momp
proA
neuA
Occurrence of this strain in other regions according to the EWGLI/ESGLI Database (as by 7.10.2015 Related to clinical cases, not isolated from environmental sources not related to disease
(N = colonies tested)
(N = colonies tested)
1 Knoxville1
62
8
10
3
15
18
1
6
ce
pt
8 patients from the outbreak
Serogroup / mAb subtype
ed
Source
M
315
92500
1 Knoxville1 (n=19)
62 (n=4)
8
10
3
15
18
1
6
1. CT 1A
92500
1 OLDA2 (n=1)
1 (n=1)
1
4
3
1
1
1
1
Commonly found among clinical and environmental isolates worldwide
92500
10 (n=1)
908 (n=1)
1
4
3
5
1
1
6
Unique
1. CT 1A
92500
8 (n=3)
1373 (n=2)
5
2
3
10
6
25
203
Unique
1. CT 1A
92500
3 (n=8)
984 (n=1)
12
29
2
20
50
20
15
Unique
1. CT 1A
92500
L. rubrilucens3 (n=1)
NA5
2. CT 1B
70 000
8 (n=94)
1373 (n=2)
5
2
3
10
6
25
203
Unique
2. CT 1B
70 000
L. rubrilucens3 (n=2)
NA5
1. CT 1A
Ac
1. CT 1A
14
Page 14 of 20
cr us 300
1 Bellingham2 (n=1)
4. CT
3000
4 (n=1)
4. CT
3000
5. CT
1
6. CT
1
7. CT
130
an
3. CT
2
6
17
6
13
11
11
Isolated from clinical and environmental sources in Germany and the Netherlands
681 (n=1)
11
14
16
25
7
13
6
Isolated once in France
6 (n=1)
956 (n=1)
6
10
3
3
9
1
9
Isolated once in Poland
1 OLDA2 (n=1)
1 (n=1)
1
4
3
1
1
1
1
Commonly found among clinical and environmental isolates worldwide
pt
ed
M
334 (n=1)
NA5
1 Bellingham2 (n=1)
334 (n=1)
2
6
17
6
13
11
11
Isolated from clinical and environmental sources in Germany and the Netherlands
15 000
1 OLDA2 (n=2)
595 (n=1)
2
14
16
16
15
13
2
Isolated once in Japan
8. CT
6
4 (n=1)
1369 (n=1)
8
14
16
25
7
13
207
Unique
9. CT
5
1 OLDA2 (n=1)
1 (n=1)
1
4
3
1
1
1
1
Commonly found among clinical and environmental isolates worldwide
10. CT
1
1 OLDA2 (n=1)
104 (n=1)
3
10
1
1
14
9
1
Isolated 9x from clinical and environmental Sources in the UK
Ac
8. CT
ce
Putative new Legionella species (n=1)3, 4
15
Page 15 of 20
cr us an
1
mAb 3-1 positive
318
2
mAb 3-1 negative
319
3
based on mip (macrophage infectivity potentiator) sequence (Ratcliff et al., 1998)
320 321
4
mip sequence Accession number HE613854; 80% related to L. brunensis , Acc Nr U92227; 99% identity to Legionella brunensis like strain Tver-868.1 isolated in Russia, NCBI Acc Nr. FJ357014.1;
322
5
ed
M
317
pt
NA, non applicable for non-pneumophila strains
Ac
ce
323
16
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324
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