Short report: impact of selective reporting of antibiotic susceptibility testing on antibiotic use in patients with bloodstream infection with Enterococcus faecalis.

INTRODUCTION: Bloodstream infections with Enterococcus faecalis are associated with relevant morbidity and mortality. Targeted antimicrobial therapy is essential. The choice of an adequate treatment may be challenging when susceptibility testing offers different options. Selective reporting of antibiotic susceptibility test results might lead to a more tailored antibiotic therapy and could therefore be an important antimicrobial stewardship program intervention. The aim of this study was to analyse whether the introduction of selective reporting of antibiotic test results leads to a more targeted antibiotic therapy in patients with bloodstream infection with Enterococcus faecalis. METHODS: This study was performed as a retrospective cohort study at the University Hospital Regensburg, Germany. All patients with blood cultures positive for Enterococcus faecalis between March 2003 and March 2022 were analysed. In February 2014 selective reporting of antibiotic susceptibility test results omitting sensitivity results for agents not recommended was introduced. RESULTS: 263 patients with blood cultures positive for Enterococcus faecalis were included. After introduction of selective reporting of antibiotic tests (AI) significantly more patients received ampicillin than before introduction of selective reporting (BI) (9.6% BI vs. 34.6% AI, p < 0.001). CONCLUSION: Selective reporting of antibiotic susceptibility test results led to a significantly higher use of ampicillin.

Antimicrobial coatings for environmental surfaces in hospitals: a potential new pillar for prevention strategies in hygiene.

Recent reports provide evidence that contaminated healthcare environments represent major sources for the acquisition and transmission of pathogens. Antimicrobial coatings (AMC) may permanently and autonomously reduce the contamination of such environmental surfaces complementing standard hygiene procedures. This review provides an overview of the current status of AMC and the demands to enable a rational application of AMC in health care settings. Firstly, a suitable laboratory test norm is required that adequately quantifies the efficacy of AMC. In particular, the frequently used wet testing (e.g. ISO 22196) must be replaced by testing under realistic, dry surface conditions. Secondly, field studies should be mandatory to provide evidence for antimicrobial efficacy under real-life conditions. The antimicrobial efficacy should be correlated to the rate of nosocomial transmission at least. Thirdly, the respective AMC technology should not add additional bacterial resistance development induced by the biocidal agents and co- or cross-resistance with antibiotic substances. Lastly, the biocidal substances used in AMC should be safe for humans and the environment. These measures should help to achieve a broader acceptance for AMC in healthcare settings and beyond. Technologies like the photodynamic approach already fulfil most of these AMC requirements.

[Hygiene in ENT examination: Fluorescence study with overview of current recommendations]. / Hygiene bei der HNO-Untersuchung: Fluoreszenzstudie mit Übersicht der aktuellen Empfehlungen.

Increasing normative demands on patient safety and quality assurance measures, but also the problem of multi-resistant germs and pathogens with a high potential for infection, increase the need for hygienically correct work.In this study, hygienically relevant potential sources of contamination in ENT examinations were to be identified and possible improvement strategies examined.A complete ENT examination was performed by 5 examiners with different professional experience, whose gloves were wetted with fluorescent lotion prior to the examination. Contaminations especially on the examination unit and on the instruments were identified. The potential risk of transmission of pathogens to subsequent patients was assessed using a specially developed score. Various strategies to reduce identified contamination possibilities were developed and thought through.The score of the investigators was very high with an average of 87.4 points (±3.6). The implementation of individual hygiene measures during the examination process would lead to a significant reduction of the score and thus to an improvement in hygiene: No shaking of hands (81.8), additional disinfection of patient's chair (79.8), disinfection of important surfaces (69.8), provision of standard instruments (60.2) or all instruments (32.2), disinfection of all relevant surfaces and provision of all instruments (7.4).The results show very clearly that an ENT examination is a complex procedure from the point of view of hygiene. For reliable protection against possible transmission events, a structured bundling of hygiene measures is therefore necessary.

Regulation of TNFR1 and CD95 signalling by receptor compartmentalization.

