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In this study, we evaluated the Sofia Streptococcus pneumoniae FIA test (Quidel Corporation, San Diego, CA, USA), a new immunofluorescence-based lateral flow test for the qualitative detection of S. pneumoniae antigen in urine or cerebrospinal fluid specimens. The analyses of 100 non-concentrated urine samples (including 50 samples from S. pneumoniae cases) showed a sensitivity and specificity (95 % CI) of, respectively, 66.0 % (52.2–77.6) and 100.0 % (92.9–100.0) for the Sofia test, and 62.0 % (48.2–74.1) and 98.0 % (89.5–99.7) for the BinaxNOW SPN Antigen Card. There were no significant differences in sensitivity and specificity between the tests (McNemar’s tests, P=0.625 and P=1.000). In conclusion, this study indicates that the Streptococcus pneumoniae FIA test shows similar sensitivity and specificity rates compared to the BinaxNOW SPN Antigen Card.

Previous analysis of the Dutch National Legionella Outbreak Detection Program 2002–2012 has shown that buildings required to maintain a Legionella control plan for their drinking water installation are more likely to test positive for Legionella spp. Than buildings without such a plan (38% versus 22% of samples). To clarify this discrepancy, we analysed the results of mandatory water sample testing conducted as part of risk assessments in 206 buildings in the Netherlands from 2011 to 2015. Of the 6171 samples analysed, 16.2% exceeded the Dutch drinking water standard for Legionella spp. of 100 CFU/litre. In buildings with ≤50 tap points, the average percentage of samples containing ≥100 CFU/litre was 28.2%, and from buildings with >50 tap points, it was 12.2%. Analysis of serial samples (taken every 6 months) from each building showed that 33.2% of all buildings tested positive for at least one sample every 6 months. The overall increase was 4.4% per year. Analysis of Legionella subgroups showed that while the majority of positive samples contained L. non-pneumophila (96.9%), some samples did contain L. pneumophila serogroup 1 (1.0%) and serogroups 2–14 (2.1%). Our data suggest that the Dutch mandatory risk assessment and drinking water management plan is not sufficiently effective in preventing the proliferation of Legionella spp. and may even contribute to proliferation. This analysis should now be expanded to include other areas of the Netherlands in order to understand the geographical differences that we observed in our results, and why smaller buildings appear to be more likely to test positive for Legionella spp.

In this study, we compared the bioNexia test (bioMérieux, Marcy-l'Étoile, France), a new immunochromatographic assay for the detection of Legionella pneumophila serogroup 1 in urine, with the BinaxNOW urinary antigen test (Alere, Waltham, Massachusetts, USA). After 15 min of incubation (in accordance with the manufacturers' instructions), the sensitivities and specificities were, respectively, 76.5% and 97.2% for the bioNexia test and 87.1% and 100% for the BinaxNOW test. After a prolonged incubation time of 60 min, the sensitivities and specificities increased to, respectively, 89.4% and 97.2% for the bioNexia test and 91.8% and 100% for the BinaxNOW test. When the tests were read after 15 min, the concentration of discrepant urine samples increased the sensitivities to 94.1% for both tests. In conclusion, we found that although the bioNexia test showed lower sensitivity for the detection of L. pneumophila antigen in nonconcentrated urine compared to the BinaxNOW test, a prolonged incubation time as well as the use of concentrated samples showed comparable sensitivities for both tests.

Legionnaires' disease (LD) is an acute pneumonia caused by the inhalation or aspiration of aerosols contaminated with the Legionella bacteria. In the Netherlands, around 300 LD cases per year were reported between 2000 and 2008, but in 2009, the number dropped to 251, which was the lowest number in the previous 5 years of surveillance. We investigated if this decrease could be explained by the number of performed Legionella diagnostic tests in this year. We analyzed the number of tests performed between 2007 and 2009 in three large microbiological laboratories in different geographical regions in the Netherlands. Our data showed that there was no decrease in the number of patients for whom a diagnostic test for Legionella was performed in this period. These results are not in line with our hypothesis that the decrease in reported Legionella pneumonia patients in 2009 would be due to a decrease in patients for whom a diagnostic test was performed. We conclude that it is more likely that other factors such as the influence of weather patterns might explain the sudden drop in reported Legionella pneumonia patients in 2009 compared to the previous years, and it would be interesting to investigate this for the period described.

