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Invasive fungal infections (IFIs) are commonly observed in patients, who are at high risk of severe infections during the neutropenic phase. The aim of this retrospective single-center study was to evaluate the efficacy and safety of voriconazole as a fungal prophylaxis after induction chemotherapy for acute myeloid leukemia (AML) in adult patients. Six proven/probable IFIs were diagnosed in 213 patients with AML (median age 61 years, range 18-85), who received a total of 377 induction chemotherapies. This yielded an incidence rate of 1.6% based on all induction cycles administered. Voriconazole prophylaxis was administered as intended in 317 out of 377 (84%) induction cycles until the end of neutropenia with a median duration of 20 days (range: 2-101 days). In conclusion, voriconazole demonstrates efficacy and safety as a first-line IFI prophylaxis comparable to published data on posaconazole, which is the standard fungal prophylaxis recommendation for AML patients in international guidelines today.

BackgroundMorganellaare opportunistic pathogens involved in various infections. InMorganella, intrinsic resistance to multiple antibiotics including colistin combined with the emergence of carbapenemase-producers (CP) strongly limits the antimicrobial armamentarium.MethodsFrom 2013 to 2021, 172 highly drug-resistant (XDR)Morganellaisolates from 8 European countries and Canada, two reference strains from the Pasteur Institute collection and two susceptible isolates were characterized by WGS, antimicrobial susceptibility testing and biochemical tests. Complete genomes from Genbank (n=103) were included for genomic analysis. Intrinsic resistance mechanism to polymyxins was deciphered by combining genetic analysis with mass spectrometry on the lipid A.FindingsMorganellacould be separated into 4 species namedM. psychrotolerans, M. sibonii, M. morganiiand a new species represented by a unique strain.Morganella morganiiincluded two subspecies:M. morganiisubsp.morganii(the most prevalent) andM. morganiisubsp.intermedius. Intrinsic resistance to tetracycline and conservation of metabolic pathways correlated this refined taxonomy. CP were mostly identified among five ‘high-risk’ clones ofM. morganiisubsp.morganii. A single nucleotide polymorphism (SNP) cut-off of 100 was used to decipher outbreaks involving this species. Cefepime-zidebactam and ceftazidime-avibactam were the most potent antimicrobials towards the 172 XDRMorganellaspp. isolates of our collection (including 145 CP) except for metallo-β-lactamase-producers. The intrinsic resistance to polymyxins corresponds to the addition of L-Ara4N on the lipid A.InterpretationThis global characterization of the widest collection of XDRMorganellaspp. highlighted the need to clarify the taxonomy, deciphered intrinsic resistance mechanisms and paved the way for further genomic comparisons.FundingNoneRESEARCH IN CONTEXTEvidence before this studyOn January 28th2022, we have searched for the terms “Morganella” and “carbapenemase” in all published reports available in PubMed with no language restriction. We identified a total of 43 articles and most of them (41/43) corresponded to a report of a single isolate of carbapenemase-producingMorganella morganii. Only one article aimed to decipher the antimicrobial susceptibility on a collection ofProteus, ProvidenciaandMorganellaisolated from global hospitalized patients with intra-abdominal and urinary tract infections. However, this collection only included 7M. morganiiisolates. On March 2021, when we finished the inclusions in our collection, only 104 genomes ofMorganellaspp. were available in the NCBI database.Since September 2021, very few reports were published on carbapenemase-producing Morganella with the exception of a study from Xiang Get al. reported 40 multi-drug resistantM. morganiiisolates recovered from three hospitals in China from 2014 to 2020. Unfortunately, this collection included only two carbapenemase-producingM. morganiiisolates (one OXA-48 and one IMP-1). A report of KPC-producingM. morganiiin Japan and a longitudinal study of carbapemase-producing Enterobactrales in Taiwan that did not focused on Morganella.We also searched in PubMed for the terms ‘Morganella sibonii” or “Morganella psychrotolerans” in all published reports with no language restrictions. Our search identified a total of 20 articles. None of them was related to antimicrobial resistance and no study deciphered theMorganellaspp. epidemiology on clinical isolates.Added values of this studyThis global characterization involved the widest collection ofMorganellaspp. isolates ever reported (barely doubling the number ofMorganellaspp. genomes in Genbank). In addition, 145 isolates of this worldwide collection made of 172 multidrug resistantMorganellaspp. were carbapenemase producers for which therapeutic alternatives are scarce due to intrinsic resistance to last resort molecules, such as polymyxin.First, we found that cefepime-zidebactam and ceftazidime-avibactam were the most potent antimicrobials towards XDRMorganellaspp. isolates except for metallo-β-lactamase-producers.Then, we observed that carbapenemase-encoding genes were present in differentMorganellaspecies highlighting necessary changes in the taxonomy.Morganellagenus could be divided into 4 species namedM. psychrotolerans, M. sibonii, M. morganiiand a new species represented by a unique strain.Morganella morganiiincludes two subspecies:M. morganiisubsp.morganii(the most prevalent) andM. morganiisubsp.intermedius. We demonstrated that this refined taxonomy correlated with the intrinsic resistance to tetracycline, which was found only inM. sibonii, as well as several metabolic pathways (e.g. trehalose assimilation, type III (T3SS) and type IV secretion system (T6SS), etc.…).In addition, we highlighted five “high-risk” clones of carbapenemase-producingM. morganiisubsp.morganiithat have already disseminated worldwide. Combining whole genome sequencing (WGS) data with epidemiological investigations, we demonstrated that a cut-off of 100 single nucleotide polymorphisms (SNPs) could be used to discriminate clonally-related from sporadic independent isolates. This information is of the utmost importance since WGS is now considered as the reference method to identify and follow outbreaks.The intrinsic resistance ofMorganellaspp. to polymyxins was well-known but the underlying mechanism was unclear. Here, we demonstrated that the addition of L-Ara4N on the lipid A ofMorganellais involved.Implications of all the available evidenceThe identification of “high-risk” clones among highly-drug resistantMorganellaspp. paves the way of future investigations to better understand and hopefully limit the spread of these bugs. Additionally, our results identified new components and virulence factors of someMorganellaspecies (e.g. T6SS and T3SS inM. sibonii) that deserve further investigation since they might be implicated in the bacterial lifestyle of this genus.

