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Importance: The association of Lyme neuroborreliosis with the development of psychiatric disease is unknown and remains a subject of debate.Objective: To investigate the risk of psychiatric disease, the percentage of psychiatric hospital inpatient and outpatient contacts, and the receipt of prescribed psychiatric medications among patients with Lyme neuroborreliosis compared with individuals in a matched comparison cohort.Design, Setting, and Participants: This nationwide population-based matched cohort study included all residents of Denmark who received a positive result on an intrathecal antibody index test for Borrelia burgdorferi (patient cohort) between January 1, 1995, and December 31, 2015. Patients were matched by age and sex to a comparison cohort of individuals without Lyme neuroborreliosis from the general population of Denmark. Data were analyzed from February 2019 to March 2020.Exposures: Diagnosis of Lyme neuroborreliosis, defined as a positive result on an intrathecal antibody index test for B burgdorferi.Main Outcomes and Measures: The 0- to 15-year hazard ratios for the assignment of psychiatric diagnostic codes, the difference in the percentage of psychiatric inpatient and outpatient hospital contacts, and the difference in the percentage of prescribed psychiatric medications received among the patient cohort vs the comparison cohort.Results: Among 2897 patients with Lyme neuroborreliosis (1646 men [56.8%]) and 28 970 individuals in the matched comparison cohort (16 460 men [56.8%]), the median age was 45.7 years (interquartile range [IQR], 11.5-62.0 years) for both groups. The risk of a psychiatric disease diagnosis and the percentage of hospital contacts for psychiatric disease were not higher among patients with Lyme neuroborreliosis compared with individuals in the comparison cohort. A higher percentage of patients with Lyme neuroborreliosis compared with individuals in the comparison cohort received anxiolytic (7.2% vs 4.7%; difference, 2.6%; 95% CI, 1.6%-3.5%), hypnotic and sedative (11.0% vs 5.3%; difference, 5.7%; 95% CI, 4.5%-6.8%), and antidepressant (11.4% vs 6.0%; difference, 5.4%; 95% CI, 4.3%-6.6%) medications within the first year after diagnosis, after which the receipt of psychiatric medication returned to the same level as the comparison cohort.Conclusions and Relevance: In this population-based matched cohort study, patients with Lyme neuroborreliosis did not have an increased risk of developing psychiatric diseases that required hospital care or treatment with prescription medication. The increased receipt of psychiatric medication among patients with Lyme neuroborreliosis within the first year after diagnosis, but not thereafter, suggests that most symptoms associated with the diagnosis subside within a short period.

Lyme neuroborreliosis (LNB) has recently been added to the list of diseases under the European Union epidemiological surveillance in order to obtain updated information on incidence. The goal of this study was to identify temporal (yearly) variation, high risk geographical regions and risk groups, and seasonal variation for LNB in Denmark. This cohort-study investigated Danish patients (n = 2791) diagnosed with LNB (defined as a positive Borrelia burgdorferi sensu lato (s.l.) intrathecal antibody test) between 1996–2015. We calculated incidence and incidence ratios of LNB by comparing 4-yr groups of calendar-years, area of residency, sex and age, income and education groups, and the number of new LNB cases per month. The incidence of LNB was 2.2 per 100,000 individuals and year in 1996−1999, 2.7 in 2004−2007 and 1.1 per 100,000 individuals in 2012−2015. Yearly variations in LNB incidence were similar for most calendar-year groups. LNB incidence was highest in Eastern Denmark and among males and individuals who were 0–14 yrs old, who had a yearly income of >449,000 DKK, and who had a Master's degree or higher education. The number of LNB cases was highest from July to November (p < 0.001). In conclusion, based on Danish nationwide data of patients with positive B. burgdorferi s.l. intrathecal antibody index (1996–2015) the incidence of LNB was found to increase until 2004−2007 but thereafter to decline. European surveillance studies of Lyme borreliosis should be encouraged to monitor the incidence trend.