Polymorphisms leading to deficiency of mannose-binding lectin (MBL) are associated with predisposition to infection. However, MBL deficiency can be protective against intracellular pathogens that use MBL to enter host cells. The role of MBL genotype and activity in infection with the intracellular pathogen Legionella pneumophila was studied in a large outbreak of legionellosis at a Dutch flower show. A total of 141 patients, 65 exposed asymptomatic exhibition staff members and 670 unexposed blood bank donors were included for the study of MBL2 genotypes and MBL-mediated complement activation. Genotypic MBL deficiency was equally prevalent in patients and controls. Deficient MBL-mediated complement activation was more prevalent in patients. Even in patients with genotypes that confer MBL sufficiency, 20.6% lacked MBL-mediated complement activation. In most patients with MBL-sufficient genotypes who lacked MBL-mediated activation at the acute phase of disease, lectin pathway functionality was restored at convalescence. In conclusion, genotypic MBL deficiency was not a risk factor for legionellosis. However, patients with legionellosis displayed deficient MBL-mediated complement activation even with MBL-sufficient genotypes. Together, these genotypical and functional data suggest that the observed deficiency of lectin pathway activation is an effect of legionellosis rather than a risk factor for acquiring it.

Legionella longbeachae was cultured from the sputum of a patient suffering from Legionnaires’ disease. Source identification efforts included analysis of samples of potting soil from the patient's garden, and a genotypically indistinguishable strain of L. longbeachae was cultured from this material. Following examination of a national collection of Legionella isolates, two more patients with indistinguishable genotypes were identified. One of these patients had visited a garden centre in the same municipality in which the index patient had acquired his potting soil. The study demonstrated the value of systematic collection of identification data and patient isolates over a prolonged period.

The authors discuss the case of a 79-year-old patient who suffered from a swallowing disorder and developed Legionnaires' disease 2 days after her dismissal from an orthopedics ward, where she had recovered from hip surgery. To determine the source of the Legionnaires' disease, the authors performed an environmental investigation, which included a national, standardized questionnaire and a microbiological investigation of suspected sources. The investigation revealed ice from an ice-making machine in the hospital as the most probable source of the infection through aspiration, even though the hospital had rigorously adhered to strict assessment and decontamination schedules. The infectious serogroup was one that was not common to the area. From the data available, the authors inferred that a dose of 1-2000 colony-forming units might have caused Legionnaires' disease in this patient.

After a large outbreak of Legionnaires' disease in the Netherlands, we determined risk factors for intensive care unit (ICU) admission and death and the impact of adequate therapy on ICU-free survival among 141 hospitalized patients. Overall mortality rate was 13%, and ICU mortality rate was 36%. Smoking, temperature >38.5 degrees C, and bilateral infiltrates shown on chest x-ray were independent risk factors for ICU admission or death (all p

Legionellae are part of the microbial community of aquatic ecosystems, natural as well as man-made, which explains why legionellosis occurs worldwide. In many countries Legionnaires’ disease is a notifiable disease. This chapter presents the preliminary results of the distribution of Legionella genotypes cultured from patients and environmental sources. Between August 2002 and September 2005, sero- and genotyping of 130 patient isolates and 220 environmental isolates showed that 98% of the patient strains were from the Legionella pneumophila genus. The genotypes 004 Lyon and 010 London, responsible for almost one-third of all Legionnaires’ disease patients in the authors' study period in The Netherlands, were not found in the environmental samples collected from the potential sources that resulted from the structured interviews according to the questionnaires. The preliminary results of the study indicate that systematic collection and sampling gives insight to the distribution of the L. pneumophila genus and to serotypes and genotypes in humans and in the environment. Based on the findings, actions in The Netherlands should be more aggressive for the European Working Group for Legionella Infections (EWGLI) genotypes 004 Lyon and 010 London. Legionellae are capable of infecting humans by aerosol inhalation or by drinking and subsequent aspiration of water.

