Axel Hamprecht

Emergence of azole-resistant invasive aspergillosis in HSCT recipients in Germany

OBJECTIVES Aspergillus fumigatus is the most common agent of invasive aspergillosis (IA). In recent years, resistance to triazoles, the mainstay of IA therapy, has emerged in different countries worldwide. IA caused by azole-resistant A. fumigatus (ARAF) shows an exceedingly high mortality. In this study, IA due to ARAF isolates in HSCT recipients in Germany was investigated. METHODS The epidemiology of azole resistance in IA was analysed in two German haematology departments. Between 2012 and 2013, 762 patients received HSCT in Essen (n = 388) and Cologne (n = 374). Susceptibility testing of A. fumigatus isolates was performed by Etest, followed by EUCAST broth microdilution testing if elevated MICs were recorded. In all ARAF isolates the cyp51A gene was sequenced and the genotype was determined by microsatellite typing using nine short tandem repeats. RESULTS In total, A. fumigatus was recovered from 27 HSCT recipients. Eight patients had azole-resistant IA after HSCT, and seven of the cases were fatal (88%). All except one patient received antifungal prophylaxis (in five cases triazoles). TR34/L98H was the most common mutation (n = 5), followed by TR46/Y121F/T289A (n = 2). In one resistant isolate no cyp51A mutation was detected. Genotyping revealed genetic diversity within the German ARAF isolates and no clustering with resistant isolates from the Netherlands, India and France. CONCLUSIONS This report highlights the emergence of azole-resistant IA with TR34/L98H and TR46/Y121F/T289A mutations in HSCT patients in Germany and underscores the need for systematic antifungal susceptibility testing of A. fumigatus.

Detection of NDM-7 in Germany, a new variant of the New Delhi metallo-β-lactamase with increased carbapenemase activity

OBJECTIVES This study characterized a new variant of the New Delhi metallo-β-lactamase (NDM). METHODS A multidrug-resistant Escherichia coli isolate was recovered from the wounds, throat and rectum of a Yemeni patient who presented at the Frankfurt University Hospital in Germany. The presence of β-lactamase genes was analysed by PCR and sequencing. The isolate was further characterized by susceptibility testing, conjugation and transformation assays and plasmid analysis. RESULTS The E. coli isolate was resistant to all β-lactams including carbapenems. By PCR analysis, the β-lactamase genes blaCMY-2, blaCTX-M-15, blaTEM-1 and blaNDM were identified. Sequencing revealed a blaNDM gene that differed from blaNDM-1 by two point mutations at positions 388 (G→A) and 460 (A→C) corresponding to amino acid substitutions Asp130Asn and Met154Leu, respectively. This NDM variant was identified as NDM-7. The blaNDM-7 gene was located on a self-transferable IncX3 plasmid of 60 kb. E. coli TOP10 transformants harbouring NDM-7 showed higher MICs of β-lactams including carbapenems compared with transformants harbouring NDM-1. Multilocus sequence typing analysis revealed that the E. coli isolate belonged to a novel sequence type (ST599). CONCLUSIONS This study identified a novel NDM variant in E. coli, NDM-7, possessing a high ability to hydrolyse β-lactam antibiotics. Given the diversity of NDM variants located on self-transferable plasmids found in different Gram-negative species and isolated in different countries, the blaNDM gene will most likely efficiently disseminate worldwide.

Diagnosis and antimicrobial therapy of lung infiltrates in febrile neutropenic patients (allogeneic SCT excluded): updated guidelines of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Medical Oncology (DGHO)