Background Lyme neuroborreliosis (LNB), caused by the tick-borne spirochetes of the Borrelia burgdorferi sensu lato species complex, has been suggested to be associated with a range of neurological disorders. In a nationwide, population-based cohort study, we examined the associations between LNB and dementia, Alzheimer’s disease, Parkinson’s disease, motor neuron disease, epilepsy, and Guillain-Barré syndrome. Methods We used national registers to identify all Danish residents diagnosed during 1986–2016 with LNB (n = 2067), created a gender- and age-matched comparison cohort from the general population (n = 20 670), and calculated risk estimates and hazard ratios. Results We observed no long-term increased risks of dementia, Alzheimer’s disease, Parkinson’s disease, motor neuron diseases, or epilepsy. However, within the first year, 8 (0.4%) of the LNB patients developed epilepsy, compared with 20 (0.1%) of the comparison cohort (difference, 0.3%; 95% confidence interval, .02–.6%). In the LNB group, 11 (0.5%) patients were diagnosed with Guillain-Barré syndrome within the first year after LNB diagnosis, compared with 0 (0.0%) in the comparison cohort. After the first year, the risk of Guillain-Barré was not increased. Conclusions LNB patients did not have increased long-term risks of dementia, Alzheimer’s disease, Parkinson’s disease, motor neuron diseases, epilepsy, or Guillain-Barré. Although the absolute risk is low, LNB patients might have an increased short-term risk of epilepsy and Guillain-Barré syndrome.

Background: The impact of P. aeruginosa on long-term prognosis in COPD remains unknown. Objective: To determine whether P. aeruginosa is associated with increased risk of exacerbation or death in patients with COPD. Methods: Observational cohort study on 22.053 COPD outpatients (Fig.1). Time-dependent Cox proportional hazard regression model was used to estimate a) hospitalisation for exacerbation or all-cause mortality and b) all-cause mortality after 2 years. Results:P. aeruginosa was found in 4.1% of the patients. Hospital admissions (80% and 48%,p Conclusions:P. aeruginosa is associated with substantially worsened long-term prognosis in COPD. A randomized controlled trial is needed to conclude whether targeted antibiotic interventions can improve the prognosis in this highly vulnerable group of patients.

OBJECTIVE: To estimate long term survival, health, and educational/social functioning in patients with Lyme neuroborreliosis compared with the general population.DESIGN: Nationwide population based cohort study using national registers.SETTING: Denmark.PARTICIPANTS: All Danish residents diagnosed during 1986-2016 as having Lyme neuroborreliosis (n=2067), defined as a positive Borrelia burgdorferi intrathecal antibody test and a clinical diagnosis of Lyme borreliosis, and a comparison cohort from the general population matched on sex and date of birth (n=20 670).MAIN OUTCOME MEASURES: Mortality rate ratios, incidence rate ratios of comorbidities, and differences in educational and social outcomes.RESULTS: Mortality among patients with Lyme neuroborreliosis was not higher than in the general population (mortality rate ratio 0.90, 95% confidence interval 0.79 to 1.03). Lyme neuroborreliosis patients had increased risk of haematological (incidence rate ratio 3.07, 2.03 to 4.66) and non-melanoma skin cancers (1.49, 1.18 to 1.88). At diagnosis, Lyme neuroborreliosis patients had slightly higher employment and lower disability pension rates. After five years, patients and comparison cohort members had similar numbers of hospital contacts (difference -0.22, 95% confidence interval -0.45 to 0.02, in-hospital days/year; 0.37, -0.10 to 0.83, outpatient visits/year), employment rates (difference 1.5%, -2.1% to 5.1%), income (difference -1000, -20 000 to 18 000, Danish kroner), days of sick leave (difference -0.3, -3.5 to 3.0, per year), rates of receipt of a disability pension (difference -0.9%, -3.2% to 1.3%), and number of children (difference -0.10, -0.27 to 0.08). More patients were married (difference 4.8%, 2.2% to 7.4%) and had completed high school education (difference 7%, 1% to 12%).CONCLUSION: A verified diagnosis of Lyme neuroborreliosis had no substantial effect on long term survival, health, or educational/social functioning. Nevertheless, the diagnosis decreased labour market involvement marginally and was associated with increased risk of haematological and non-melanoma skin cancers.