The genetic mechanisms associated with acquisition of linezolid (LZD) resistance are diverse, including point mutations in the V domain of the 23S rRNA and the 50S ribosomal proteins as well as cfr...

Growing evidence shows diminished response to mRNA-based SARS-CoV‑2 vaccination in kidney transplant recipients. We aimed to investigate the seroconversion rate after a 3rd and 4th dose of mRNA vaccination in kidney transplant recipients without prior antibody response to two or three vaccination doses.This retrospective study included 324 prevalent kidney transplant recipients of a single tertiary transplantation center of which 157 remained seronegative, defined as anti-spike-RBD-IgG antibody titer 7.1 BAU/ml, after two doses of mRNA-based SARS-CoV‑2 vaccination. Maintenance immunosuppression was not changed. The median patient age was 60.6 years (IQR 51.4-68.1 years), 66.9% were male. Positivity for anti-spike-RBD-IgG (≥ 7.1 BAU/ml) was measured 4-5 weeks after administration of a 3rd and 4th vaccine dose.Seroconversion rates were 63.9% after a 3rd dose and 29.3% after a 4th dose of vaccine. Cumulative prevalence of seropositivity was 51.5% after 2 doses, 80.5% after 3 doses and 84.2% after 4 doses.In conclusion, seroconversion can be achieved in the majority of the kidney transplant recipients by administrating three or four doses of mRNA vaccine without changing maintenance immunosuppression.

X.D. was funded by a grant from the National Institute for Health Research (NIHR) Health Protection Research Unit in Genomics and Enabling Data (no. NIHR200892). M.A.H. was supported by grants from the Medical Research Council (nos. G1001787/1, MR/N002660/1 and MR/P007201/1) and the Economic and Social Research Council (no. ES/S000186/1). E.M.H. was supported by a UK Research and Innovation (UKRI) Fellowship (no. MR/S00291X/1). The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus—a notorious human pathogen—appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two β-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development. Publisher PDF

BACKGROUND Candida-associated infections put a significant burden on western healthcare systems. Development of (multi-)resistant fungi can become untreatable and threaten especially vulnerable target groups, such as the immunocompromised. OBJECTIVES We assessed antifungal susceptibility and explored possible influence factors of clinical Candida isolates collected from Austrian hospitals between 2007 and 2016. METHODS Thousand three hundred and sixty clinical Candida spp. isolated from blood cultures were subjected to antifungal susceptibility testing (AFST) in a liquid-handling aided continuous microdilution assay. We tested against fluconazole, voriconazole, posaconazole, itraconazole, isavuconazole, anidulafungin, caspofungin and micafungin according to EUCAST with additional recording of growth curves. We performed rigid quality control on each assay via growth curve assessment and included two standard reference strains. Minimal inhibitory concentrations (MIC) were quantified according to EUCAST guideline E.DEF 7.3.1, and susceptibility was evaluated using EUCAST clinical breakpoints. RESULTS The isolate collection consisted of Candida albicans (59%), C. glabrata (19%), C. parapsilosis (9%), C. tropicalis (5%) and C. krusei (3%) and few other Candida species and fungi (5%). During the observed time period, species abundance and antifungal resistance rates remained constant. Multi-resistance was rare and we found no single isolate which was resistant to both azoles and echinocandins. Within the antifungal resistance profile of our strain collection, we observed clusters along species boundaries. CONCLUSIONS Over the last decade, the distribution of Candida species and its level of antifungal resistance remained constant in Austria. Our data compare well with other European countries. Principal component analysis of the susceptibility profile of this collection revealed species-specific clusters and substantial intra-species variation, especially for C. glabrata.

The genetic mechanisms associated with acquisition of linezolid (LZD) resistance are diverse, including point mutations in the V domain of the 23S rRNA and the 50S ribosomal proteins as well as

Enterobacterales belonging to the genus Enterobacter are well-known human pathogens and blaIMI carrying strains have been described in several countries. From Austria, we report on the first human Enterobacter mori isolate, typically a plant pathogen, which showed an undescribed multilocus sequence type and carried a blaIMI-2 carbapenemase.

Background Enterococcus faecium is part of the human gastrointestinal flora but may act as opportunistic pathogen. Environmental persistence, high colonization capability and diverse intrinsic and acquired resistance mechanisms make it especially successful in nosocomial high-risk settings. In March 2014, an outbreak of Linezolid resistant Enterococcus faecium (LREfm) was observed at the hematooncology department of a tertiary care center in Upper Austria. Methods We report on the outbreak investigation together with the whole genome sequencing (WGS)-based typing results including also non-outbreak LREfm and susceptible isolates. Results The 54 investigated isolates could be divided in six clusters based on cgMLST. Cluster one comprised LREfm isolates of genotype ST117 and CT24, which was identified as the causative clone of the outbreak. In addition, the detection of four other clusters comprising isolates originating from hematooncology patients but also at other hospitals, pointed to LREfm transmission between local healthcare facilities. LREfm patients (n = 36) were typically at risk for acquisition of nosocomial pathogens because of immunosuppression, frequent hospitalization and antibiotic therapies. Seven of these 36 patients developed LREfm infection but were successfully treated. After termination of the initial outbreak, sporadic cases occurred despite a bundle of applied outbreak control interventions. Conclusions WGS proved to be an effective tool to differentiate several LREfm clusters in an outbreak. Active screening for LREfm is important in a high-risk setting such as hematooncology, where multiple introductions are possible and occur despite intensified infection control measures.