The death receptors tumour-necrosis factor receptor-1 (TNFR1) and CD95 (also known as FAS and APO-1) transduce signals that promote cell death by apoptosis. However, these receptors are also capable of inducing anti-apoptotic signals through the activation of the transcription factor nuclear factor-kappaB (NF-kappaB) or through activation of the proliferative mitogen-activated protein kinase (MAPK) cascade. Recent findings reveal a role for receptor internalization and endosomal trafficking in selectively transmitting the signals that lead either to apoptosis or to the survival of the cell.

An epidemic CC1-MRSA-IV clone yields false-negative test results in molecular MRSA identification assays: a note of caution, Austria, Germany, Ireland, 2020.

We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX/SCCmec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCCmec types and junction sites.

Palmitoylation is required for TNF-R1 signaling.

BACKGROUND: Binding of tumor necrosis factor (TNF) to TNF-receptor 1 (TNF-R1) can induce either cell survival or cell death. The selection between these diametrically opposed effects depends on the subcellular location of TNF-R1: plasma membrane retention leads to survival, while endocytosis leads to cell death. How the respective TNF-R1 associated signaling complexes are recruited to the distinct subcellular location is not known. Here, we identify palmitoylation of TNF-R1 as a molecular mechanism to achieve signal diversification. METHODS: Human monocytic U937 cells were analyzed. Palmitoylated proteins were enriched by acyl resin assisted capture (AcylRAC) and analyzed by western blot and mass spectrometry. Palmitoylation of TNF-R1 was validated by metabolic labeling. TNF induced depalmitoylation and involvement of APT2 was analyzed by enzyme activity assays, pharmacological inhibition and shRNA mediated knock-down. TNF-R1 palmitoylation site analysis was done by mutated TNF-R1 expression in TNF-R1 knock-out cells. Apoptosis (nuclear DNA fragmentation, caspase 3 assays), NF-κB activation and TNF-R1 internalization were used as biological readouts. RESULTS: We identify dynamic S-palmitoylation as a new mechanism that controls selective TNF signaling. TNF-R1 itself is constitutively palmitoylated and depalmitoylated upon ligand binding. We identified the palmitoyl thioesterase APT2 to be involved in TNF-R1 depalmitoylation and TNF induced NF-κB activation. Mutation of the putative palmitoylation site C248 interferes with TNF-R1 localization to the plasma membrane and thus, proper signal transduction. CONCLUSIONS: Our results introduce palmitoylation as a new layer of dynamic regulation of TNF-R1 induced signal transduction at a very early step of the TNF induced signaling cascade. Understanding the underlying mechanism may allow novel therapeutic options for disease treatment in future.

KRAS, HRAS and EGFR Mutations in Sporadic Sebaceous Gland Hyperplasia.

Sporadic sebaceous gland hyperplasia (SGH) is a benign skin lesion, with a high prevalence in the general population. Although SGH has been attributed to both extrinsic and intrinsic factors, the underlying genetic changes have not yet been characterized. Recently, HRAS and KRAS mutations have been identified in sebaceous naevus, a hamartoma sharing histological characteristics with SGH. Therefore we screened 43 SGH for activating mutations in RAS genes and other oncogenes. We identified a wide spectrum of mutually exclusive activating HRAS (8/43), KRAS (11/43) and EGFR mutations (7/31) in altogether 60% of the lesions investigated. A RAS and EGFR wildtype status was found in 15 normal sebaceous glands in the head and neck area. Our findings indicate that activating HRAS, KRAS and EGFR mutations play a major role in the pathogenesis of sporadic SGH. These results support the concept that SGH is a true benign neoplasm rather than a reactive hyperplasia.

Lipid-labeling facilitates a novel magnetic isolation procedure to characterize pathogen-containing phagosomes.

Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.

Pathogen-triggered activation of plasmacytoid dendritic cells induces IL-10-producing B cells in response to Staphylococcus aureus.

Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell-independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the V(H)3(+) BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A-bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scid(Prkdc)/γc(-/-) mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell-derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell-mediated immune tolerance.

Introduction and spread of vancomycin-resistant Enterococcus faecium (VREfm) at a German tertiary care medical center from 2004 until 2010: a retrospective whole-genome sequencing (WGS) study of the molecular epidemiology of VREfm.