Episodes of adult bacterial meningitis (ABM) at a Danish hospital in 1991–2000 were identified from the databases of the Department of Clinical Microbiology, and compared with data from the Danish National Patient Register and the Danish National Notification System. Reduced penicillin susceptibility occurred in 21 (23%) of 92 cases of known aetiology, compared to an estimated 6%in nationally notified cases (p < 0.001). Ceftriaxone plus penicillin as empirical treatment was appropriate in 97% of ABM cases in the study population, and in 99.6% of nationally notified cases. The notification rate was 75% for penicillin-susceptible episodes, and 24% for penicillin-non-susceptible episodes (p < 0.001). Cases involving staphylococci, Pseudomonas spp. and Enterobacteriaceae were under-reported. Among 51 ABM cases with no identified risk factors, nine of 11 cases with penicillin-non-susceptible bacteria were community-acquired. Severe sequelae correlated independently with age, penicillin non-susceptibility, mechanical ventilation and non-transferral to a tertiary hospital (p < 0.05; logistic regression). Other factors that correlated with severe sequelae by univariate analysis only were inappropriate clinical handling, abnormal consciousness, convulsions and nosocomial infection. Overall, the data indicated that neither age alone, community-acquired infection nor absence of identified risk factors can predict susceptibility to penicillin accurately. Recommendations for empirical antibiotic treatment for ABM should not be based exclusively on clinical notification systems with possible unbalanced under-reporting.

The aims of this work were to assess (i) the intercentre reproducibility and epidemiological concordance of amplified fragment length polymorphism analysis for epidemiological typing of Legionella pneumophila serogroup 1, and (ii) the suitability of the method for standardisation and implementation by members of the European Working Group on Legionella Infections. Fifty coded isolates comprising two panels of well-characterised strains, a "reproducibility" panel (n=20) and an "epidemiologically related" panel (n=30), were sent to 13 centres in 12 European countries. Analysis was undertaken in each centre following a previously determined standard protocol. Results were analysed by the participants, using gel analysis software where available, and submitted to the coordinating centre. The coordinating centre reanalysed all results visually and selected data-sets with gel analysis software. Data analysis by participants yielded reproducibility (R) values of 0.20-1.00 and epidemiological concordance (E) values of 0.11-1.00, with 6 to 34 types. Following visual analysis by the coordinating centre, R=0.78-1.00, and E=0.67-1.00, with 10-20 types. Analysis of three data-sets by the coordinating centre using gel analysis software yielded R=1.00 and E=1.00, with 12, 13 or 14 types. This method can be used as a simple, rapid screening tool for epidemiological typing of isolates of Legionella pneumophila serogroup 1. Results demonstrate that the method can be highly reproducible (R=1.00) and epidemiologically concordant (E=1.00), with good discrimination. The electropherograms generated are amenable to computer-aided analysis, but strict adherence to a previously defined laboratory protocol is required. Following designation of representative type strains and patterns, this method will be adopted by the European Working Group on Legionella Infections as the first internationally standardised typing method for use in the investigation of travel-associated Legionella infections.

Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis , and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae . Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis , was sequenced from 18 rifampin-resistant Legionella isolates representing four species ( L. bozemanii , L. longbeachae , L. micdadei , and L. pneumophila ), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae .

Objectives To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. Methods Coded isolates (114) of L. pneumophila sg 1 comprising one epidemiologically ‘unrelated' (79) and one ‘related' panel of isolates (35) were sent to 12 laboratories in 11 European countries. Analysis was undertaken in each laboratory using one or more of the following methods: ribotyping, restriction fragment length polymorphism analysis, restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), PCR using arbitrary/repeat sequence primers (AP-, AP/rep-PCR), and amplified fragment length polymorphism (AFLP) analysis. Results were analyzed visually or using gel analysis software. Each method was assessed for its: index of discrimination ( D ), epidemiologic concordance ( E ), speed of application and ease of use. In addition, phenotypic analysis was performed in two laboratories using monoclonal antibodies (mAbs). Results The D of each of the genotypic methods ranged from 0.840 for ribotyping to 0.990 for PFGE using Sfi I : E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using Pst I /Eco RI and AFLP: in general, E was inversely related to D. Although offering only limited discrimination ( D =0.838), mAb typing was both rapid and highly epidemiologically concordant ( E =1.00). Conclusions Two methods, PFGE using Sfi l and AFLP, were selected for further study. AFLP is rapid and highly epidemiologically concordant ( E =1.00), but is not highly discriminatory. This method will be developed as a rapid screening tool. PFGE using Sfi l is highly discriminatory but, in the present study, yielded low values of E (0.12–0.71). Attempts will be made to rigorously standardize this method for use as the reference method. Primary screening of isolates by mAb subgrouping is recommended.

