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Background: Critically ill COVID-19 patients have proven to be at risk for developing invasive fungal infections. However, the incidence and impact of possible/probable COVID-19-associated pulmonary aspergillosis (CAPA) in severe COVID-19 patients varies between cohorts. We aimed to assess the incidence, risk factors, and clinical outcome of invasive pulmonary aspergillosis in a regional cohort of COVID-19 intensive care patients. Methods: We performed a regional, multicentre, retrospective cohort study in the intensive care units (ICUs) in North Brabant, The Netherlands. We included adult patients with rt-PCR-confirmed SARS-CoV-2 infection (COVID-19), requiring mechanical ventilation for acute respiratory distress syndrome. Demographics, clinical course, biomarker value, and treatment outcomes were compared between the groups with possible/probable CAPA from the main study centre and the regional centres, and without signs of CAPA from the main study centre as controls. The primary aim was to assess the regional impact of possible/probable CAPA in COVID-19 ICU patients, measured as all-cause mortality at 30 days after ICU admission. Secondary outcomes were risk factors for developing CAPA, based on underlying host factors and to identify the value of the mycological arguments for the diagnosing of CAPA. Results: Between 1 March and 30 April 2020, we included 123 patients with severe COVID-19: 29 patients (30.9%) in the main ICU with possible/probable CAPA, and 65 (69.1%) with no signs of CAPA; 29 patients in the regional ICUs with signs of CAPA. Patients’ characteristics and risk factors did not differ for CAPA and non-CAPA patients. Patients with COPD and/or chronic steroid medication developed CAPA more frequently, although this was not statistically significant. CAPA patients were admitted to the ICU earlier, had lower PF-ratios, and more often required renal replacement therapy. All-cause 30-day mortality was significantly higher in mechanically ventilated COVID-19 patients with possible/probable CAPA 39.7% (23/58) compared to patients without evidence for CAPA 16.9% (11/65) (OR 3.2 [95% CI 1.4–7.4] p = 0.005). Conclusion: The high incidence of possible and probable CAPA in critically ill COVID-19 patients is alarming. The increase in 30-day mortality in CAPA highlights the need for active surveillance and management strategies in critically ill COVID-19 patients.

Background: 10 days after the first reported case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the Netherlands (on Feb 27, 2020), 55 (4%) of 1497 health-care workers in nine hospitals located in the south of the Netherlands had tested positive for SARS-CoV-2 RNA. We aimed to gain insight in possible sources of infection in health-care workers. Methods: We did a cross-sectional study at three of the nine hospitals located in the south of the Netherlands. We screened health-care workers at the participating hospitals for SARS-CoV-2 infection, based on clinical symptoms (fever or mild respiratory symptoms) in the 10 days before screening. We obtained epidemiological data through structured interviews with health-care workers and combined this information with data from whole-genome sequencing of SARS-CoV-2 in clinical samples taken from health-care workers and patients. We did an in-depth analysis of sources and modes of transmission of SARS-CoV-2 in health-care workers and patients. Findings: Between March 2 and March 12, 2020, 1796 (15%) of 12 022 health-care workers were screened, of whom 96 (5%) tested positive for SARS-CoV-2. We obtained complete and near-complete genome sequences from 50 health-care workers and ten patients. Most sequences were grouped in three clusters, with two clusters showing local circulation within the region. The noted patterns were consistent with multiple introductions into the hospitals through community-acquired infections and local amplification in the community. Interpretation: Although direct transmission in the hospitals cannot be ruled out, our data do not support widespread nosocomial transmission as the source of infection in patients or health-care workers. Funding: EU Horizon 2020 (RECoVer, VEO, and the European Joint Programme One Health METASTAVA), and the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Objectives Fosfomycin susceptibility testing is complicated and prone to error. Before using fosfomycin widely in patients with serious infections, acquisition of WT distribution data and reliable susceptibility testing methods are crucial. In this study, the performance of five methods for fosfomycin testing in the routine laboratory against the reference method was evaluated. Methods Ten laboratories collected up to 100 ESBL-producing isolates each (80 Escherichia coli and 20 Klebsiella pneumoniae). Isolates were tested using Etest, MIC test strip (MTS), Vitek2, Phoenix and disc diffusion. Agar dilution was performed as the reference method in a central laboratory. Epidemiological cut-off values (ECOFFs) were determined for each species and susceptibility and error rates were calculated. Results In total, 775 E. coli and 201 K. pneumoniae isolates were tested by agar dilution. The ECOFF was 2 mg/L for E. coli and 64 mg/L for K. pneumoniae. Susceptibility rates based on the EUCAST breakpoint of ≤32 mg/L were 95.9% for E. coli and 87.6% for K. pneumoniae. Despite high categorical agreement rates for all methods, notably in E. coli, none of the alternative antimicrobial susceptibility testing methods performed satisfactorily. Due to poor detection of resistant isolates, very high error rates of 23.3% (Etest), 18.5% (MTS), 18.8% (Vitek2), 12.5% (Phoenix) and 12.9% (disc diffusion) for E. coli and 22.7% (Etest and MTS), 16.0% (Vitek2) and 12% (Phoenix) for K. pneumoniae were found. None of the methods adequately differentiated between WT and non-WT populations. Conclusions Overall, it was concluded that none of the test methods is suitable as an alternative to agar dilution in the routine laboratory.

Item does not contain fulltext To rapidly assess possible community transmission in Noord-Brabant, the Netherlands, healthcare workers (HCW) with mild respiratory complaints and without epidemiological link (contact with confirmed case or visited areas with active circulation) were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Within 2 days, 1,097 HCW in nine hospitals were tested; 45 (4.1%) were positive. Of six hospitals with positive HCW, two accounted for 38 positive HCW. The results informed local and national risk management.