Objectives: High-level β-lactam resistance in methicillin-resistant Staphylococcus aureus is mediated in the majority of strains by a mecA or mecC gene. In this study, we identified ten mec gene negative MRSA human isolates from Austria and eleven bovine isolates from the UK showing high levels of β-lactam resistance and sought to understand the molecular basis of the resistance observed. Methods: Different antimicrobial resistance testing methods (disk diffusion, Etest and Vitek®2) were used to establish the β-lactam resistance profiles for the isolates and the isolates were further investigated by whole-genome sequencing. Results: A number of mutations (including novel ones) in penicillin-binding proteins, AcrB, YjbH and pbp4-promoter were identified in the resistant isolates but not in closely related susceptible isolates. Importantly, a truncation in the c-di-AMP phosphodiesterase enzyme, GdpP, was identified in seven of the ten Austrian isolates and ten of the eleven UK isolates. Complementation of four representative isolates with an intact copy of gdpP gene restored susceptibility to penicillins and abolished the growth defects caused by the truncation. Conclusion: This study reports naturally occurring inactivation of GdpP protein in S. aureus of both human and animal origins, and demonstrated clinical relevance to a previously reported association between this truncation and increased β-lactam resistance and impaired bacterial growth in laboratory generated mutants. It also highlights possible limitations of genomic determination of antibiotic susceptibility based on single gene presence or absence when choosing the appropriate antimicrobial treatment for patients.

This five-site study was performed to assess the reproducibility of ceftaroline MIC and disk results for Staphylococcus aureus. Three commercial broth microdilution, three gradient diffusion and ceftaroline 5μg disk diffusion methods were compared to a reference broth microdilution method against challenge isolates (n = 41) and isolates collected at four European sites (n = 30/site). For four MIC methods (Sensititre and three gradient diffusion methods), 99.0% of consolidated MIC results were within +/- 1 dilution of the reference MIC. Categorical agreement rates based on EUCAST breakpoints for the challenge isolates were 75.6-100% and for disk testing were 78.0-92.7%. There was no clear distinction between isolates with MIC results of 1 and 2mg/L with regard to variation in MIC or molecular genotyping results. The addition of an intermediate category for isolates with MIC results of 2mg/L would help to identify these isolates as borderline susceptible/non-susceptible isolates.

Enterobacterales belonging to the genus Enterobacter are well-known human pathogens and bla

Since colistin resistance based on the plasmid-encoded mcr-1 gene was first described, this resistance gene in Enterobacteriaceae has been found worldwide. These organisms are typically of heterogeneous genetic background and show exceptional clonal diversity. We describe the first confirmation of mcr-1 in a human Escherichia coli strain cultured from a surveillance stool sample of an Austrian oncology patient.

The members of the European Survey on Carbapenemase-Producing Enterobacteriaceae, (EuSCAPE) Working Group are: Portugal—Manuela Caniça and Vera Manageiro BACKGROUND: Gaps in the diagnostic capacity and heterogeneity of national surveillance and reporting standards in Europe make it difficult to contain carbapenemase-producing Enterobacteriaceae. We report the development of a consistent sampling framework and the results of the first structured survey on the occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in European hospitals. METHODS: National expert laboratories recruited hospitals with diagnostic capacities, who collected the first ten carbapenem non-susceptible clinical isolates of K pneumoniae or E coli and ten susceptible same-species comparator isolates and pertinent patient and hospital information. Isolates and data were relayed back to national expert laboratories, which made laboratory-substantiated information available for central analysis. FINDINGS: Between Nov 1, 2013, and April 30, 2014, 455 sentinel hospitals in 36 countries submitted 2703 clinical isolates (2301 [85%] K pneumoniae and 402 (15%) E coli). 850 (37%) of 2301 K pneumoniae samples and 77 (19%) of 402 E coli samples were carbapenemase (KPC, NDM, OXA-48-like, or VIM) producers. The ratio of K pneumoniae to E coli was 11:1. 1·3 patients per 10 000 hospital admissions had positive clinical specimens. Prevalence differed greatly, with the highest rates in Mediterranean and Balkan countries. Carbapenemase-producing K pneumoniae isolates showed high resistance to last-line antibiotics. INTERPRETATION: This initiative shows an encouraging commitment by all participants, and suggests that challenges in the establishment of a continent-wide enhanced sentinel surveillance for carbapenemase-producing Enterobacteriaeceae can be overcome. Strengthening infection control efforts in hospitals is crucial for controlling spread through local and national health care networks. European Centre for Disease Prevention and Control info:eu-repo/semantics/publishedVersion

Background Respiratory tract infections are widespread and may cause significant morbidity and mortality in immunosuppressed populations such as oncological patients. Objectives The RealAccurate Respiratory RT PCR Kit covering 14 respiratory viruses was compared to the RespiFinder Smart22, a broad-spectrum multiplex ligation-dependent probe amplification (MLPA) test, targeting 22 viral and bacterial respiratory pathogens. Study design After verification of its analytical performance, the clinical performance of the RespiFinder Smart22 was evaluated by re-analysis of 96 respiratory samples from oncological patients. Additionally, the time to result (TTR) of both methods was compared. Results The analytical performance of the RespiFinder Smart22 fulfilled all previously specified criteria. Concordant results in both assays were achieved in 74.0% of all clinical specimens and in 91.2% when only positive results were taken into account. The RespiFinder Smart22 yielded additional results in a total of 22 (22.9% of 96) samples due to higher test sensitivity and a broader, highly multiplexed spectrum of pathogens. The TTR of a typical routine test consisting of three samples were 206 and 356 min for the RealAccurate Respiratory RT PCR Kit and the RespiFinder Smart22, respectively. However, hands-on time was reduced by 59.0% applying the MLPA method. Conclusions In our hands, the RespiFinder Smart22 showed excellent analytical performance while hands-on time was halved in comparison to the RT PCR method. Regarding the clinical evaluation, the MLPA method provided additional results in 22.9% (22/96) of specimens due to its comprehensive format, higher test sensitivity and the capability to detect 22 pathogens compared to 14 with the RealAccurate Respiratory RT PCR Kit.

Since colistin resistance based on the plasmid-encoded mcr-1 gene was first described, this resistance gene in Enterobacteriaceae has been found worldwide. These organisms are typically of heterogeneous genetic background and show exceptional clonal diversity. We describe the first confirmation of mcr-1 in a human Escherichia coli strain cultured from a surveillance stool sample of an Austrian oncology patient.