BACKGROUND: In most of Europe and especially in Germany, there is currently a concerning rise in the number of hospital-acquired infections due to vancomycin-resistant Enterococcus faecium (VREfm). Therefore, there is a need to improve our understanding of the way VREfm spreads in hospitals. In this study, we investigated the molecular epidemiology of VREfm isolates from the first appearance at our university hospital in 2004 until 2010. There is only very scarce information about the molecular epidemiology of VREfm from this early time in Germany. METHODS: Our analysis includes all available first VREfm isolates of each patient at our tertiary care center collected during the years 2004-2010. If available, additional consecutive VREfm isolates from some patients were analyzed. We used multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) for the analysis and description of nosocomial transmission pathways as well as the detection of outbreaks. RESULTS: VREfm isolates from 158 patients and 76 additional subsequent patient isolates were included in the analysis. Until 2006, detections of VREfm remained singular cases, followed by a peak in the number of VREfm cases in 2007 and 2008 with a subsequent decline to baseline in 2010. MLST and cgMLST analysis show significant changes in the dominant sequence types (STs) and complex types (CTs) over the study period, with ST192 and ST17 being responsible for the peak in VREfm cases in 2007 and 2008. The four largest clusters detected during the study period are comprised of these two STs. Cluster analysis shows a focus on specific wards and departments for each cluster. In the early years of this study (2004-2006), all analyzed VREfm stemmed from clinical specimens, whereas since 2007, approximately half of the VREfm were detected by screening. Of the 234 VREfm isolates analyzed, 96% had a vanB and only 4% had a vanA resistance genotype. CONCLUSIONS: This retrospective study contributes significant knowledge about regional VREfm epidemiology from this early VREfm period in Germany. One remarkable finding is the striking dominance of vanB-positive VREfm isolates over the entire study period, which is in contrast with countrywide data. Analysis of cgMLST shows the transition from sporadic VRE cases at our institution to a sharp increase in VRE numbers triggered by oligoclonal spread and specific outbreak clusters with the dominance of ST192 and ST17.

Dirty hands: photodynamic killing of human pathogens like EHEC, MRSA and Candida within seconds.

Hand hygiene is one of the most important interventions for reducing transmission of nosocomial life-threatening microorganisms, like methicillin resistant Staphylococcus aureus (MRSA), enterohemorrhagic Escherichia coli (EHEC) or Candida albicans. All three pathogens have become a leading cause of infections in hospitals. Especially EHEC is causing severe diarrhoea and, in a small percentage of cases, haemolytic-uremic syndrome (HUS) as reported for E. coli 104:H4 in Germany 2011. We revealed the possibility to inactivate very fast and efficiently MRSA, EHEC and C. albicans using the photodynamic approach. MRSA, EHEC and C. albicans were incubated in vitro with different concentrations of TMPyP for 10 s and illuminated with visible light (50 mW cm(-2)) for 10 and 60 s. 1 µmol l(-1) of TMPyP and an applied radiant exposure of 0.5 J cm(-2) achieved a photodynamic killing of ≥99.9% of MRSA and EHEC. Incubation with higher concentrations (up to 100 µmol l(-1)) of TMPyP caused bacteria killing of >5 log(10) (≥99.999%) after illumination. Efficient Candida killing (≥99.999%) was achieved first at a higher light dose of 12 J cm(-2). Different rise and decay times of singlet oxygen luminescence signals could be detected in Candida cell suspensions for the first time, indicating different oxygen concentrations in the surrounding for the photosensitizer and singlet oxygen, respectively. This confirms that TMPyP is not only found in the water-dominated cell surrounding, but also within the C. albicans cells. Applying a water-ethanol solution of TMPyP on ex vivo porcine skin, fluorescence microscopy of histology showed that the photosensitizer was exclusively localized in the stratum corneum regardless of the incubation time. TMPyP exhibited a fast and very effective killing rate of life-threatening pathogens within a couple of seconds that encourages further testing in an in vivo setting. Being fast and effective, antimicrobial photodynamic applications might become acceptable as a tool for hand hygiene procedures and also in other skin areas.