Highlights • Naso/oropharygeal swabs and deep respiratory samples, but not feces, show good clinical performance in Cobas®6800 SARS-CoV-2 assay. • No significant difference was found after one freeze-thaw cycler with or without lysis buffer. • Time-to-result is lower using Cobas®6800 SARS-CoV-2 assay compared to LDA., Introduction Studies describing the performance characteristics of the cobas®6800 system for SARS-CoV-2 detection in deep respiratory specimens and freeze-thaw stability are limited. The current study compares the clinical performance of the automated SARS-CoV-2 assay on the cobas®6800 system to a lab-developed assay (LDA) and the cobas impact of freeze-thawing combined with lysis buffer. Methods Both retrospective and prospectively selected deep respiratory samples and oro- and nasopharyngeal samples in either E-swab® or GLY- were tested using the SARS-CoV-2 assay on the cobas®6800 System and compared to a lab developed assay. Additonally, SARS-CoV-2 RNA stability was assessed after one freeze-thaw cycle with or without lysis buffer. Results In total, 221 (58.3 %) oro- and nasopharyngeal swabs, 131 (34.6 %) deep respiratory specimens, and n = 25 (6.6 %) swabs of unknown origin were included to study clinical performance. Only 4 samples gave discrepant results, all being positive in the LDA and not the cobas®6800 system. For stability testing, 66 samples without and 110 with lysis buffer were included. No clinically significant difference was found in test results after one freeze-thaw cycle and addition of lysis buffer. Conclusion Based on our findings, the cobas®6800 SARS-CoV-2 RNA assay yielded similar results as the LDA in oro-/nasopharyngeal swabs and deep respiratory specimens. Moreover, the cobas®6800 SARS-CoV-2 RNA assay yielded similar results before and after a freeze-thaw cycle, with better preservation of low viral loads in lysis buffer.

Rapid and accurate detection of carbapenemase-producing Enterobacteriaceae is pivotal for adequate antibiotic therapy and infection control. The Cepheid Xpert Carba-R assay detects and identifies the most prevalent carbapenemases (KPC, VIM, IMP, NDM and OXA-48), using automated real-time PCR. The test performance of the Xpert Carba-R was evaluated with 129 well-characterized non-repeat Enterobacteriaceae isolates, suspected for carbapenemase production, i.e. with meropenem MICs >0.25 mg l−1. The isolate collection contained 100 carbapenemase-producing isolates (36 KPC-2 or KPC-3, 20 VIM-1, 4 KPC-2 plus VIM-1, 5 NDM-1, 2 IMP-1, 1 IMP-28, 1 IMP-1 plus VIM-1 and 31 OXA-48 like) and 29 negative control isolates producing extended-spectrum β-lactamase and/or AmpC β-lactamases. PCR and sequencing of β-lactamase genes were used as reference tests. The sensitivity of the Xpert Carba-R was 100 % (100/100), with a 100 % (29/29) specificity. The time to result was approximately 55 min with a hands-on time of only 1 min per isolate. In conclusion, the Carba-R assay is a rapid and accurate instrument for the confirmation and identification of the bla KPC, bla VIM, bla IMP, bla NDM and bla OXA-48 genes.

The aim of this study was to analyze the epidemiology in the bacteriological profile and susceptibility of clinically relevant bacterial pathogens in a burn center in the Netherlands over a 7-year period. The swab results of 693 patients of the period 2005 to 2008 and 539 patients of the period 2009 to 2011 were studied for change of microorganisms and antibiotic resistance. Definitions according to the Working Party on Infection Prevention were used as a tool for assessing the scale of the resistance problem at a local level. Between the studied periods only small changes were found in the bacteriological profile. Staphylococcus aureus showed a slight increase of prevalence in inventory swabs during the second period. In both inventory and wound swabs, S. aureus is the most frequently isolated clinically relevant bacterial pathogen. Resistance for ciprofloxacin in Escherichia coli increased from 3% in 2005 to 2008 to 7% in 2009 to 2011 (P = .028). Resistance for cefotaxim in E. coli increased from 4% in 2005 to 2008 to 14% in 2009 to 2011, although this decrease was not statistically significant (P = .24). The prevalence of highly resistant microorganisms (HRMOs) remained low in both time periods, 4.9% in 2005 to 2008 and 7.4% in 2009 to 2011 (P = .063). The Netherlands is considered a low-prevalence country for antimicrobial resistance, and the occurrence of HRMOs in our center is relatively rare. A large percentage of HRMO isolates were extended spectrum β-lactamase producers, indicating the rapid increase in the production of this resistance mechanism in recent years. The transmission of HRMOs in our center is controlled effectively, using well-established transmission-based precautions.

Generally accepted laboratory methods that have been used for decades do not reliably distinguish between H. influenzae and H. haemolyticus isolates. H. haemolyticus strains are often incorrectly identified as nontypeable Haemophilus influenzae (NTHi). To distinguish H. influenzae from H. haemolyticus we have created a new database on the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) bio-typer 2 and compared the results with routine determination of Haemophilus (growth requirement for X and V factor), and multilocus sequence typing (MLST). In total we have tested 277 isolates, 244 H. influenzae and 33 H. haemolyticus. Using MLST as the gold standard, the agreement of MALDI-TOF MS was 99.6 %. MALDI-TOF MS allows reliable and rapid discrimination between H. influenzae and H. haemolyticus.