No actual data are available on the epidemiology and morbidity of community acquired pneumonia (CAP) in youths and children in Vienna, Austria. The objective was to determine the epidemiology of CAP and morbidity of children hospitalized due to CAP in a tertiary care facility. During three winter seasons youths and children hospitalized due to CAP were enrolled. Testing for viral and bacterial pathogens of pneumonia was performed in a routine clinical setting. Blood cultures were performed; respiratory viruses, Mycoplasma pneumoniae and Chlamydia pneumoniae were searched for by an established Real Time polymerase chain reaction (PCR) panel. Clinical signs and indices of inflammation were documented. Out of 279 children and youths with CAP a causative agent could be detected in 190 (68 %). Viruses and bacteria were diagnosed in 107 (57 %) and 58 patients (30 %), respectively. Co-infection was found in 20 patients (10 %), Mycoplasma pneumoniae or Clamydia pneumoniae in 16 cases (8 %). In seven patients blood cultures were positive. C-reactive protein (CRP) was significantly higher in children with positive Streptococcus pneumoniae antigen (SPAG) than with viral infection and/or co-infection. Clinical parameters showed no statistically significant differences. C. pneumoniae and M. pneumoniae were only diagnosed in children and youths with 5 years and older. Testing for pathogens in CAP in clinical routine achieves a high recovery rate. Blood cultures are rarely helpful, but the molecular testing for viruses seemed to be helpful to establish the diagnosis.

The Carba NP test is a simple confirmation method for carbapenemase-producing Enterobacteriaceae (CPE) but reagents have to be freshly prepared as imipenem sodium salt is unstable. We evaluated the Carba NP test performance based on a commercially available 10-fold cheaper drug formulation containing cilastatin against 217 CPE and 78 non-CPE isolates with reduced meropenem susceptibility. Specificity and sensitivity were 100 % and 98.6 %, respectively and 3 false negative results of blaVIM-1-producing Proteus mirabilis were reproducible with the RAPIDEC® Carba NP test. Cilastatin does not disturb test performance provided that the imipenem drug quantity is doubled.

Objective To determine the presence of chlamydial species including recently described chlamydial agents as well as the human pathogen Chlamydophila pneumoniae in feline conjunctivitis. Animal studied Twenty five cats without and 49 cats with conjunctivitis were tested for chlamydia using a Chlamydiaceae real time (RT) PCR (targeting the 23S rRNA gene sequence), a Chlamydiales PCR (targeting the 16S rRNA gene sequence), and cell culture. The PCR products of all positive samples were sequenced and subsequently analyzed using a basic local alignment search tool search. Results Chlamydiaceae RT PCR and subsequent sequence analyses identified C. pneumoniae in five cats in the conjunctivitis group. The presence of Chlamydophila felis was shown in two cats with conjunctivitis. Chlamydiae related to uncultured members of Chlamydiales were detected in three conjunctivitis cases and in one cat without clinical symptoms. Conclusion This study detects for the first time, the known human pathogen C. pneumoniae in feline conjunctivitis cases using Chlamydiaceae RT PCR and sequence analyses.

Objective Persistent Chlamydia trachomatis infections are associated with tubal pathology. We studied whether sampling from multiple sites would increase the identification of the infections. Design Prospective cohort study. Setting Tertiary care facility. Patient(s) Two hundred two infertile women. Intervention(s) Smears were taken from the cervix, urethra, high vagina, fimbriae and the Douglas cavity. Blood samples were collected and tubal patency was assessed by pertubation with lipiodol and methylene blue. Main Outcome Measure(s) Detection of C. trachomatis DNA, detection of IgA and IgG antibodies against C. trachomatis, and antibodies against chlamydial heat-shock protein 60, tubal patency. Result(s) Chlamydia trachomatis was detected in 2 of 202 patients, for an overall prevalence of 1%. In both patients PCR results were positive in the cervical, vaginal, and urethral specimens. Chlamydia trachomatis IgG, IgA, and chlamydial heat-shock protein 60 IgG were significantly more prevalent in women with distal tubal pathology than in those without (26/40 [65.0%] vs. 16/162 [9.9%], 9/40 [22.5%] vs. 7/162 [4.3%], and 34/40 [85.0%] vs. 34/162 [21.0%]). Bacterial colonization was found in 1 of 202 samples from the Douglas cavity. Conclusion(s) Routine DNA testing for C. trachomatis should be confined to cervical sampling. The association between tubal pathology and seropositivity of IgG, IgA, and cHSP60 IgG was confirmed but did not add clinically valuable information during the diagnostic workup of infertility patients.

Background No recent Austrian data are available on the epidemiology and morbidity of Chlamydophila pneumoniae (C. pneumoniae) as causative agent in youths and children hospitalized with community-acquired pneumonia (CAP). These data would serve as a rationale for empiric antimicrobial therapy. Methods During winter months 2006/7 112 immunocompetent youths and children between 2 months and 18 years hospitalized consecutively with CAP were prospectively enrolled. Microbiological detection of conventional bacteria, Mycoplasma pneumoniae as well as respiratory viruses was performed, and clinical signs as well as indices of inflammation were documented. Diagnosis of C. pneumoniae was performed by means of species-specific real-time PCR as well as cell culture from respiratory secretions. Results PCR for C. pneumoniae was performed in 60 patients. In 4 cases (6.7%) C. pneumoniae was found as causative agent. In 1 case C. pneumoniae could be confirmed by cell-culture. All children with C. pneumonia were ≥5 years of age. All but 1 child received clarithromycin together with cefuroxime. The number of days with fever >38,5°C was between 1 and 7, the number of days in hospital was between 4 and 7, all children recovered completely. Conclusion C. pneumoniae was found in 6.7% of children and youths hospitalized with CAP in a community hospital during the winter months 2006/07. Children and youths with lower respiratory infections should further be tested for C. pneumoniae to elucidate the actual epidemiological situation and to guide empiric antimicrobiotic therapy. Pediatr Pulmonol. 2009; 44:873–876. © 2009 Wiley-Liss, Inc.

The purpose of this study was to investigate methicillin-resistant Staphylococcus aureus (MRSA) isolates with antibiotic resistance restricted to beta-lactam antibiotics and variable resistance to fusidic acid for the presence of Panton-Valentine leukocidin (PVL) genes. Our data show that the selected resistance pattern is found rarely among MRSA isolates in our hospital, but it appears that this phenotype consistent with typical community-acquired (ca) MRSA is indicative of PVL-positive MRSA.