An open study was carried out on 16 patients with hospital-acquired, bacteraemic Staphylococcus aureus infections to evaluate the safety and efficacy of teicoplanin plus rifampicin. Patients received teicoplanin 400 mg bd for the first 24 h followed by 400 mg od thereafter, and rifampicin 600 mg bd. Both agents were given intravenously. Serum samples were collected to determine trough and peak antibiotic concentrations. The MIC of teicoplanin and rifampicin and the MBC of teicoplanin were determined for all S. aureus isolates. Time-kill curves were performed for the drugs individually and in combination. Clinical efficacy was assessed by the APACHE II scoring system. Bacteriological success was evaluated by elimination, persistence or recurrence of S. aureus. Safety was carefully monitored by regular biochemical and haematological testing and recording of adverse events. Fifteen patients were evaluable, of whom 13 (86.7%) were clinically cured with elimination of S. aureus. One patient died, but death was not attributed to the study drugs. Treatment failed in another patient who relapsed with a high fever. S. aureus was recovered from blood cultures from this patient, and resistance to rifampicin had developed. Time-kill curves all showed adequate killing of S. aureus at the drug concentrations measured in vivo. Neither synergy nor antagonism between teicoplanin and rifampicin was demonstrated. The combination of teicoplanin and rifampicin is an effective and well-tolerated treatment for bacteraemic S. aureus infections, but in deep-seated foci of infection resistance to rifampicin may develop.

To predict at an early phase the clinical course and outcome of nosocomial Staphylococcus aureus bacteremia, the APACHE II score at onset of bacteremia was calculated in 99 patients. A delta APACHE II score (i.e., the difference between this score and one calculated for the day before the bacteremia) was also determined. This delta APACHE II score was highly significantly correlated with clinical course (P

With continuous arteriovenous haemodiafiltration (CAVHD), time-averaged clearance rates are higher than with intermittent haemodialysis. Indeed, drug removal rates may be high enough to warrant dose adjustment. In this study we measured the rate of drug clearance by CAVHD for 7 commonly used antibiotics: cefuroxime, cefotaxime, ceftazidime, imipenem, ciprofloxacin, tobramycin and vancomycin. By combining our data on clearance rates by CAVHD with literature data on drug distribution volumes and nonrenal clearance rates, we developed guidelines for drug dosage in intensive care patients treated by CAVHD. Dose adjustments during CAVHD treatment were deemed necessary for cefuroxime, ceftazidime, tobramycin and vancomycin.

Study objectives: Given an observed geographical variation in Legionnaires’ disease incidence in The Netherlands, the aim of the study was to test the hypothesis that the type of drinking water production was an independent determinant of the incidence of Legionnaires’ disease. Design: For the 1987–2005 period, the incidence of Legionnaires’ disease in The Netherlands and the price of water as a proxy for production type was studied at the municipal level. The data on the price of water were available at the municipal level. Methods: For each of the 466 municipalities in The Netherlands a mean standardised incidence rate per 100 000 inhabitants over the 1987–2005 period was calculated, excluding patients with the most probable source of infection abroad or in hospital. Logistic regression was used to assess the relation of the price of water to the incidence rates. In order to control for diagnostic and inclusion bias, they were estimated using questionnaire data collected from all 62 medical microbiology laboratories in the country. Main results: The incidence of Legionnaires’ disease varied between municipalities from 0.0 to 5.6 per 100 000 person-years. In univariate analysis high versus low water price was positively associated with a high municipal incidence rate (odds ratio (OR) 1.9; 95% CI 1.5–2.6). The association persisted (OR 5.1; 95% CI 3.2–8.0) after correction for diagnostic and inclusion bias. Conclusions: The price of water as a proxy for the type of water production was an independent risk factor for high municipal Legionnaires’ disease incidence in The Netherlands. This can guide future prevention policies.