Item does not contain fulltext OBJECTIVES: Routine use of disk diffusion tests for detecting antibiotic resistance in Legionella pneumophila has not been described. The goal of this study was to determine the correlation of MIC values and inhibition zone diameter (MDcorr) in clinical L. pneumophila isolates. METHODS: Inhibition zone diameter of 183 L. pneumophila clinical isolates were determined for ten antimicrobials. Disk diffusion results were correlated with MICs as determined earlier with E-tests. RESULTS: Overall the correlation of MIC values and inhibition zone diameters (MDcorr) of the tested antimicrobials is good, and all antimicrobials showed a WT distribution. Of the tested fluoroquinolones levofloxacin showed the best MDcorr. All macrolides showed a wide MIC distribution and good MDcorr. The MDcorr for cefotaxim, doxycycline and tigecycline was good, while for rifampicin and moxifloxacin, they were not. CONCLUSION: Overall good correlation between MIC value and disk inhibition zone were found for the fluoroquinolones, macrolides and cefotaxim.

This paper describes (1) the Highly Resistant Gram Negative Rod (HR-GNR) prevalence rate, (2) their genotypes, acquired resistance genes and (3) associated risk factors of HR-GNR colonization among the hospitalized population in the Dutch region Kennemerland. Between 1 October 2013 and 31 March 2014, cross-sectional prevalence measurements were performed in three regional hospitals as part of each hospitals infection control program. Rectal swabs were analyzed at the Regional Public Health Laboratory Kennemerland by direct culturing. Genotypes and acquired resistance genes of positive isolates were determined using Whole Genome Sequencing with the MiSeq instrument (Illumina). Association between several independent variables and HR-GNR positivity was examined using logistic regression models. Out of 427 patients, 24 HR-GNR positive isolates were recovered from 22 patients, resulting in a regional HR-GNR colonization prevalence (95 % CI) of 5.2 % (3.6–7.9). Of these 22 positive patients, 15 were Extended Spectrum Beta-Lactamase (ESBL) positive (3.5 % (2.1–5.7)), 7 patients were positive for a Fluoroquinolones and Aminoglycosides (Q&A) resistant Enterobacteriaceae (1.6 % (0.8–3.3)) and from one patient (0.2 % (0–1.3)) a Stenotrophomonas maltophilia resistant towards co-trimoxazole was isolated. No carbapenemase producing Enterobacteriaceae (CPE), multi-resistant Acinetobacter species or multi-resistant Pseudomonas aeruginosa were isolated. The ESBL genes found were bla CTX-M-1 (n = 4, 25.0 %), bla CTX-M-15 (n = 3, 18.8 %), bla CTX-M-27 (n = 2, 12.5 %), bla CTX-M-14b (n = 2, 12.5 %), bla CTX-M-9 (n = 2, 12.5 %), bla CTX-M-14 (n = 1, 6.3 %), bla CTX-M-3 (n = 1, 6.3 %), bla SHV-11 (n = 1, 6.3 %) and bla SHV-12 (n = 1, 6.3 %). Being known HR-GNR positive in the past was the only significant associated risk factor for HR-GNR positivity, odds ratio (95 % CI): 7.32 (1.82–29.35), p-value = 0.005. Similar ESBL prevalence rates and genotypes (3.5 %) were found in comparison to other Dutch studies. When previously HR-GNR positive patients are readmitted, they should be screened for HR-GNR colonization since colonization with GR-GNRs could be prolonged. We recommend for future studies to include all defined HR-GNRs in addition to ESBLs in prevalence studies, in order to obtain a more comprehensive overview of colonization with HR-GNRs.

We conducted a study on throat swabs obtained from a group of hospitalized patients with community acquired pneumonia (CAP). Throat swab specimens from 242 adults admitted to hospital with CAP were tested. In total, 1 or more aetiological agents were identified by real-time PCR in 55 (23%) patients. The most frequently detected pathogens were coronavirus (17%), parainfluenza virus (6%) and influenza virus (4%). Overall, viral pathogens were identified by conventional techniques in 7 (2%) patients, and real-time PCR in 50 (21%) patients (p

Increasing antibiotic resistance in gram-negative bacteria has recently renewed interest in colistin as a therapeutic option. The increasing use of colistin necessitates the availability of rapid and reliable methods for colistin susceptibility testing. We compared seven methods of colistin susceptibility testing (disk diffusion, agar dilution on Mueller-Hinton [MH] and Isosensitest agar, Etest on MH and Isosensitest agar, broth microdilution, and VITEK 2) on 102 clinical isolates collected from patient materials during a selective digestive decontamination or selective oral decontamination trial in an intensive-care unit. Disk diffusion is an unreliable method to measure susceptibility to colistin. High error rates and low levels of reproducibility were observed in the disk diffusion test. The colistin Etest, agar dilution, and the VITEK 2 showed a high level of agreement with the broth microdilution reference method. Heteroresistance for colistin was observed in six Enterobacter cloacae isolates and in one Acinetobacter baumannii isolate. This is the first report of heteroresistance to colistin in E. cloacae isolates. Resistance to colistin in these isolates seemed to be induced upon exposure to colistin rather than being caused by stable mutations. Heteroresistant isolates could be detected in the broth microdilution, agar dilution, Etest, or disk diffusion test. The VITEK 2 displayed low sensitivity in the detection of heteroresistant subpopulations of E. cloacae . The VITEK 2 colistin susceptibility test can therefore be considered to be a reliable tool to determine susceptibility to colistin in isolates of genera that are known not to exhibit resistant subpopulations. In isolates of genera known to (occasionally) exhibit heteroresistance, an alternative susceptibility testing method capable of detecting heteroresistance should be used.