Aim: Introduction of a protocol for broad-range diagnosis of bacterial infections, which remain negative in culture. Methods and Results: The new TaqMan real-time PCR assay amplifies part of the 16S rRNA gene. Species are identified by subsequent sequencing and phylogenetic blast analysis. The analytical sensitivity showed to be 50 fg DNA per PCR. The lowest detectable bacterial cell concentration in blood was 1000 CFU per 200 μl EDTA-blood. The utility in clinical routine diagnosis was evaluated by testing 136 clinical specimens. Bacterial pathogens were detected in 33 samples (24·3%) either by culture or molecular diagnosis. In 10 culture negative cases, pathogens such as Mycoplasma timone/orale, Ureaplasma parvum/urealyticum, Treponema pallidum, different streptococci and staphylococci were identified by molecular diagnosis only. Conclusions: The introduced broad-range real-time PCR protocol showed to be useful in the clinical routine in cases where bacterial infection was highly anticipated but culture remained negative. However, the obtained data have to be always interpreted with caution and in conjunction with the clinical data, crossing-point values and with the Blast result of both the sample and the controls. Significance and Impact of the Study: This work introduces a new and well-evaluated broad-range real-time PCR protocol for diagnosis of bacterial infections.

Objectives: The aims of this study were to determine the prevalence of extended-spectrum b-lactamases (ESBLs) in AmpC-carrying Enterobacter spp. in a tertiary care university hospital in Vienna, Austria, and to implement a cost-effective strategy to detect ESBLs in this particular genus on a routine basis. Methods: Clinical Enterobacter isolates (n 5 208) were investigated by means of (i) an inhibitor-potentiated diffusion test using cefpodoxime, (ii) an expanded double disc diffusion synergy test (discs of cefotaxime, ceftazidime, cefpodoxime and cefepime placed around amoxicillin/clavulanic acid), (iii) the Etest ESBL screening method and (iv) the cefoxitin–cefotaxime antagonist test. Cefepime MICs were determined by separate Etests. Results: Of 208 isolates, 76 (37%), 18 (9%) and 92 (44%) were derepressed, partially derepressed and inducible AmpC producers, respectively. Eight (4%) ESBL-producing Enterobacter strains could be detected, all of which would have been detected using disc-based tests. Six out of eight strains were genetically not related, as assessed by random amplification of polymorphic DNA. Typing results were confirmed by means of enterobacterial repetitive intergenic consensus PCR. The MIC90 of cefepime was not different in ESBL carriers (range 2–4 mg/L), and was especially low in inducible AmpC producers (0.125 mg/L). More than half of all Enterobacter isolates (n 5 110; 53%) were partly derepressed or fully inducible AmpC producers. In the absence of cefoxitin, they appeared susceptible or intermediately susceptible to cefazolin (n 5 8; 9%), cefuroxime (n 5 75; 81.5%), ceftazidime (n 5 91; 99%), cefotaxime (n 5 92; 100%), cefpodoxime (n 5 75; 81.5%) and cefepime (n 5 91; 99%). Conclusions: Susceptibility to third-generation cephalosporins would have been falsely assumed in more than half of all Enterobacter isolates, but ESBL in Enterobacter is currently rare in our institution. Integration of multiple double disc tests into the routine antibiogram seems a reliable approach to screen for emerging resistance mechanisms. Etests did not provide additional information in this study.

Background: The aim of the present study was to assess the prevalence and resistance of commensal S. aureus in the nasal microbiota of community-dwelling persons in Austria, as well as to identify possible associations with socio-demographic factors. Multi-drug resistance in this population was additionally studied.Method: This cross-sectional study was conducted within the context of the European APRES project. In nine European countries, nasal swabs were collected from 32,206 general practice patients who received care for non-infectious reasons. In Austria, 20 GPs attempted to recruit 200 consecutive patients without infectious diseases, with each patient completing demographic questionnaires as well as providing a nose swab sample. Isolation, identification, and resistance testing of S. aureus were performed. Statistical analyses included subgroup analyses and logistic regression models.Results: 3309 nose swabs and corresponding questionnaires from Austrian subjects were analyzed. S. aureus was identified in 16.6 % (n = 549) of nose swabs, of which 70.1 % were resistant against one or more antibiotics, mainly penicillin. S. aureus carrier status was significantly associated with male sex (OR 1.6; 1.3-2.0), younger age (OR 1.3; 1.0-1.8), living in a rural area (OR 1.4; 1.1-1.7) and working in the healthcare sector (OR 1.5; 1.0-2.1). Multi-drug resistances were identified in 13.7 % (n = 75) of the S. aureus carriers and 1.5 % (n = 8) tested positive for MRSA. The highest resistance rate was observed against penicillin (64.8 %), followed by azithromycin (13.5 %) and erythromycin with 13.3 %.Conclusion: This study describes the prevalence and resistance patterns of commensal S. aureus in community-dwelling persons in Austria and shows that differences exist between socio-demographic groups. Demographic associations have been found for S. aureus carriers but not for carriers of resistant S. aureus strains. Only two thirds of S. aureus strains were found to be resistant against small spectrum penicillin. As it is recognized that one of the corner stones for the containment of antibiotic resistance is the appropriate prescription of antibiotics in the outpatient sector, this finding lends support to the avoidance of prescription of broad-spectrum antibiotics to treat S. aureus infections in the community.

In order to evaluate the suitability of fosfomycin in combination with other agents for the treatment of Helicobacter pylori infections, the susceptibility profiles of 65 H. pylori strains were determined against multiple antimicrobial agents and combinations thereof using the agar dilution method. For fosfomycin alone, the range of minimum inhibitory concentration (MIC) results and the MICs at which 50% and 90% of strains were inhibited were 0.5-32 microg/ml and 2 and 4 microg/ml, respectively. For the combination of fosfomycin with amoxicillin, clarithromycin or metronidazole, the means calculated for the minimum and maximum fractional inhibitory concentration index were 0.70-1.17 and 1.15-2.03, respectively, suggesting partial synergy or indifference in the majority of strains. The combination of clarithromycin and metronidazole showed synergistic activity against 14 of 28 H. pylori strains tested. The in vitro activity results suggest the combination of fosfomycin with either amoxicillin or clarithromycin may be a promising alternative for the treatment of H. pylori infection. However, the clinical efficacy of these regimens remains to be investigated.