Membrane trafficking of death receptors: implications on signalling.

Death receptors were initially recognised as potent inducers of apoptotic cell death and soon ambitious attempts were made to exploit selective ignition of controlled cellular suicide as therapeutic strategy in malignant diseases. However, the complexity of death receptor signalling has increased substantially during recent years. Beyond activation of the apoptotic cascade, involvement in a variety of cellular processes including inflammation, proliferation and immune response was recognised. Mechanistically, these findings raised the question how multipurpose receptors can ensure selective activation of a particular pathway. A growing body of evidence points to an elegant spatiotemporal regulation of composition and assembly of the receptor-associated signalling complex. Upon ligand binding, receptor recruitment in specialized membrane compartments, formation of receptor-ligand clusters and internalisation processes constitute key regulatory elements. In this review, we will summarise the current concepts of death receptor trafficking and its implications on receptor-associated signalling events.

Prognostic factors for favorable outcomes after veno-venous extracorporeal membrane oxygenation in critical care patients with COVID-19.

BACKGROUND: Patients with COVID-19 and severe acute respiratory failure may require veno-venous extracorporeal membrane oxygenation (VV ECMO). Yet, this procedure is resource-intensive and high mortality rates have been reported. Thus, predictors for identifying patients who will benefit from VV ECMO would be helpful. METHODS: This retrospective study included 129 patients with COVID-19 and severe acute respiratory failure, who had received VV ECMO at the University Medical Center Regensburg, Germany, between 1 March 2020 and 31 December 2021. Patient-specific factors and relevant intensive-care parameters at the time of the decision to start VV ECMO were investigated regarding their value as predictors of patient survival. In addition, the intensive-care course of the first 10 days of VV ECMO was compared between survivors and patients who had died in the intensive care unit. RESULTS: The most important parameters for predicting outcome were patient age and platelet count, which differed significantly between survivors and non-survivors (age: 52.6±8.1 vs. 57.4±10.1 years, p<0.001; platelet count before VV ECMO: 321.3±132.2 vs. 262.0±121.0 /nL, p = 0.006; average on day 10: 199.2±88.0 vs. 147.1±57.9 /nL, p = 0.002). A linear regression model derived from parameters collected before the start of VV ECMO only included age and platelet count. Patients were divided into two groups by using receiver operating characteristics (ROC) analysis: group 1: 78% of patients, mortality 26%; group 2: 22% of patients, mortality 75%. A second linear regression model included average blood pH, minimum paO2, and average pump flow on day 10 of VV ECMO in addition to age and platelet count. The ROC curve resulted in two cut-off values and thus in three groups: group 1: 25% of patients, mortality 93%; group 2: 45% of patients, mortality 31%; group 3: 30% of patients, mortality 0%.

Detection and characterization of putative hypervirulent Klebsiella pneumoniae isolates in microbiological diagnostics.

Hypervirulent Klebsiella pneumoniae strains (hvKp) can cause invasive community-acquired infections in healthy patients of all ages. In this study, the prevalence of putative hvKp in a German tertiary center was investigated and hvKp were characterized by phenotypic and molecular assays. All K. pneumoniae isolates in routine microbiological diagnostics from a single center were screened by string-testing over a period of 6 months. String-test positive (≥ 0.5 mm) isolates were re-evaluated on different media and under various conditions (aerobe, anaerobe). For string-test positive isolates, genes (magA, iutA, rmpA and rmpA2) associated with hypermucoviscosity and hypervirulence were amplified by multiplex PCR. PCR-positive isolates were subjected to whole-genome sequencing and sedimentation and biofilm formation assays. From 1310 screened K. pneumoniae isolates in clinical routine 100 isolates (7.6%) were string test positive. From these, 9% (n = 9) were defined as putative hvKp (string-test+/PCR+). Highest rate of string-test-positive isolates was observed on MacConkey agar under aerobic conditions. Amongst these nine putative hvKp isolates, the international lineage ST23 carrying hvKp-plasmid pKpVP-1 was the most common, but also a rare ST86 with pKpVP-2 was identified. All nine isolates showed hypermucoviscosity and weak biofilm formation. In conclusion, 9% of string-positive, respectively 0.69% of all K. pneumoniae isolates from routine were defined as putative hypervirulent. MacConkey agar was the best medium for hvKp screening.