The aetiology of acute exacerbations of chronic obstructive pulmonary disease (COPD) is heterogeneous and still under discussion. Serological studies have suggested that Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila may play a role in acute exacerbations of COPD. The presence of these atypical pathogens in sputum samples was investigated in patients with stable COPD and with acute exacerbations of COPD using real-time PCR. The present study was part of a randomised, double-blind, single-centre study and a total of 248 sputum samples from 104 COPD patients were included. In total, 122 samples obtained during stable disease (stable-state sputa) and 126 samples obtained during acute exacerbations of COPD (exacerbation sputa) were tested. Of the 122 stable-state sputa, all samples were negative for M. pneumoniae and C. pneumoniae DNA, whereas one sample was positive for Legionella non-pneumophila DNA. Of the 126 exacerbation sputa, all samples were negative for M. pneumoniae and C. pneumoniae DNA, whereas one sample was positive for Legionella non-pneumophila DNA. The possible relationship between the presence of atypical pathogens and the aetiology of acute exacerbations in chronic obstructive pulmonary disease was investigated in patients with stable disease and in those with acute exacerbations using real-time PCR. No indication was found of a role for Legionella spp., Chlamydia pneumoniae or Mycoplasma pneumoniae in stable, moderately severe chronic obstructive pulmonary disease and in its exacerbations.

Bartonella (B.) henselae is the causative agent of cat-scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. This study reports the development and evaluation of an internally controlled real-time polymerase chain reaction targeting the groEL gene for detection of Bartonella spp. DNA was extracted using the MagNA Pure system. The lower detection limit was 10-100 fg DNA and the in vitro sensitivity of the assay was not affected by duplexing with an internal control PCR. The real-time PCR assay detected DNA from all five B. henselae strains tested, and from B. birtlesii, B. vinsonii subsp. vinsonii, B. vinsonii subsp. arupensis and B. doshiae. The assay generated negative results with a selection of other bacteria, including several Mycobacterium spp., Streptococcus pyogenes and Staphylococcus aureus. Results of real-time PCR in clinical samples were compared with those of a conventional 16S rDNA-based PCR assay. During the period described in the Material and methods section, real-time PCR and conventional 16S PCR were performed on 73 clinical samples. Of these samples, 29 (40%) were found to give positive results and 44 (60%) gave negative results, both by real-time PCR and by conventional PCR, with a 100% agreement between the two tests. The PCR developed in this study is a rapid, sensitive, and simple method for the detection of Bartonella spp. in CSD and is suitable for implementation in the diagnostic laboratory.

Our aim was to investigate the occurrence and identity of Legionella spp. in Dutch tap water installations using culture, real-time PCR and sequence analysis. The PCR assays used were a 16S rRNA gene based PCR with both a Legionella species specific probe and a L. pneumophila specific probe and a L. pneumophila-specific PCR based on the sequence of the mip gene. A total of 357 water samples from 250 locations in The Netherlands was investigated. The detection rates of Legionella spp. were 2,2% (8 of 357) by culture, and 87,1% (311 of 357) by PCR. The majority of samples was found to contain Legionella species other than L. pneumophila. These comprised of Legionella Like Amoebal Pathogens (LLAPs), L. busanensis, L. worsliensis and others. Fourteen (3,9%) samples were positive for L. pneumophila by either culture, 16S rRNA based PCR and/or mip based PCR. It is apparent from this study that Legionella spp. DNA is ubiquitous in Dutch potable water samples. Our findings further suggest that LLAPs and viable but nonculturable (VBNC) Legionella represent a large proportion of the population in man-made environments.

Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially in the ability to diagnose Legionnaires' disease (LD) at an early stage of the disease in a specimen that is readily obtainable. The aim of this study was to assess the performance of PCR as a rapid diagnostic method and to compare the results of different PCR assays of serum samples from patients with LD. Samples included 151 serum samples from 68 patients with proven LD and 60 serum samples from 36 patients with respiratory tract infections other than Legionella. PCR assays were based on the 5S rRNA gene, 16S rRNA gene and the mip gene. The samples from patients with infections caused by pathogens other than Legionella all tested negative in PCR. Among the patients with proven LD 54.4 % (37/68) tested positive in 5S rRNA PCR, 52.9 % (36/68) in mip gene PCR and 30.9 % (21/68) in 16S rRNA PCR in the first available serum sample. The association between threshold cycle value in 5S PCR positive serum samples (n=49) and C-reactive protein value was determined, and showed a strong negative correlation (Pearson correlation coefficient r=−0.63, PLegionella DNA in serum could be a useful tool for early diagnosis of LD caused by any Legionella species and serogroup, and has the potential to provide a diagnosis in a time frame that could affect initial infection management.

We evaluated the abilities of the Vircell immunoglobulin G (IgG) and IgM indirect immunofluorescence assay (IFA) for Legionella pneumophila serogroup 1, the IgM and IgG enzyme-linked immunosorbent assay (ELISA) for Legionella pneumophila serogroup 1, and the IgM-plus-IgG ELISA for Legionella pneumophila serogroups 1 to 6 to diagnose Legionnaires' disease (LD) in a well-described sample of patients with and without LD. Also, we determined the agreements, sensitivities, and specificities of the different Vircell assays in comparison to a validated ELISA (Serion classic ELISA). Clinical sensitivity and specificity were 74.6% and 96.6%, respectively, for the IgM IFA, 65.1% and 88.0% for the IgG IFA, 92.3% and 100% for the IgM ELISA, 43.3% and 96.6% for the IgG ELISA, and 90.8% and 100% for the IgM-plus-IgG ELISA. Compared to Serion classic ELISA, agreement, sensitivity, and specificity were 80.0%, 83.1%, and 78.4%, respectively, for the IgM IFA, 75.2%, 66.0%, and 79.5% for the IgG IFA, 89.5%, 82.0%, and 97.6% for the IgM ELISA, 81.9%, 88.9%, and 78.0% for the IgG ELISA, and 93.5%, 90.0%, and 96.6% for the IgM-plus-IgG ELISA. The value of a positive diagnostic result obtained by the Vircell IgM IFA, the Vircell IgG IFA, and the Vircell IgG ELISA might not be acceptable for a diagnostic assay. Both the high specificities and sensitivities of the Vircell IgM ELISA and the IgM-plus-IgG ELISA and the high correlation with the Serion classic ELISA indicate that they are useful in the diagnosis of LD.