The raising frequency of occurrence of multi-resistant Gram-positive pathogens has led to difficult to treat infections not only in hospitals but also in outpatient settings. Methicillin-resistant Staphylococcus aureus, the glycopeptide-susceptibility of which one cannot be sure of any more, vancomycin-resistant enterococci and penicillin-resistant pneumococci are on the top of the list of Gram-positive problem organisms nowadays. Clinical microbiology laboratories are faced with the challenge of accurately detecting emerging antibiotic resistance, which might be difficult and purpose of reporting data should be twofold: First to report adequately identified, relevant organisms as well as their susceptibility profiles to clinicians in context with adequate patient management and secondly to report continuously epidemiological data in form of statistics to the community in general and within a given setting as could be a specified hospital in particular. For reliable detection, laboratories may need to employ special screening- and susceptibility testing methods. Certain of these tests are highly specific, while others may require additional confirmatory testing for definite results. This article reports on current resistance mechanisms of gram-positive pathogens and subsequently resulting consequences for clinicians. Moreover, the frequency of occurrence in Austria in general as well as at the Vienna General Hospital in particular will be referred to.

The present multicenter study was designed to find explanations for the discrepancies in the reported rates of detection of Chlamydia pneumoniae DNA in endarterectomy specimens. Coded identical sets of (i) a C. pneumoniae DNA dilution series (panel 1; n = 10), (ii) spiked control tissue specimens (panel 2; n = 10 specimens, including 5 negative controls), and (iii) endarterectomy specimens (panel 3; 15 atheromas, 5 negative controls) were analyzed at four laboratories by three standardized DNA extraction methods in each laboratory and a nested touchdown PCR protocol targeting the ompA gene of C. pneumoniae . Panel 1 samples were correctly identified as positive to levels of 0.3 inclusion-forming units (IFU)/PCR mixture (100%) and 0.03 IFU/PCR mixture (50%). All negative controls were correctly reported as negative. Panel 2 samples were identified as C. pneumoniae positive to levels of 0.01 IFU/PCR mixture (100%) and 0.005 IFU/PCR mixture (91%), independent of the DNA extraction method used, and only one false-positive result was reported. For panel 3 samples, 5 of 240 (2%) analyses (in which DNA extractions and PCR were performed at the same laboratory) were positive; the positive specimens were from three endarterectomy specimens and two negative controls. After exchange of DNA extracts between laboratories, 13 of 15 atheroma samples were C. pneumoniae DNA positive in at least 1 of a series of 48 analyses per atheroma sample; however, the overall positivity rate did not exceed 5% (33 of 720 analyses) and therefore was lower than that for the negative controls (8%; 19 of 240 analyses). Not a single positive result could be achieved when all panel 3 extracts ( n = 240 analyses) were reamplified by a 16S rRNA PCR followed by hybridization with a C. pneumoniae -specific probe. Statistical analyses demonstrated that positive results did not occur in an independent and random fashion and could most likely be explained by amplicon carryover at the nested PCR level as well as amplicon introduction during DNA extraction, but not by the patterns of distribution of very low target levels or a certain DNA extraction protocol. The results of studies by nested PCR for detection of the prevalence of C. pneumoniae will always be questionable.

In order to elucidate trends in the incidence and susceptibility profiles of causative agents of bacteremia/fungemia in nine surgical intensive care units, a total of 744 isolates obtained during a 5-year period (1996-2000) were studied. The isolates included 698 bacteria and 46 fungi obtained from 523 positive blood cultures, representing 317 episodes of bacteremia/fungemia. Methicillin-resistant Staphylococcus aureus accounted for 2.3 episodes per 1000 surgical ICU admissions in 1996, 1.6 in 1997, 0.3 in 1998, 0.6 in 1999, and 1.7 in 2000. One Enterococcus faecalis (VanA) isolate resistant to both vancomycin and teicoplanin was recovered in 1996. Ciprofloxacin resistance in Pseudomonas aeruginosa decreased from 36% in 1996 to 20% in 2000, and resistance to third-generation cephalosporins decreased from 40% in 1996 to 9% in 2000. In light of differences between these results and those found elsewhere, these findings might prove useful for making infection control policy decisions in intensive care units.

The role of urease in Helicobacter pylori adherence to and internalization by Kato III cells was investigated. Kato III cells were incubated with wild-type strains (N6 or P1), with isogenic mutants lacking urease (N6ureB::TnKm or P1ureA::TnMax5) or producing the inactive apoprotein (N6ureG::TnKm), and with urease-positive clones recovered after complementation of N6ureB::TnKm with ureAB. Bacteria were stained with the green fluorescent dye PKH2, and the bacteria load of cells was analyzed by flow cytometry. With mutants lacking urease, the bacteria load was considerably increased, in comparison with the corresponding parental strains (P

This study of pediatric patients was intended to determine the suitability of stool PCR and two antigen enzyme immunoassays (EIAs; Premier Platinum HpSA and the novel FemtoLab H. pylori), which detect Helicobacter pylori antigens in feces, as pretreatment diagnostic tools and especially as posttreatment control. Forty-nine H. pylori -infected children with dyspepsia received eradication therapy. Successful treatment was determined by a negative [ 13 C]urea breath test 4 and 12 weeks after discontinuation of therapy. Fecal specimens were collected prior to eradication therapy as well as 4 weeks after the end of treatment. Successfully treated children delivered stool samples at 6, 8, and 12 weeks posttreatment also. Specimens were examined by seminested PCR and Premier Platinum HpSA and were reexamined by both EIAs as soon as FemtoLab H. pylori was available. In the first test series, the overall sensitivities of PCR and Premier Platinum HpSA were 93.0 and 91.1%, respectively. With specimens collected at 4 weeks after treatment, the respective specificities were 68.8 and 79.3%. After longer follow-up periods, however, they gradually increased to 100 and 96.9%, respectively. In the new test series, Premier Platinum HpSA delivered a considerably lower number of false-positive results (4 versus 18), indicating intertest variations. The overall test sensitivity was 94.6%, and the overall specificity was 97.5%. FemtoLab H. pylori showed an excellent performance with an overall sensitivity and specificity of 98.2 and 98.1%, respectively. Thus, in contrast to PCR, both EIAs were shown to be suitable for early posttreatment control.