In 1999, an outbreak involving 188 patients with Legionnaires' disease (LD) occurred at a flower show in the Netherlands. This large outbreak provided the opportunity to evaluate serum antibody tests to assay anti-Legionella pneumophila, since limited data are available on the sensitivity of these tests. The sensitivities of an indirect serotype 1-6 immunofluorescence antibody test (IFAT), a rapid micro-agglutination test (RMAT) IgM serotype 1 antibody assay, and an ELISA to detect IgM and IgG serotype 1-7 antibodies, were evaluated using serum samples from LD patients related to the 1999 outbreak. Sensitivity was calculated using positive culture and/or a positive urinary antigen test as the gold standard in outbreak-related patients with radiographically confirmed pneumonia who fulfilled the epidemiological criteria. The IFAT, RMAT and ELISA showed sensitivities of 61, 44 and 64%, respectively. The sensitivity of the three tests combined was 67%. In epidemic situations, however, high standing titres may be included in the laboratory evidence of LD cases. In the study population, high standing titres were found in 16% of cases. If the presence of high standing antibody titres was added to the criteria of a positive test, the sensitivities of IFAT, RMAT and ELISA were 86, 48 and 75%, respectively. The sensitivity was 91% for all tests combined. The higher sensitivity for the combined use of tests is offset by a reduction in specificity to 97.6%. The results of this study indicate that using a combination of serologic tests in pneumonia patients suspected to have LD does not substantially improve sensitivity. The results suggest that in the microbiological diagnosis of LD, both IFAT and ELISA are reasonably sensitive assays. In an epidemic situation, both tests are highly sensitive, the IFAT more so than the ELISA.

Background To stop a possible outbreak of Legionnaires’ disease (LD) at an early stage an outbreak detection programme was installed in The Netherlands. Methods The programme consisted of sampling and controlling of potential sources to which LD patients had been exposed during their incubation period. Potential sources were considered to be true sources of infection if two or more LD patients (cluster) had visited them, or if available patients’ isolates and environmental Legionella spp. were indistinguishable by amplified fragment length polymorphism genotyping. Results Rapid sampling and genotyping as well as cluster recognition helped to target control measures. Despite these measures, two small outbreaks were only stopped after renewal of the water system. The combination of genotyping and cluster recognition lead to 29 of 190 (15%) patient–source associations. Conclusion Systematic sampling and cluster recognition can contribute to outbreak detection and lead to cost-effective secondary prevention of Legionnaires’ disease.

Since 1977, the diagnostic tools for Legionnaires' disease have been culture and serological investigation. Both methods require considerable time to produce results and have low to reasonable sensitivity. Since the introduction of urinary antigen tests in the mid 1990s, underdiagnosis has diminished and mortality has declined, thanks to early diagnosis. To obtain the most accurate diagnosis, culture, serological investigation, and urinary antigen testing should all be performed. In the last decade, much effort has been directed toward the development of assays detecting Legionella nucleic acid. Thus far, only widely varying results with small patient series have been reported. Furthermore, these assays are labour intensive and complicated. As a result, these assays are not yet suitable for the average medical microbiological laboratory.

In 1999 an outbreak involving 188 patients with Legionnaires' disease (LD) occurred among visitors to a flower show in the Netherlands. Two enzyme immunoassays (Binax and Biotest) and one immunochromatographic assay (Binax NOW) were tested, using urine samples from LD patients from the 1999 outbreak. Sensitivity was calculated using positive culture and/or seroconversion as the “gold standard” in outbreak-related patients with radiographically confirmed pneumonia who fulfilled the epidemiological critera. The Binax EIA, Biotest EIA, and Binax NOW assay showed overall sensitivities of 69, 71, and 72%, respectively. When the tests were performed with concentrated urine samples, the overall sensitivities increased to 79, 74, and 81%, respectively. Using multiple logistic regression analysis with backward elimination, a statistically significant association was found between clinical severity and test sensitivity for all tests. For patients with mild LD, the test sensitivities ranged from 40 to 53%, whereas for patients with severe LD who needed immediate special medical care, the sensitivities reached 88 to 100%. These findings have major implications for the diagnostic process in patients with mild pneumonia and suggest that patients with mild pneumonia may go underdiagnosed if urine antigen tests alone are used.