BACKGROUND: Little is known about the clinical presentation and outcome of pneumococcal lower respiratory tract infection (LRTI) without positive chest X-ray findings and blood cultures. We investigated the prognostic impact of a pulmonary infiltrate and bacteraemia on the clinical course of hospitalized patients with confirmed pneumococcal LRTI. METHODS: We studied a population-based multi-centre cohort of 705 adults hospitalized with LRTI and Streptococcus pneumoniae in LRT specimens or blood: 193 without pulmonary infiltrate or bacteraemia, 250 with X-ray confirmed pneumonia, and 262 with bacteraemia. We compared adverse outcomes in the three groups and used multiple regression analyses to adjust for differences in age, sex, comorbidity, and lifestyle factors. RESULTS: Patients with no infiltrate and no bacteraemia were of similar age but had more comorbidity than the other groups (Charlson index score >=1: no infiltrate and no bacteraemia 81% vs. infiltrate without bacteraemia 72% vs. bacteraemia 61%), smoked more tobacco, and had more respiratory symptoms. In contrast, patients with a pulmonary infiltrate or bacteraemia had more inflammation (median C-reactive protein: no infiltrate and no bacteraemia 82 mg/L vs. infiltrate without bacteraemia 163 mg/L vs. bacteraemia 316 mg/L) and higher acute disease severity scores. All adverse outcomes increased from patients with no infiltrate and no bacteraemia to those with an infiltrate and to those with bacteraemia: Length of hospital stay (5 vs. 6 vs. 8 days); intensive care admission (7% vs. 20% vs. 23%); pulmonary complications (1% vs. 5% vs. 14%); and 30-day mortality (5% vs. 11% vs. 21%). Compared with patients with no infiltrate and no bacteraemia, the adjusted 30-day mortality rate ratio was 1.9 (95% confidence interval (CI) 0.9-4.1) in patients with an infiltrate without bacteraemia and 4.1 (95% CI 2.0-8.5) in bacteraemia patients. Adjustment for acute disease severity and inflammatory markers weakened these associations. CONCLUSIONS: Hospitalization with confirmed pneumococcal LRTI is associated with substantial morbidity and mortality even without positive chest X-ray findings and blood cultures. Still, there is a clinically important outcome gradient from LRTI patients with pneumococcal isolation only to those with detected pulmonary infiltrate or bacteraemia which is partly mediated by higher acute disease severity and inflammation.

BackgroundThere is limited knowledge of serotypes that cause non-bacteremic pneumococcal pneumonia (NBP). Here we report serotypes, their associated disease potential and coverage of pneumococcal conjugate vaccines (PCV) in adults with NBP and compare these to bacteremic pneumonia (BP).MethodsAdults with pneumonia and Streptococcus pneumoniae isolated from the lower respiratory tract or blood were included 1 year in a population-based design in Denmark. Pneumonia was defined as a new infiltrate on chest radiograph in combination with clinical symptoms or elevated white blood count or plasma C-reactive protein. All isolates were serotyped using type-specific pneumococcal rabbit antisera. All values are medians with interquartile ranges.ResultsThere were 272 cases of NBP and 192 cases of BP. Ninety-nine percent were hospitalized. NBP and BP cases were of comparable age and sex but NBP cases had more respiratory symptoms and less severe disease compared to BP cases. In total, 46 different serotypes were identified. Among NBP cases, 5 serotypes accounted for nearly a third of isolates. PCV10 and -13 types covered 17% (95% confidence interval (CI): 11-23%) and 34% (95% CI: 25-43%) of NBP isolates, respectively. In contrast, the five most frequent serotypes accounted for two-thirds of BP isolates. PCV10 and -13 types covered 39% (95% CI: 30-48%) and 64% (95% CI: 48-79) of BP isolates, respectively. More severe NBP disease was associated with infection with invasive serotypes while there was an inverse relationship for BP.ConclusionsOnly a third of cases of adult non-bacteremic pneumococcal pneumonia would potentially be preventable with the use of PCV13 and just one sixth of cases with the use of PCV10 indicating that PCVs with increased valency are needed to increase vaccine coverage for NBP in adults. PCV13 could potentially prevent two-thirds of adult bacteremic pneumococcal pneumonia.