There are now more arguments against than for a causal relationship between C. pneumoniae and atherosclerosis. Seroepidemiologic results are largely technique-dependent; PCR results show intra- and interlaboratory variability; methodological factors contribute to bias; and detection of C. pneumoniae fails when the specificity of the reaction is optimized. Immunohistochemical staining detects nonspecific compounds in atherosclerotic lesions, and secondary prevention trials are unsuccessful. The evidence for an association between a pathogen and a chronic disease should be based on concordance of evidence arising from different approaches applied by different groups, at different times, in different places, and under different circumstances. None of these conditions have been fulfilled in the case of C. pneumoniae and atherosclerosis.1 Citing the concluding paragraph of Margareta Ievens’ excellent review on Chlamydia pneumoniae and atherosclerosis, published in 2005 by the American Society of Micobiology (ASM) in their flagship journal for clinical microbiologists, the Journal of Clinical Microbiology , probably best summarizes the problems arising from diagnostic shortcomings of C pneumoniae , a microorganism that has been associated with well over 30 various diseases of markedly different pathologies and pathogenic mechanisms. Even though there is now “more evidence of no evidence” of the particular association on “ C pneumoniae and atherosclerosis” than ever, every month articles continue to be published based on methods for which there is evidence that they are inadequate at various levels to define a patients’ C pneumoniae status. C pneumoniae serology is one of these methods. In this issue of Stroke , Elkind et al2 studied whether evidence of C pneumoniae infection is associated with risk of first ischemic stroke. Again, a study based on serology, using the microimmunofluorescence technique (MIF), found elevated C pneumoniae IgA titers were associated with in-creased risk of first ischemic stroke after adjusting for conventional risk factors. The …

Disseminated infection with the coelomycetous fungus Nattrassia mangiferae is a very rare disease affecting only the immunocompromised host. We report the first case of a disseminated infection with spondylodiscitis and granular skin lesions due to N. mangiferae in a renal transplant patient.

A new chromogenic Oxacillin Resistance Screen Agar (ORSA; Oxoid) for the presumptive detection of methicillin-resistant Staphylococcus aureus (MRSA) was compared to a phenyl-mannitol-salt-oxacillin medium (MS-Oxa), blood agar and brain heart-infusion (BHI) on 579 clinical specimens. After 24 h [48h] sensitivity and specificity for ORSA vs. MS-Oxa were 50.8% [68.2%] vs. 53.8% [65.7%] and 95.6% [94.5%] vs. 92.7% [91.8], respectively. Within 24 h, ORSA and MS-Oxa identified 51% and 54% MRSA. It is not feasible to omit BHI in MRSA-screening protocols since 33% MRSA grew in BHI only.

To determine the presence of chlamydial species including recently described chlamydial agents as well as the human pathogen Chlamydophila pneumoniae in feline conjunctivitis.Twenty five cats without and 49 cats with conjunctivitis were tested for chlamydia using a Chlamydiaceae real time (RT) PCR (targeting the 23S rRNA gene sequence), a Chlamydiales PCR (targeting the 16S rRNA gene sequence), and cell culture. The PCR products of all positive samples were sequenced and subsequently analyzed using a basic local alignment search tool search.Chlamydiaceae RT PCR and subsequent sequence analyses identified C. pneumoniae in five cats in the conjunctivitis group. The presence of Chlamydophila felis was shown in two cats with conjunctivitis. Chlamydiae related to uncultured members of Chlamydiales were detected in three conjunctivitis cases and in one cat without clinical symptoms.This study detects for the first time, the known human pathogen C. pneumoniae in feline conjunctivitis cases using Chlamydiaceae RT PCR and sequence analyses.

No recent Austrian data are available on the epidemiology and morbidity of Chlamydophila pneumoniae (C. pneumoniae) as causative agent in youths and children hospitalized with community-acquired pneumonia (CAP). These data would serve as a rationale for empiric antimicrobial therapy.During winter months 2006/7 112 immunocompetent youths and children between 2 months and 18 years hospitalized consecutively with CAP were prospectively enrolled. Microbiological detection of conventional bacteria, Mycoplasma pneumoniae as well as respiratory viruses was performed, and clinical signs as well as indices of inflammation were documented. Diagnosis of C. pneumoniae was performed by means of species-specific real-time PCR as well as cell culture from respiratory secretions.PCR for C. pneumoniae was performed in 60 patients. In 4 cases (6.7%) C. pneumoniae was found as causative agent. In 1 case C. pneumoniae could be confirmed by cell-culture. All children with C. pneumonia wereor =5 years of age. All but 1 child received clarithromycin together with cefuroxime. The number of days with fever38,5 degrees C was between 1 and 7, the number of days in hospital was between 4 and 7, all children recovered completely.C. pneumoniae was found in 6.7% of children and youths hospitalized with CAP in a community hospital during the winter months 2006/07. Children and youths with lower respiratory infections should further be tested for C. pneumoniae to elucidate the actual epidemiological situation and to guide empiric antimicrobiotic therapy.

Summary Objective Human Granulocytic Anaplasmosis (HGA) is an emerging disease caused by the gram-negative bacterium Anaplasma phagocytophilum which is transmitted by ticks of the genus Ixodes ricinus . For molecular detection of the pathogen by PCR, a conserved portion of the groEL gene within the groESL operon is frequently used as a target. A single G/A polymorphism in this region allows to discriminate between two genotypes, groEL -G and groEL -A. Methods Total DNA from peripheral blood samples of two HGA patients was analysed by RealTime PCR, employing a protocol designed for genotyping groEL- G- and groEL- A variants of A. phagocytophilum . Results We confirmed two clinical cases of HGA by PCR; in one patient, and for the first time in a human host, the groEL -A variant was detected, in the other case the pathogen was recognised as the groEL -G variant, up to now representing the only genotype reported in man. Conclusions It is documented that HGA infections can be caused by two A. phagocytophilum groEL genotypes. At present, the preference of the A. phagocytophilum groEL -G genotype in humans remains unclear, as we describe the first patient with HGA caused by the groEL -A variant. For a conclusive interpretation, more data from HGA patients will be required.