Studies of Staphylococcus aureus nasal carriage have distinguished three carriage patterns: persistent, intermittent, and noncarriage. The criteria used to identify these carriage patterns have been inconsistent. In 1988 the S. aureus nasal carrier index, i.e., the proportion of nasal swab specimen cultures yielding S. aureus , was determined for 91 staff members of various departments of a large university hospital by obtaining weekly nasal swab specimens for culture over a 12-week period. Thirty-three (36%) persons had carrier indices of 0.80 or higher, 15 (17%) had indices between 0.1 and 0.7, and 43 (47%) had indices of zero. In 1995, 17 individuals with carrier indices of 0.80 or higher in 1988 were available for reexamination. For 12 (71%) of these individuals, S. aureus was again isolated from a single nasal swab, i.e., from each individual with a 1988 carrier index of 1.0 but from only half of those with indices below 1.0. Genotyping (by randomly amplified polymorphic DNA analysis and pulsed-field gel electrophoresis) of all S. aureus strains showed that strains isolated from only three individuals, all with 1988 carrier indices of 1.0, in 1988 and 1995 showed genetic similarity. In conclusion, persistent S. aureus nasal carriage is a unique characteristic of a fraction of the population, and the attribute “persistent” should be confined to those individuals for whom serial nasal swab specimen cultures consistently yield S. aureus .

In January, 1996, a 64-year-old man was admitted with pneumonia to a regional hospital in Haarlem, Netherlands. Culture of bronchial lavage was positive for Legionella pneumophila serogroup 1. He was treated with ciprofloxacin, because of hypersensitivity to combined rifampicin and erythromycin. He recovered and left hospital after 7 weeks. His disease had started with flu-like symptoms 5 days after visiting a sauna. Water samples were taken from the sauna water-heating system, showerheads, swimming pool, and footbaths. In one sample, taken from an air-perfused footbath, L pneumophila was found. Genetic typing of this strain by arbitrarily primed PCR 1 with multiple DNA primers showed the strain was the same as that of the patient. Looking back over 1991–96 health-surveillance data on Legionnaire’s disease, five more cases (two with a fatal outcome) were found to be related to the same sauna. Five of the six patients were men (mean age of 50 years); the one woman was aged 28 years. She had had a renal transplantation and died 10 days after visiting the sauna of complicated pneumonia due to the same strain of L pneumophila as the index patient. Disturbed immune status was also found in a 55-year-old man with a history of hairy-cell leukaemia. The other fatal case was a 59-year-old man with no history of immunosuppression. All six cases were regular visitors to the sauna, mostly on a monthly basis. All but one were heavy smokers. The first case of Legionnaire’s disease related to the sauna was in December, 1992. For two recent cases, the L pneumophila strains (obtained 1 month and 14 months before the index case was culture positive) were still available. The genetic profiles of these L pneumophila strains were identical to that of the strain isolated from the sauna’s footbath. The hot-water installation of the sauna was altered by the removal of all elements that could possibly cause standing or slow-flowing water. Since then, cultures of water samples taken over 3 months have remained negative for L pneumophila, and no new cases have been reported. It has been suggested that regular sauna bathing reduces the incidence of certain infections, although our report describes the first documented cases of lethal sauna-associated Legionnaire’s disease, and confirms existing knowledge of long-lasting spread of L pneumophila due to a persistent source of infection.