Studies on the value of culture-independent molecular identification of bacteria in cardiac valves are mostly restricted to comparing agreement of identification to what is obtained by culture to the number of identified bacteria in culture-negative cases. However, evaluation of the usefulness of direct molecular identification should also address weaknesses, their relevance in the given setting, and possible improvements. In this study cardiac valves from 56 Danish patients referred for surgery for infective endocarditis were analysed by microscopy and culture as well as by PCR targeting part of the bacterial 16S rRNA gene followed by DNA sequencing of the PCR product. PCR and DNA sequencing identified significant bacteria in 49 samples from 43 patients, including five out of 13 culture-negative cases. No rare, exotic, or intracellular bacteria were identified. There was a general agreement between bacterial identity obtained by ribosomal PCR and DNA sequencing from the valves and bacterial isolates from blood culture. However, DNA sequencing of the 16S rRNA gene did not discriminate well among non-haemolytic streptococci, especially within the Streptococcus mitis group. Ribosomal PCR with subsequent DNA sequencing is an efficient and reliable method of identifying the cause of IE, but exact species identification of some of the most common causes, i.e. non-haemolytic streptococci, may be improved with other molecular methods.

Organ transplant recipients are at high risk of contracting Legionnaires' disease in a hospital environment contaminated with legionellae. This study describes the first cases of culture-verified Legionella infections with an established link to potable hospital water in Denmark; three patients operated on at the Cardiopulmonary Transplant Unit, Rigshospitalet, Copenhagen, became infected with legionellae. Environmental and clinical isolates of Legionella pneumophila serogroups 1 and 6 were investigated by restriction enzyme analysis and ribotyping. An ice machine located in the kitchen of the intensive care unit was implicated as a source of infection in two of the three cases.

Denmark experienced two waves of Mycoplasma pneumoniae infection during autumn and early winter in 2010 and 2011, respectively. Both affected the whole country. The proportion of positive results was almost the same for both, indicating that the two waves were probably of equal size. High macrolide consumption during the epidemics did not seem to affect levels of macrolide resistance in M. pneumoniae, which remain low in Demark (1% to 3%).

Currently recommended methods in Legionnaires' disease serology are based upon crude whole-cell antigenic preparations. To investigate whether purified antigens would perform better in a given diagnostic test for antibodies against Legionella pneumophila, we compared the performance of three antigenic preparations of L. pneumophila serogroup 1 consisting of outer membrane protein (OMP), flagellin (FLA), and lipopolysaccharide (LPS) to a sonic extract (SON) in indirect immunosorbent assay (ELISA) measuring both IgG, IgA, and IgM. The reactivity of sera from 20 patients with culture-verified Legionnaires' disease and sera from 12 patients with pneumonia and a diagnostic rise in titre by a microagglutination test (MA) was studied. Our results indicated that the SON IgA assay was the most sensitive test in both groups of patients. The LPS IgG and IgM assays, however, were the most specific tests, closely followed by the corresponding SON tests. By combining two individual assays, a maximum nosographic sensitivity of 85% could be obtained. Whereas no benefit of using purified outer membrane protein or flagella instead of a sonic extract in the indirect ELISAs was found, the LPS antigen provided a sensitive and specific alternative to the sonic extract.

Cardiobacterium valvarum is a newly recognized human pathogen related to infective endocarditis. Cardiobacterium species are, however, only rarely the aetiology of infective endocarditis. An infective endocarditis case is presented and, additionally, phenotypic and phylogenetic comparison of a further 10 collection strains, representing the two species within the genus, was performed. C. valvarum was isolated from the blood and DNA was present in valvular tissue (partial 16S rRNA gene analysis) from a 64-year-old man with infective endocarditis of the mitral valve, rupture of chordae and prolapse of pulmonary valves in addition to a fluttering excrescence. A mechanical mitral valve and neochordae were inserted successfully. Phenotypically, the two species within the genus Cardiobacterium resemble each other greatly. When using the Vitek 2 Neisseria–Haemophilus identification card, the reaction for phenylphosphonate was positive for all Cardiobacterium hominis strains, but negative for all C. valvarum strains, thereby separating the two species. The two species made up two separate clusters by phylogenetic examination using 16S rRNA gene sequence analysis.