Recently there have been reports indicating an increased incidence of MRSA infections, afflicting individuals with no apparent risk factors for hospital acquisition. Patients with community-associated (CA) MRSA are significantly younger and had different distributions of clinical infections compared with HA-MRSA patients. CA-MRSA infections have mostly been associated with staphylococcal strains bearing the SCCmec type IV element and PVL genes. These strains are more frequently susceptible to a variety of non-beta-lactam antibiotics. Clinicians must be aware of the wide and, in some cases, unique spectrum of disease caused by CA-MRSA. Continued emergence of MRSA in the community is a public-health problem that warrants increased vigilance in the diagnosis and management of suspected and confirmed staphylococcal infections.

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Reported here is the case of a previously healthy 67-year-old man who was admitted to the intensive care unit with pneumonia caused by Legionella longbeachae. The organism was identified in sputum and serum by 16S rRNA-based PCR assay and sequence-based typing. One acute serum sample produced a single elevated IgM antibody titer of 1:512 against non-pneumophila Legionella spp. The patient fully recovered following the initiation of appropriate antibiotic treatment. Since most current laboratory tests for Legionella spp. cannot detect infections caused by non-pneumophila Legionella spp., culture on Legionella-selective media or PCR should be considered when diagnosing severe pneumonia of unknown etiology.

Diagnosis of Legionnaires’ disease (LD) in patients with pneumonia is based on phenotypic (culture, serologic testing, antigen detection in urine) and genotypic PCR methods. This chapter assesses the sensitivity and specificity of Legionella DNA detection using PCR on serum samples from patients with proven LD and patients with infections other than Legionella. Serum samples from two healthy volunteers were included as negative controls after every four samples. One patient was admitted to the emergency department of our hospital with proven LD caused by Legionella pneumophila serogroup 1 (urinary antigen test positive, culture positive, and sputum PCR positive). From this patient, consecutive serum samples were collected for Legionella-specific, real-time PCR. The results of PCR showed an increase in Ct values (corresponding with a logarithmic decrease of bacterial DNA) in the course of time and were found to mirror the clinical condition of the patient and c-reactive protein values during the acute stage of infection. Serologic methods to diagnose L. pneumophila infections are highly sensitive, but their utility is generally limited to epidemiologic studies due to the time lag needed to detect seroconversion. The advantages of PCR for the detection of L. pneumophila DNA in serum are evident; serum samples are readily obtainable and can be processed within a working day.

A new methicillin-resistant Staphylococcus aureus (MRSA) clone related to pig and cattle farming was detected in the Netherlands. We investigated the extent of S. aureus presence in meat and found 36 S. aureus strains in 79 samples. Two strains were MRSA; 1 was multilocus sequence type 398, the clone related to farming.

Since antigen detection in urine has proved to be a sensitive and rapid method for detecting L. pneumophila serogroup 1, this technique has become one of the most frequently used tools for diagnosing Legionnaires’ disease (LD) [1]. Currently, there are several commercially available tests for detecting L. pneumophila antigen in urine [2, 3]. Commercial kits that use enzyme immunoassay (EIA) methodologies have been available for several years and perform similarly. One immunochromatographic (ICT) assay (Binax NOW Legionella Urinary Antigen Test; Binax, Portland, Maine, USA) has similar sensitivity and specificity to EIA [2]. ICT assays have important advantages over conventional EIAs: the tests are simple to perform, they do not require special laboratory equipment, and results can be obtained within 15 min. The aim of the study presented here was to evaluate a newly developed ICT urine antigen test (the Rapid U Legionella Plus Test; Diamondial, Sees, France), for its ability to detect L. pneumophila serogroup 1 in urine. We evaluated the ability of the new antigen test to detect LD in a well-described sample of patients with and without LD using frozen urine samples. The samples had been collected between 1997 and 2005 and stored at −70°C until processing. We included 71 urine samples obtained from 71 patients with LD (cases). A case of LD was defined as a patient with pneumonia who had radiological signs of infiltration and laboratory evidence of infection with L. pneumophila. Laboratory evidence included one or more of the following criteria: isolation of L. pneumophila from a lower respiratory tract (LRT) sample; a positive PCR result on a LRT sample using a 16S rRNA-based assay [4]; seroconversion to positive IgM and/or IgG antibodies against L. pneumophila in paired acute-phase and convalescentphase sera using a commercial ELISA (Serion ELISA; Institut Virion\Serion, Wurzburg, Germany). All LD-positive samples tested negative for pneumococcal antigen in urine (PAG; Binax NOW, Portland, Maine, USA). Urine samples of 91 patients with respiratory tract infections caused by pathogens other than Legionella were tested in a similar manner to test specificity. All patients were tested for LD using an immunochromatographic assay (Binax NOW); all samples tested negative. The laboratory results for these patients tested with blood cultures (blood), PAG detection in urine (PAG) and sputum culture (sputum) were as follows: Streptococcus pneumoniae (n=52 patients: detected in sputum in 8 patients, in blood and PAG in 23, and in blood alone in 21), Haemophilus influenzae (n=10: blood, 3; sputum, 7), Moraxella catarralis (n=3: all in sputum), Staphylococcus aureus (n=4: blood and sputum, 2; sputum, 2), Escherichia coli (n=2: blood and sputum, 1; sputum alone, 1), Acinetobacter baumannii (n=1: blood and sputum), Streptococcus pyogenes (n=2: both blood and sputum), Klebsiella pneumoniae (n=1: sputum), Mycobacterium tuberculosis (n=3: all sputum), Pneumocystis jirovecii (n=1: Giemsa and silver-stain positive). Twelve patients with a fourfold or greater rise in (complement-fixating) antibodies against influenza A virus (n=3), adenovirus (n=1), Chlamydia psittaci (n=3), Mycoplasma pneumoniae (n=4) and parainfluenza virus (n=1) were included. The presence of L. pneumophila antigen in urine samples was investigated using Rapid U Legionella Plus Test, a qualitative ICT assay. We compared the sensitivity and specificity of this test with that of a widely used ICT assay, the Binax Now urinary antigen test (Binax NOW). Eur J Clin Microbiol Infect Dis (2006) 25:733–735 DOI 10.1007/s10096-006-0213-0