Background Discrimination between clinical and environmental strains within many bacterial species is currently underexplored. Genomic analyses have clearly shown the enormous variability in genome composition between different strains of a bacterial species. In this study we have used Legionella pneumophila, the causative agent of Legionnaire's disease, to search for genomic markers related to pathogenicity. During a large surveillance study in The Netherlands well-characterized patient-derived strains and environmental strains were collected. We have used a mixed-genome microarray to perform comparative-genome analysis of 257 strains from this collection. Results Microarray analysis indicated that 480 DNA markers (out of in total 3360 markers) showed clear variation in presence between individual strains and these were therefore selected for further analysis. Unsupervised statistical analysis of these markers showed the enormous genomic variation within the species but did not show any correlation with a pathogenic phenotype. We therefore used supervised statistical analysis to identify discriminating markers. Genetic programming was used both to identify predictive markers and to define their interrelationships. A model consisting of five markers was developed that together correctly predicted 100% of the clinical strains and 69% of the environmental strains. Conclusions A novel approach for identifying predictive markers enabling discrimination between clinical and environmental isolates of L. pneumophila is presented. Out of over 3000 possible markers, five were selected that together enabled correct prediction of all the clinical strains included in this study. This novel approach for identifying predictive markers can be applied to all bacterial species, allowing for better discrimination between strains well equipped to cause human disease and relatively harmless strains.

In patients with acute renal failure, who were treated with continuous arteriovenous hemofiltration (CAVH) or continuous arteriovenous hemodiafiltration (CAVHD), we measured clearance rates of imipenem and cilastatin (Tienam-500®). Literature data on volume of distribution and on the endogenous clearance in normals and in anuric patients and the observed clearance rates by CAVH/CAVHD were used to develop guidelines for dose adaptations. Based on the desired peak levels of imipenem, normal subjects should receive the fixed imipenem/cilastatin dose combination (500 mg/500 mg) q.i.d. and patients with acute renal failure should receive the same dose b.i.d. After starting treatment with either CAVH, CAVHD or continuous venovenous hemofiltration (CVVH), no further dose adjustment is necessary. The non-renal clearance rate of cilastatin is very low compared to that of imipenem. If a patient develops anuria, the clearance rate of imipenem decreases from the normal value of 245 ml/min to 116 ml/min. Clearance rate of cilastatin, however, decreases from 230 ml/min to 3 ml/min. Therefore, in patients with renal failure accumulation of cilastatin will occur. On the other hand, if the patient is treated by CAVHD, the relative contribution of the dialyser clearance to the total drug clearance is much greater for cilastatin than for imipenem. As a result, the accumulation of cilastatin is reversed. During treatment by CAVHD, the clearance rate of imipenem raises 15%–25% and that of cilastatin 335%–600%. For this reason, we conclude that the use of the fixed dose combination (500 mg/500 mg) b.i.d. in patients with acute renal failure treated by CAVHD may be justified.

A new immunochromatographic assay for rapid qualitative detection of Legionella pneumophila serogroup 1 antigen in urine specimens was used during an outbreak of legionellosis in The Netherlands. The assay seems of the utmost value in providing a rapid diagnosis of Legionnaires' disease in patients with severe community-acquired pneumonia in an outbreak setting.

The aim of this study is to evaluate by means of the kinesiographic test, how the muscular alteration, linked with mouth breathing, modifies mandibular dynamics. From a sample of children aged between 8-11 years, on the basis of increased nasal airways resistance measured with the forced oscillation technique, we selected a group of 14 subjects. These children underwent kinesiographic examination (K-System Biotronic) and they were compared to a control group. Certainly, the speeds reduction in the pathological group emerged as the most significant data. The mastication tracings on the frontal plane, in the group with increased nasal airways resistance, moreover appeared to be reduced in width, elongated in shape and sometimes irregularly square, compared to the bell shape typical of subjects without any dysfunctional pathologies. Considering that each subject responds in a personal manner to an increase of the nasal resistances, due to the extremely variable individual adaptability, the kinesiographic test, together with the electromyographic one, in our opinion, can assist anamnesis, clinical examination and other instrumental investigations, in the identification of those subjects in which the increase of the nasal airways resistance interferes with muscular function, laying the basis for possible modifications in the craniofacial growth.