Objective To investigate the impact of HIV co-infection on mortality in patients infected with hepatitis C virus (HCV). Methods From a nationwide Danish database of HCV-infected patients, we identified individuals diagnosed with HCV subsequent to an HIV diagnosis. For each co-infected patient, four control HCV patients without HIV were matched on age, gender and year of HCV diagnosis. Data on comorbidity, drug abuse, alcoholism and date of death were extracted from two healthcare databases. We constructed Kaplan–Meier curves and used Cox regression analyses to estimate mortality rate ratios (MRRs), controlling for comorbidity. Results We identified 483 HCV–HIV co-infected and 1932 HCV mono-infected patients, yielding 2192 and 9894 person-years of observation with 129 and 271 deaths, respectively. The 5-year probability of survival was 0.74 [95% confidence interval (CI) 0.69–0.80] for HCV–HIV co-infected patients and 0.87 (95% CI 0.85–0.89) for HCV mono-infected patients. Co-infection was associated with substantially increased mortality (MRR 2.1, 95% CI 1.7–2.6). However, prior to the first observed decrease in CD4 counts to below 300 cells/μL, HIV infection did not increase mortality in HCV-infected patients (MRR 0.9, 95% CI 0.5–1.50). Conclusions HIV infection has a substantial impact on mortality among HCV-infected individuals, mainly because of HIV-induced immunodeficiency.

A 60-year old man was admitted with sepsis and meningitis of unknown aetiology. Underlying aortic valve endocarditis was diagnosed by echocardiography and severe insufficiency led to aortic valve replacement. Application of broad-range PCR to cusp tissue revealed a DNA product, and a diagnosis of Streptococcus pneumoniae endocarditis was obtained by DNA sequencing.

The first reported case of peritonitis caused by Capnocytophaga cynodegmi is presented. The patient was treated with peritoneal dialysis and had contact with a cat. C. cynodegmi is part of the normal oral flora of dogs and cats but is very rarely isolated in clinical specimens from humans.

The laboratory diagnosis of Lyme disease in Denmark is reviewed with recommendations for serological testing. In Denmark the laboratory testing is performed with an ELISA technique. Most laboratories use an assay based on purified flagella antigen. The two-tier approach with Western Blot as confirmatory testing is not recommended since the contribution to the diagnostic specificity is only marginal. Predictive values of Lyme serology are presented, based on the estimated prevalence of the different stages of Lyme disease in Denmark.

OBJECTIVES: To undertake a multicenter study to evaluate the Biotest legionella urinary antigen enzyme immunoassay (EIA) performance against those EIAs already in use in 14 European laboratories. METHODS: Each laboratory examined urine specimens from appropriate patients using both their current assay and the Biotest EIA. Each examined: a standard panel of 12 coded urine samples (distributed by Biotest); a panel of 10 coded urine samples provided as part of a European external quality assurance (EQA) scheme; urine samples from patients with proven legionnaires' disease (LD); urine samples from patients with pneumonia of microbiologically proven cause other than LD; and urine samples submitted for routine examination. Thus, the performance of the Biotest assay (in comparison with current EIAs), its specificity and utility, and the inter-laboratory agreement were assessed. RESULTS: Inter-laboratory agreement was excellent, with all participants obtaining the expected results for 20 of 22 coded urine specimens. Specificity, determined using 123 specimens from patients with infections of known etiology, was 100%. The Biotest EIA gave positive results in 86% of specimens which had been positive in the laboratories' current EIAs, and in 94.6% of those specimens which were positive for Legionella pneumophila serogroup 1. CONCLUSION: The Biotest EIA is simple to use and specific and the results obtained in different laboratories show excellent agreement. The assay compares well existing EIAs, at least for L. pneumophila serogroup 1