The purpose of the present study was to assess the in vitro activity of daptomycin against a well-defined collection of methicillin-resistant Staphylococcus aureus (MRSA) isolates ( n = 98), including heterogeneously glycopeptide-resistant MRSA (hGISA) strains. Susceptibility testing was performed by using the Etest system. Daptomycin was potent against both glycopeptide-susceptible and hGISA strains.

MRSA ID was evaluated for its ability to identify methicillin-resistant Staphylococcus aureus . A well-defined collection of staphylococci was used ( n = 998). The sensitivity after 24 h was 96.4%, increasing to 98.8% after 48 h. The specificity was 98.2% after 24 h and decreased to 89.7% after 48 h.

The role of Legionella spp. in the aetiology of acute respiratory infections (ARIs) is largely unknown. In this case-control study, conducted in a general practitioner setting during 2000 and 2001, nose and throat samples from patients presenting with ARIs (n = 230) and controls (n = 200) were analysed for the presence of Legionella spp. by real-time PCR. Legionella DNA was not detected in any of the cases or controls. Thus, Legionella spp. do not seem to play a role in patients presenting with ARIs, nor were they present in patients who visited their general practitioner for complaints other than ARIs.

Item does not contain fulltext OBJECTIVES: The purpose of this study was to establish wild-type (WT) distributions and determine the epidemiological cut-off values (ECOFF) in clinical L. pneumophila serogroup 1 isolates for 10 antimicrobials commonly used for the treatment of Legionella infections using a method feasible in a routine clinical laboratory. METHODS: MICs of 183 clinical L. pneumophila serogroup 1 isolates, collected as part of an outbreak detection program, were tested using E-test methodology on buffered charcoal yeast extract agar supplemented with alpha-ketoglutarate (BCYE-alpha). The MICs were read after 2 days of incubation at 35 degrees C with increased humidity and without CO(2). ECOFFs were determined according to EUCAST methodology and expressed as WT

In 271 Enterobacter blood culture isolates from 12 hospitals, extended-spectrum beta-lactamase (ESBL) prevalence varied between 0% and 30% per hospital. High prevalence was associated with dissemination, indicating the potential relevance of infection control measures. Screening with cefepime or Vitek 2, followed by a cefepime/cefepime-clavulanate Etest, was an accurate strategy for ESBL detection in Enterobacter isolates (positive predictive value, 100%; negative predictive value, 99%).

Here we report an outbreak among 17 patients caused by a single strain of a Multiresistant Methicillin-Susceptible Staphylococcus aureus (MR-MSSA) in a burn centre. The MR-MSSA strains were resistant to penicillin, ciprofloxacin, erythromycin, clindamycin and co-trimoxazole. Further analysis showed an increased prevalence of MR-MSSA carriership in S. aureus colonized patients admitted to the burn centre, from 0% in 2005 (0/118), 3.3% in 2006 (4/121), 6.1% in 2007 (6/99), to 7.8% in 2008 (7/90). Molecular typing with Amplified Fragment Length Polymorphism showed that all MR-MSSA isolates derived from burn centre patients had a unique genotype, and was different compared to isolates derived from other hospital patients. All healthcare workers (HCWs) who worked in the burn centre during the outbreak were screened for nasal carriage with MR-MSSA. One HCW tested positive for a genotype of MR-MSSA that was indistinguishable from the genotype found in samples of the burned patients. No new cases of MR-MSSA colonization or infection were identified after the colonized HCW stopped working at the burn centre. The routine practice of molecular typing of collected S. aureus strains from both patients and HCWs will help to detect nosocomial spread in a burn centre, and opens the possibility of a rapid, almost pre-emptive response.