Antimicrobial Photodynamic Coatings Reduce the Microbial Burden on Environmental Surfaces in Public Transportation-A Field Study in Buses.

Millions of people use public transportation daily worldwide and frequently touch surfaces, thereby producing a reservoir of microorganisms on surfaces increasing the risk of transmission. Constant occupation makes sufficient cleaning difficult to achieve. Thus, an autonomous, permanent, antimicrobial coating (AMC) could keep down the microbial burden on such surfaces. A photodynamic AMC was applied to frequently touched surfaces in buses. The microbial burden (colony forming units, cfu) was determined weekly and compared to equivalent surfaces in buses without AMC (references). The microbial burden ranged from 0-209 cfu/cm2 on references and from 0-54 cfu/cm2 on AMC. The means were 13.4 ± 29.6 cfu/cm2 on references and 4.5 ± 8.4 cfu/cm2 on AMC (p < 0.001). The difference in microbial burden on AMC and references was almost constant throughout the study. Considering a hygiene benchmark of 5 cfu/cm2, the data yield an absolute risk reduction of 22.6% and a relative risk reduction of 50.7%. In conclusion, photodynamic AMC kept down the microbial burden, reducing the risk of transmission of microorganisms. AMC permanently and autonomously contributes to hygienic conditions on surfaces in public transportation. Photodynamic AMC therefore are suitable for reducing the microbial load and closing hygiene gaps in public transportation.

Characterization of PVL-Positive MRSA Isolates in Northern Bavaria, Germany over an Eight-Year Period.

PURPOSE: Community-acquired methicillin-resistant Staphylococcus aureus strains (CA-MRSA) are spread worldwide and often cause recurring and persistent infections in humans. CA-MRSA strains frequently carry Panton-Valentine leukocidin (PVL) as a distinctive virulence factor. This study investigates the molecular epidemiology, antibiotic resistance and clinical characteristics of PVL-positive MRSA strains in Northern Bavaria, Germany, isolated over an eight-year period. METHODS: Strains were identified by MALDI-TOF MS and antibiotic susceptibility was tested by automated microdilution (VITEK 2) or disk diffusion. PVL-encoding genes and mecA were detected by PCR. MRSA clonal complexes (CC) and lineages were assigned by genotyping via DNA microarray and spa-typing. RESULTS: In total, 131 PVL-positive MRSA were collected from five hospital sites between 2009 and 2016. Predominant lineages were CC8-MRSA-[IV+ACME], USA300 (27/131; 20.6%); CC30-MRSA-IV, Southwest Pacific Clone (26/131; 19.8%) and CC80-MRSA-IV (25/131; 19.1%). Other CCs were detected less frequently. Resistance against erythromycin and clindamycin was prevalent, whereas all strains were sensitive towards vancomycin and linezolid. In total, 100 cases (76.3%) were causally linked to an infection. The majority (102/131; 77.9%) of isolates were detected in skin swabs or swabs from surgical sites. CONCLUSIONS: During the sample period we found an increase in the PVL-positive MRSA lineages CC30 and CC1. Compared to less-abundant lineages CC1 or CC22, the predominant lineages CC8, CC30 and CC80 harbored a broader resistance spectrum. Furthermore, these lineages are probably associated with a travel and migration background. In the spatio-temporal setting we investigated, these were arguably drivers of diversification and change in the landscape of PVL-positive MRSA.

Inhibition of ADAM17 impairs endothelial cell necroptosis and blocks metastasis.

Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell-induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the γ-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies.

Lymphotoxin-ß receptor activation by lymphotoxin-α(1)ß(2) and LIGHT promotes tumor growth in an NFκB-dependent manner.