The utility of amplified fragment length polymorphism (AFLP) analysis as a genotyping method for the epidemiological typing of Legionella pneumophila serogroup 1 has been previously demonstrated. This study (i). reports recommendations for the designation of the European Working Group on Legionella Infections (EWGLI) AFLP types, (ii). describes the EWGLI AFLP types identified for the 130 strains in the EWGLI culture collection, and (iii). reports the results of a newly introduced international programme of proficiency testing. Following preliminary analysis of 20 epidemiologically unrelated isolates, 16 major AFLP types were identified. A coded proficiency panel, comprising 12 additional isolates representing 9 of these 16 AFLP types, was sent to 17 centres in 14 European countries where it was analysed following a previously determined standard protocol. The identity of each coded strain (recorded as AFLP type 001-016 or untypeable) was determined by participants with reference to these 16 AFLP types, either visually or using gel analysis software where available, and reported to the coordinating centre. Nine of the 12 strains, including an epidemiologically related pair and two pairs of unrelated isolates of the same type, were correctly identified to the correct AFLP type by all or all but one of the participants. Seven laboratories correctly identified all 12 isolates, and a further seven laboratories correctly identified 11. Type identification scores ranged from 75% (1 centre), 83% (2 centres), and 92% (7 centres) to 100% (7 centres). The AFLP method as described is robust and rapid and allows the genotypic comparison of isolates of Legionella pneumophila between different testing centres without the need for exchange of the strains studied.

Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis, and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae. Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis, was sequenced from 18 rifampin-resistant Legionella isolates representing four species (L. bozemanii, L. longbeachae, L. micdadei, and L. pneumophila), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae.

The minimal inhibitory concentration (MIC) values of erythromycin, ciprofloxacin, ofloxacin, rifampicin, and clindamycin were determined for 56 strains of Legionella pneumophila (38 patient, 3 environmental, and 15 reference strains) and 37 strains of other Legionella species (7 patient, 2 environmental, and 28 reference strains) using the epsilon-test system on BCYEalpha agar plates. High-level resistance (MICor = 4 microg/mL) was found only for clindamycin (57%), with MIC values ranging from 0.25-32 microg/mL. Low-level resistance was found for erythromycin (18%) (0.5MIC8), ciprofloxacin (1%) (1MIC4), and clindamycin (40%) (0.5MIC4), but not for ofloxacin and rifampicin. MIC50 for the 45 Danish clinical Legionella strains were 0.25 microg/mL (erythromycin), 0.25 microg/mL (ciprofloxacin), 0.19 microg/mL (ofloxacin), below 0.016 microg/mL (rifampicin), and 4 microg/mL (clindamycin). Of the clinical isolates, 64% were resistant to clindamycin. There were no significant differences between the MIC50 values obtained for clinical and nonclinical Legionella strains. Selected susceptible strains were exposed to increasing concentrations of either erythromycin, ciprofloxacin, or rifampicin to select for resistance. Isolates resistant to erythromycin (MIC 0.75-32 microg/mL) or ciprofloxacin (MIC 2-3 microg/mL) could be selected by a two-step procedure. One single strain recovered from media containing 50 microg/mL of erythromycin had an MIC value higher than 256 microg/mL to erythromycin. In contrast, high-level resistance toward rifampicin with MICor = 256 microg/mL developed as a one-step phenomenon in several strains.

Crossed immunoelectrophoresis (XIE) reference systems were established for Fluoribacter (Legionella) (containing Fluoribacter bozemanae, Fluoribacter dumoffii, and Fluoribacter gormanii) and for Tatlockia (Legionella) micdadei. The Fluoribacter and Tatlockia XIE reference systems contained 54 and 72 anodemigrating antigens, respectively. These two systems, together with the previously described polyvalent Legionella pneumophila (serogroups 1 through 6) XIE reference system, were used to study the crossreactivities of antigens from organisms comprising the three proposed genera in the family Legionellaceae. Antigenic homology was expressed as the matching coefficient (MC), the ratio of the number of cross-reactive antigens to the total number of antigens. The MCs for individual L. pneumophila serogroups when the polyvalent L. pneumophila antibody was used were 0.98 ± 0.05, which was significantly higher than the MCs determined by using Fluoribacter or Tatlockia antibodies (0.50 ± 0.13) (P ≪ 0.001). The MCs for the three species of Fluoribacter when polyvalent Fluoribacter antibody was used were 0.93 ± 0.10, which was also significantly higher than the MCs when heterologous antibodies were used (0.40 ± 0.04) (P ≪ 0.001). The MCs for T. micdadei with the two heterologous antibody preparations were similar to each other (0.32 and 0.46) and to all other heterologous MCs among members of the Legionellaceae. The MCs for organisms representing three other families of bacteria were 0.16 ± 0.04 in all three XIE reference systems and were significantly lower than the MCs among members of the Legionellaceae (P ≪ 0.001). When a priori criteria for MC interpretation established in previous serotaxonomic studies of other bacterial species by XIE were used, our results from studies on the antigenic relationships among Legionella, Fluoribacter, and Tatlockia supported the proposal that there are multiple genera in the family Legionellaceae.