Objective.To provide a novel way to predict the likelihood that antibiotic therapy will result in prompt, adequate therapy on the basis of local microbiological data.Design and Setting.Prospective study conducted at 3 medical intensive care units at the Viennese General Hospital, a tertiary care medical university teaching hospital in Vienna, Austria.Patients.One hundred one patients who received mechanical ventilation and who met the criteria for having ventilator-associated pneumonia.Design.Fiberoptic bronchoscopic examination was performed, and bronchoalveolar samples were collected. Samples were analyzed immediately by a single technician. Minimum inhibitory concentrations were determined for imipenem, cephalosporins (cefepime and cefpirome), ciprofloxacin, and piperacillin-tazobactam, and drug resistance rates were calculated. These drug resistance rates were translated into the likelihood of inadequate therapy (LIT; the frequency of inadequately treated patients per antibiotic and drug-resistant strain), cumulative LIT (the cumulative frequency of inadequately treated patients), and syndrome-specific LIT.Results.Amongthe 101 bronchoalveolar samples, culture yielded significant (at least 1 × 104 colony-forming units per raL) polymicrobial findings for 34 and significant monomicrobial findings for 31; 36 culture results were negative. Of the isolates from patients with ventilator-associated pneumonia who had monomicrobial culture findings, 33% were gram-positive bacteria and 20% were gram-negative bacteria. LIT suggested that 1 of 2 patients was treated inadequately for Pseudomonas aeruginosa infection. The LIT for patients with ventilator-associated pneumonia revealed that the rank order of antibiotics for appropriate therapy was (1) imipenem, (2) cephalosporins, (3) ciprofloxacin, and (4) piperacillin-tazobactam. These calculations were based solely on microbiological data.Conclusions.The novel ratio LIT may help clinicians use microbiological data on drug resistance to predict which antimicrobial agents will provide adequate therapy. In daily practice, this new approach may be helpful for choosing adequate antimicrobial therapy.

Community-acquired pneumonia (CAP) accounts for more than two million deaths per year in children < 5 years of age. Recognition of pathogens is vital for guiding antibiotic treatment. In Gabon, no epidemiologic data on childhood CAP were available to help guide antibiotic therapy. We conducted a prospective, hospital-based, cross-sectional survey at the Albert Schweitzer Hospital, Lambarene, Gabon, to assess the importance of atypical organisms (Chlamydia pneumoniae, Mycoplasma pneumoniae, Bordetella pertussis, and Legionella pneumophila) and Streptococcus pneumoniae in the etiology of CAP in children by means of real-time polymerase chain reaction, cell culture, and serology. Collectively, atypical bacteria accounted for 11% of cases with a special emphasis on B. pertussis, accounting for 6% of cases. Clinical differentiation of atypical from typical pneumonia in children remains challenging. Molecular diagnostic methods offer fast and highly sensitive diagnostic tools and would be able to help guide antimicrobial therapy in rural areas where follow-up is difficult.

Florian Thalhammer*, Gunter Weiss**, Franz Allerberger, Petra Apfalter, Rainer Gattringer, Reinhold Glehr, Andrea Grisold, Markus Hell, Christoph Hogenauer, Oskar Janata, Robert Krause, Christian Madl, Markus Peck-Radosavljevic, Wolfgang Petritsch, Robert Sauermann, Herbert Tilg, Agnes Wechsler-Fordos und Christoph Wenisch Klinische Abteilung fur Infektionen und Tropenmedizin, Universitatskliniklinik fur Innere Medizin I, Medizinische Universitat Wien; Infektiologie, Immunologie, Rheumatologie, Pneumologie; Universitatsklinik fur Innere Medizin VI, Medizinische Universitat Innsbruck; Osterreichische Agentur fur Gesundheit und Ernahrungssicherheit (AGES), Wien; Institut fur Hygiene, Mikrobiologie und Tropenmedizin, Krankenhaus der Elisabethinen Linz; 5Niedergelassener Arzt fur Allgemeinmedizin, Hartberg; Institut fur Hygiene, Mikrobiologie und Umweltmedizin, Medizinische Universitat Graz; Zentrum fur Krankenhaushygiene und Infektionskontrolle der SALK, Universitatsklinikum der PMU, Salzburg; Klinische Abteilung fur Gastroenterologie und Hepatologie, Universitatsklinik fur Innere Medizin, Medizinische Universitat Graz; Krankenhaushygiene, Donauspital im SMZ Ost der Stadt Wien; Infektiologie, Universitatsklinik fur Innere Medizin (UKIM), Medizinische Universitat Graz; 4. Medizinische Abteilung mit Gastroenterologie und Hepatologie, Krankenanstalt Rudolfstiftung, Wien; Klinische Abteilung fur Gastroenterologie und Hepatologie, Universitatsklinik fur Innere Medizin III, Medizinische Universitat Wien; Klinische Abteilung fur Gastroenterologie und Hepatologie, Universitatsklinik fur Innere Medizin, Medizinische Universitat Graz; Abteilung Vertragspartner Medikamente, Hauptverband der osterreichischen Sozialversicherungstrager, Wien; Klinische Abteilung fur Gastroenterologie und Hepatologie, Universitatsklinik fur Innere Medizin II, Medizinische Universitat Innsbruck; Krankenhaushygiene, Krankenhaus Rudolfstiftung Wien; 4. Medizinische Abteilung mit Infektiologie, SMZ Sud – KFJ-Spital der Stadt Wien

Over the last 20 years, nucleic acid amplification tests (NAATs) have become a major tool for detection of microorganisms, for diagnostic testing, and for research purposes in the field of infectious diseases. NAATs offer significant sensitivity and speed compared to culture and do not require viable organisms. However, validated, commercially available, U.S. Food and Drug Administration-cleared assays exist for the following microorganisms: Mycobacterium tuberculosis, Chlamydia trachomatis, Neisseria gonorrhoeae, methicillin-resistant Staphylococcus aureus, group B streptococcus, Legionella pneumophila, human immunodeficiency virus, hepatitis B virus, and hepatitis C virus. Some of these tests are for very limited indications, for example, the methicillin-resistant Staphylococcus aureus assay is intended only for use with nasopharyngeal