Tigecycline is often one of the few therapeutic options for infections by multidrug-resistant Gram-negative bacteria. Therefore, accurate MIC testing is critical. Since in many laboratories tigecycline is not included in the routine panel of antibiotics for susceptibility testing, tigecycline MICs are frequently determined by Etest. However, previous studies have shown that tigecycline MICs determined by Etest may be influenced by the brand of Mueller-Hinton (MH) agar, hypothetically due to variations in the concentration of manganese (2). Furthermore, for Acinetobacter species with MICs ≥ 2 mg/liter by Etest, the MIC was at least 2 doubling dilutions lower by broth microdilution (BMD) (1). The aim of this study was to evaluate the Etest as a method to determine tigecycline MICs for Enterobacter species. Tigecycline MICs were determined by BMD and Etest for 271 Enterobacter blood culture isolates (84% Enterobacter cloacae isolates and 16% Enterobacter aerogenes isolates) obtained in 2006 and 2007 from 12 laboratories in the Netherlands. BMD was performed using Becton Dickinson (BD) MH II medium for BMD (Merlin, Germany), according to International Organization for Standardization (ISO) guideline 20776-1 (3) (the reference method of the Clinical and Laboratory Standards Institute [CLSI]) and the European Committee on Antimicrobial Susceptibility Testing [EUCAST]). Etests (bioMerieux, France) were performed on agar with the same MH medium used in the BMD method and on Iso-Sensitest agar (Oxoid, United Kingdom). In each experiment, Escherichia coli ATCC 25922 was used as a control strain. FDA and EUCAST breakpoints were used (susceptible ≤ 2 mg/liter and ≤ 1 mg/liter, respectively). The manganese concentrations of the MH broth and both agars were determined using induction-coupled plasma atomic emission spectrometry (ICP-AES) after the medium was dissolved with 65% nitric acid. Compared to BMD, MICs determined by Etest were significantly higher using either MH agar or Iso-Sensitest agar (Fig. ​(Fig.11 and Table ​Table1).1). The correlation between the results of the BMD and Etest on MH agar was high (Spearman's nonparametric correlation coefficient, 0.9; P < 0.001), and the geometric mean difference ± standard deviation (SD) was 2.4 ± 0.4 doubling dilutions, pointing toward a systematic difference between the methods. In 84% of the isolates, tigecycline MICs were 2 or more doubling dilutions higher by Etest on MH agar compared to the values found by BMD. The MICs determined by Etest on MH agar were also significantly higher than by Etest on Iso-Sensitest agar. No correlation between the manganese concentration in the test medium and the tigecycline MIC was observed (Table ​(Table11). FIG. 1. Distribution of MICs of 271 Enterobacter isolates by BMD, Etest on BD Mueller-Hinton agar (MHA), and Etest on Iso-Sensitest agar. TABLE 1. Susceptibility results for 271 Enterobacter isolates using BMD, Etest on MHA (BD), and Etest on Iso-Sensitest agar The susceptibility rates were significantly higher by the BMD method than by Etest (Table ​(Table1).1). When FDA breakpoints were used, false nonsusceptibility was observed in 18% of the isolates when performing Etest on MH agar. When EUCAST breakpoints were used, 97% of isolates were susceptible by the BMD method, whereas only 30% were susceptible by Etest on MH agar, corresponding to false nonsusceptibility in 67% of the isolates. No false-susceptible Etest results were observed using either MH agar or Iso-Sensitest agar. The MICs of the E. coli control strain were within the specified range (0.03 mg/liter to 0.25 mg/liter) for the three methods, although the MICs were 0.0325 mg/liter using BMD and 0.25 mg/liter using Etest on MH agar. In conclusion, this study shows that tigecycline MICs for Enterobacter species determined by Etest are significantly higher than MICs determined by BMD. Differences in the manganese concentration of the test media could not explain the observed differences, and further research is required to resolve this issue. We conclude that nonsusceptible tigecycline Etest results should be confirmed using a BMD method. The misclassification of isolates as tigecycline nonsusceptible may deny patients an important antibiotic option.

We evaluated a new immunochromatographic assay (Oxoid Xpect Legionella test kit) for the ability to detect Legionella pneumophila serogroup 1 antigen in urine. The results were compared with those obtained with the Binax NOW urinary antigen test by following the manufacturers’ instructions. The sensitivities and specificities were estimated to be 89 and 100%, respectively, for the Oxoid Xpect Legionella test kit and 86 and 100%, respectively, for the Binax NOW test.

The main aim of our study was to determine the added value of PCR for the diagnosis of Legionnaires’ disease (LD) in routine clinical practice. The specimens were samples submitted for routine diagnosis of pneumonia from December 2002 to November 2005. Patients were evaluated if, in addition to PCR, the results of at least one of the following diagnostic tests were available: (i) culture for Legionella spp. on buffered charcoal yeast extract agar or (ii) detection of Legionella pneumophila antigen in urine specimens. Of the 151 evaluated patients, 37 (25%) fulfilled the European Working Group on Legionella Infections criteria for a confirmed case of LD (the “gold standard”). An estimated sensitivity, specificity, and overall percent agreement of 86% (32 of 37; 95% confidence interval [CI] = 72 to 95%), 95% (107 of 112; 95% CI = 90 to 98%), and 93% (139 of 149), respectively, were found for 16S rRNA-based PCR, and corresponding values of 92% (34 of 37; 95% CI = 78 to 98%), 98% (110 of 112; 95% CI = 93 to 100%), and 97% (144 of 149), respectively, were found for the mip gene-based PCR. A total of 35 patients were diagnosed by using the urinary antigen test, and 34 were diagnosed by the 16S rRNA-based PCR. With the mip gene PCR one more case of LD ( n = 36; not significant) was detected. By combining urinary antigen test and the mip gene PCR, LD was diagnosed in an additional 4 (11%) patients versus the use of the urinary antigen test alone. The addition of a L. pneumophila -specific mip gene PCR to a urinary antigen test is useful in patients with suspected LD who produce sputum and might allow the early detection of a significant number of additional patients.

This case report describes a case of Legionnaires' disease for whom the source of infection was the campervan in which the patient had travelled for 3 months. This case shows that Legionnaires' disease can be acquired by exposure to a relatively new (not previously reported) source that is commonly used as (holiday)transportation vehicle.

We evaluated two new immunochromatographic assays for their abilities to detect Legionella pneumophila serogroup 1 antigen in urine. The results were compared with those obtained by the Binax NOW urinary antigen test. The sensitivities and specificities were estimated to be 71.2% and 96.6%, respectively, for the Rapid U test; 31.5% and 98.9%, respectively, for the SD Bioline test; and 91.8% and 100%, respectively, for the Binax NOW test.

Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially the ability to diagnose all Legionella spp. at an early stage. Detection of Legionella DNA in serum can be a valuable tool for the diagnosis of Legionnaires' disease (LD). This report describes two patients with LD diagnosed by PCR using serum samples. In addition, quantification of L. pneumophila DNA using real-time PCR during the course of illness was carried out. The results obtained mirrored both the clinical condition and C-reactive protein values during the course of the illness. Quantification of Legionella DNA in serum using real-time PCR could be a valuable tool to monitor the effects of antimicrobial therapy in patients with LD.