The objective of this study was to investigate the antigenic profiles of Legionella pneumophila serogroups 1 to 6 and antigenic relatedness among individual serogroups. A reference precipitate pattern which had 71 anodic- and 6 cathodic-migrating antigens was established with a pooled antigen preparation of sonicated L. pneumophila cells of serogroups 1 to 6 against purified homologous rabbit antibody. Sixteen antigens made heat stable by boiling for 10 min were found, of which one could be demonstrated as being the common antigen of gram-negative bacteria and another could be shown as being the serogroup-specific antigen of L. pneumophila. Precipitin patterns similar in overall appearance and precipitin numbers were demonstrated for each serogroup by testing antigen preparations of individual serogroups against reference system antibody. Crossed-line experiments revealed < 5 non-cross-reacting antigens among serogroups, with resulting matching coefficients (ratio of the number of cross-reactive to total antigens) all above 0.91. The matching coefficients found when testing antigen preparations from L. pneumophila serogroups 7 and 8 were 0.93 and 0.99, respectively. By contrast, the matching coefficients of Tatlockia micdadei, Fluoribacter bozemanae, Fluoribacter dumoffii, and Fluoribacter gormanii was 0.39, 0.45, 0.39, and 0.41, respectively. Comparison of precipitate patterns and experimental results between the previously described monovalent L. pneumophila serogroup 1 system and the polyvalent crossed immunoelectrophoresis system with L. pneumophila serogroups 1 to 6 substantiated the antigenic homogencity of L. pneumophila and the significant difference in antigenic composition between this taxon and other members of the family Legionellaceae. In addition, reproducibility of the antigenic profile of L. pneumophila by crossed immunoelectrophoresis was demonstrated.

An outbreak of listeriosis in Denmark occurred in May 2009. Multilocus variable number of tandem repeats analysis typing, later confirmed by pulsed-field gel electrophoresis typing, showed that isolates from eight patients had identical patterns and were distinguishable from Listeria monocytogenes isolates from other Danish patients. Seven out of eight patients had received a meal with beef from the same meals-on-wheels delivery catering company 3 weeks prior to onset of disease. Two patients died of their infection. Large-scale delivery of precooked meals to a vulnerable population represents a threat if proper measures against listeriosis are not taken.

OBJECTIVE: Infective endocarditis is frequently caused by oral streptococci, especially Streptococcus sanguis. In this group, many strains have recently been reclassified on the basis of new taxonomic schemes. The purpose of this study was to classify oral streptococci from patients with infective endocarditis and, further, to assess the importance of specific virulence factors for the development of streptococcal endocarditis. METHODS: Twenty-eight previously identified and 10 new streptococcal isolates from infective endocarditis were classified according to Kilian et al (1989) and compared to 30 streptococcal isolates from the oral cavities of periodontal patients without endocarditis. Subsequently, surface hydrophobicity was assessed by hydrophobic interaction chromatography, production of extracellular dextran was determined by precipitation, and non-specific proteolytic activity was evaluated by determination of hydrolysis of gelatin, and casein-precipitating activity. RESULTS: Eight streptococcal species were represented in the endocarditis isolates. Most strains were highly hydrophobic and none showed non-specific proteolytic activity. Dextran was produced with similar frequency in endocarditis and non-endocarditis isolates. CONCLUSIONS: The present study showed that infective endocarditis may be caused by a variety of oral streptococcal species. The possible virulence factors investigated were found in the same proportions in endocarditis and non-endocarditis isolates, and thus did not seem to be crucial for development of endocarditis.