Lymphotoxin beta receptor (LTßR) activation on mouse fibrosarcoma cells (BFS-1) results in enhanced solid tumor growth paralleled by increased angiogenesis induced by the expression of pro-angiogenic CXCL2. In our study, we demonstrate that both functional ligands of the LTßR, namely LTα(1) ß(2) and LIGHT, are involved in the activation of LTßR in solid fibrosarcomas. To identify whether the lymphocyte population is involved in the activation of LTßR in these fibrosarcoma tumors, we used conditional LTß-deficient mice that specifically lack LTß expression either on T cells (T-LTß(-/-)) or on B cells (B-LTß(-/-)). Solid tumor growth was reduced in both mouse strains when compared to tumor growth in wild-type mice, indicating the participation of both T and B host lymphocytes in the activation of LTßR in these tumors. Tumor growth was also reduced in LIGHT-deficient mice, suggesting a contribution of this ligand to the activation of LTßR in BFS-1 fibrosarcomas. LTßR signaling can involve IκBα and/or NFκB-inducing kinase (NIK) for subsequent NFκB activation in different types of cells. Expression of a dominant negative form of IκBα or of a dominant negative mutant of NIK resulted in decreased activation of NFκB signaling and reduced expression of pro-angiogenic CXCL2 in vitro. Moreover, expression of dominant negative form of NIK or an IκBα repressor in these fibrosarcoma cells resulted in reduced solid tumor growth in vivo, suggesting that both IκBα and NIK are involved in pro-angiogenic signaling after LTßR activation. Our data support the idea that the ablation of LTßR signaling should be considered for cancer treatment.

Rapid detection and molecular differentiation of toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans strains by LightCycler PCR.

The systemic symptoms of diphtheria are caused by the tox-encoded diphtheria toxin (DT) which is produced by toxigenic Corynebacterium spp. Besides the classical agent C. diphtheriae, the zoonotic pathogen C. ulcerans has increasingly been reported as an emerging pathogen for diphtheria. The reliable detection of toxigenic Corynebacterium spp. is of substantial importance for both diphtheria surveillance in the public health sector and the clinical workup of a patient with diphtherialike symptoms. Since the respective tox genes of C. diphtheriae and C. ulcerans differ from each other in both DNA and amino acid sequence, both tox genes should be covered by novel real-time PCR methods. We describe the development and validation of a LightCycler PCR assay which reliably recognizes tox genes from both C. diphtheriae and C. ulcerans and differentiates the respective target genes by fluorescence resonance energy transfer (FRET) hybridization probe melting curve analysis.

Randomized trial on 14 versus 7 days of esomeprazole, moxifloxacin, and amoxicillin for second-line or rescue treatment of Helicobacter pylori infection.

BACKGROUND: Triple therapy with a proton pump inhibitor, moxifloxacin, and amoxicillin has been proven effective in first-line treatment of Helicobacter pylori infection. AIM: To explore 1, the value of triple therapy with esomeprazole, moxifloxacin, and amoxicillin in second-line or rescue treatment of Caucasian patients and 2, the impact of treatment duration on eradication success. METHODS: H. pylori-infected patients with at least one previous treatment failure were randomized to oral esomeprazole 20 mg b.i.d., moxifloxacin 400 mg o.d., and amoxicillin 1000 mg b.i.d. for either 7 (EMA-7) or 14 days (EMA-14). Eradication was confirmed by 13C urea breath test. Antimicrobial susceptibility testing was performed in all patients at baseline and in patients who failed treatment. RESULTS: Eighty patients were randomized, and 60% had ≥ 2 previous treatment failures. Pretreatment resistance against clarithromycin and metronidazole was found in 70.5 and 61.5% of cases, respectively. The intention-to-treat eradication rate was significantly higher after EMA-14 compared with EMA-7 (95.0 vs 78.9%, p = .036). No independent risk factor for treatment failure could be identified. There were no serious adverse events. Five of the EMA-14 patients (12.5%) compared with none of the EMA-7 patients discontinued prematurely because of adverse events (p = .031). Post-treatment resistance against moxifloxacin was found in one of seven patients with isolated organisms (14.3%). CONCLUSION: Second-line/rescue H. pylori eradication therapy with esomeprazole, moxifloxacin, and amoxicillin is very effective and well tolerated. Fourteen days of treatment significantly increase the eradication rate but also the rate of adverse events.