A pseudo-outbreak of hepatitis B virus caused by cross-contamination from a semiautomatic cap remover for blood collection tubes is reported. The source of the outbreak was elucidated by using basic epidemiological methods. Laboratories should always be critical about their results in order to identify contamination problems.

CHROMagar MRSA was evaluated for its ability to identify methicillin-resistant Staphylococcus aureus (MRSA). A well-defined collection consisting of 216 MRSA strains and 241 methicillin-susceptible Staphylococcus aureus isolates was used. The sensitivity of CHROMagar MRSA after 24 h of incubation was 95.4%, increasing to 100% after 48 h. The specificity was already 100% after 24 h.

PCR is increasingly being used as a diagnostic test for the detection of Bordetella pertussis and Bordetella parapertussis DNA, as it has improved sensitivity and specificity in comparison to conventional techniques. The assay described here uses the two insertion sequences IS 481 and IS 1001 for B. pertussis and B. parapertussis , respectively, with detection by molecular beacons. The real-time PCR for IS 481 detects both B. pertussis and Bordetella holmesii , and the real-time PCR for IS 1001 detects both B. parapertussis and B. holmesii . By performing both assays discrimination between B. pertussis and B. parapertussis can be obtained. The sensitivity was 1 to 10 CFU/ml for B. pertussis , 10 CFU/ml for B. parapertussis , and 10 CFU/ml for B. holmesii in both assays. The clinical sensitivity of the B. pertussis assay was not affected by duplexing with an internal control PCR. Real-time PCR, conventional PCR, and culture were performed on 57 clinical samples. Eight of the 57 (14%) were found positive by culture, 19 of 57 (33%) were found positive by conventional PCR, and 22 of 57 (39%) were found positive by real-time PCR. One sample was inhibitory. When the B. pertussis assay was compared with a clinical standard for B. pertussis infection, sensitivity was 38, 83, and 100% and specificity was 100, 97, and 97% for culture, conventional PCR, and real-time PCR, respectively. The real-time PCR designed for B. pertussis and B. parapertussis provides sensitive and specific diagnosis of B. pertussis and B. parapertussis infections and is therefore suitable for implementation in the diagnostic laboratory.

To the Editor: Drexler et al. recently compared the sensitivity of the BinaxNOW Influenza A & B Rapid Test (BinaxNOW; Inverness Medical, Cologne, Germany) with that of a real-time reverse transcription–PCR (RT-PCR) assay specific for influenza A pandemic (H1N1) 2009 virus (1). Of 1,838 clinical specimens tested, 221 were confirmed as positive for pandemic (H1N1) 2009 by RT-PCR. When 144 of these 221 specimens were evaluated by using the BinaxNOW, results were positive for only 16 (11%). At onset of the pandemic, we evaluated the first 135 nasopharyngeal aspirates submitted to the Regional Laboratory of Public Health Haarlem, the Netherlands. We compared the performance of the BinaxNOW for diagnosing influenza A (H1N1) virus by using molecular detection of influenza virus as the reference standard. Samples were analyzed with a general influenza A assay targeting the matrix gene (the RespiFinder assay) (PathoFinder B.V., Maastricht, the Netherlands [2]) and a pandemic (H1N1) 2009–specific RT-PCR assay targeting the neuraminidase gene (3). We tested 135 patient samples (76 from male patients); mean age of patients was 32 years (range 0–81 years). Samples from 38 (28%) patients had positive results in both RT-PCRs, and samples from 97 (72%) patients had negative results in the matrix gene RT-PCR and neuraminidase RT-PCR assays. Sensitivity and specificity were estimated to be 47% (18/38, 95% confidence interval [CI] 32%–62%) and 95% (92/97, 95% CI 88%–98%), respectively, for the BinaxNOW antigen test. Patients’ ages did not significantly differ between rapid test–positive and –negative results. Our results largely agree with those of Vasoo et al. (4) and the Centers for Disease Control and Prevention (5). Those studies determined that the sensitivity of the BinaxNOW compared with nucleic acid amplification tests is ≈40%. The lower sensitivity observed by Drexler et al. (1) might be because of differences in study type (retrospective evaluation compared with a prospective cohort in our study), sample size, technical factors (with regard to specimen collection, specimen transport, and specimen storage), differences in the test kit, and differences between individual patients (multiple categories of age and stages of illness, differences in virus shedding). Many clinicians are not aware of the performance of specific test devices and rely on test results to make clinical decisions. Because negative results cannot rule out influenza, this test is of little use in a clinical setting without appreciation of the limitations of the test. However, because the BinaxNOW has reasonable specificity, it might prove useful in clinical or epidemiologic situations in which test sensitivity is not critical, e.g., in facility outbreaks in which multiple specimens are collected to rapidly identify the causative organism.

We evaluated a new immunochromatographic assay (Legionella V-TesT, Coris BioConcept, Gembloux, Belgium) for its ability to detect Legionella pneumophila serogroup 1 antigen in urine. Test devices were read at various time points to determine the optimum incubation time regarding performance. The results were compared with those obtained with the BinaxNOW urinary antigen test. The sensitivity and specificity were 82.2% and 98.6%, respectively, for the Legionella V-TesT and 83.9% and 100%, respectively, for the BinaxNOW urinary antigen test after 15 min of incubation. When tests were examined after 60 min, the sensitivity for both tests increased to 91.5%.

In a recent study by McDonough et al., a Legionella cluster was identified through retrospective PCR analysis of 240 throat swab samples from cases of pneumonia among young and otherwise healthy U.S. military recruits ([4][1]). Results were confirmed by sequence analysis. No diagnostic evidence