Up to 25% of patients with profound neutropenia lasting for >10 days develop lung infiltrates, which frequently do not respond to broad-spectrum antibacterial therapy. While a causative pathogen remains undetected in the majority of cases, Aspergillus spp., Pneumocystis jirovecii, multi-resistant Gram-negative pathogens, mycobacteria or respiratory viruses may be involved. In at-risk patients who have received trimethoprim-sulfamethoxazole (TMP/SMX) prophylaxis, filamentous fungal pathogens appear to be predominant, yet commonly not proven at the time of treatment initiation. Pathogens isolated from blood cultures, bronchoalveolar lavage (BAL) or respiratory secretions are not always relevant for the etiology of pulmonary infiltrates and should therefore be interpreted critically. Laboratory tests for detecting Aspergillus galactomannan, β-d-glucan or DNA from blood, BAL or tissue samples may facilitate the diagnosis; however, most polymerase chain reaction assays are not yet standardized and validated. Apart from infectious agents, pulmonary side-effects from cytotoxic drugs, radiotherapy or pulmonary involvement by the underlying malignancy should be included into differential diagnosis and eventually be clarified by invasive diagnostic procedures. Pre-emptive treatment with mold-active systemic antifungal agents improves clinical outcome, while other microorganisms are preferably treated only when microbiologically documented. High-dose TMP/SMX is first choice for treatment of Pneumocystis pneumonia, while cytomegalovirus pneumonia is treated primarily with ganciclovir or foscarnet in most patients. In a considerable number of patients, clinical outcome may be favorable despite respiratory failure, so that intensive care should be unrestrictedly provided in patients whose prognosis is not desperate due to other reasons. Abstract Up to 25% of patients with profound neutropenia lasting for >10 days develop lung infiltrates, which frequently do not respond to broad-spectrum antibacterial therapy. While a causative pathogen remains undetected in the majority of cases, Aspergillus spp., Pneumocystis jirovecii, multi-resistant Gram-negative pathogens, mycobacteria or respiratory viruses may be involved. In at-risk patients who have received trimethoprim–sulfamethoxazole (TMP/SMX) prophylaxis, filamentous fungal pathogens appear to be predominant, yet commonly not proven at the time of treatment initiation. Pathogens isolated from blood cultures, bronchoalveolar lavage (BAL) or respiratory secretions are not always relevant for the etiology of pulmonary infiltrates and should therefore be interpreted critically. Laboratory tests for detecting Aspergillus galactomannan, β-d-glucan or DNA from blood, BAL or tissue samples may facilitate the diagnosis; however, most polymerase chain reaction assays are not yet standardized and validated. Apart from infectious agents, pulmonary side-effects from cytotoxic drugs, radiotherapy or pulmonary involvement by the underlying malignancy should be included into differential diagnosis and eventually be clarified by invasive diagnostic procedures. Pre-emptive treatment with mold-active systemic antifungal agents improves clinical outcome, while other microorganisms are preferably treated only when microbiologically documented. High-dose TMP/SMX is first choice for treatment of Pneumocystis pneumonia, while cytomegalovirus pneumonia is treated primarily with ganciclovir or foscarnet in most patients. In a considerable number of patients, clinical outcome may be favorable despite respiratory failure, so that intensive care should be unrestrictedly provided in patients whose prognosis is not desperate due to other reasons.

A Multinational, Preregistered Cohort Study of β-Lactam/β-Lactamase Inhibitor Combinations for Treatment of Bloodstream Infections Due to Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae

ABSTRACT The spread of extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) is leading to increased carbapenem consumption. Alternatives to carbapenems need to be investigated. We investigated whether β-lactam/β-lactamase inhibitor (BLBLI) combinations are as effective as carbapenems in the treatment of bloodstream infections (BSI) due to ESBL-E. A multinational, retrospective cohort study was performed. Patients with monomicrobial BSI due to ESBL-E were studied; specific criteria were applied for inclusion of patients in the empirical-therapy (ET) cohort (ETC; 365 patients), targeted-therapy (TT) cohort (TTC; 601 patients), and global cohort (GC; 627 patients). The main outcome variables were cure/improvement rate at day 14 and all-cause 30-day mortality. Multivariate analysis, propensity scores (PS), and sensitivity analyses were used to control for confounding. The cure/improvement rates with BLBLIs and carbapenems were 80.0% and 78.9% in the ETC and 90.2% and 85.5% in the TTC, respectively. The 30-day mortality rates were 17.6% and 20% in the ETC and 9.8% and 13.9% in the TTC, respectively. The adjusted odds ratio (OR) (95% confidence interval [CI]) values for cure/improvement rate with ET with BLBLIs were 1.37 (0.69 to 2.76); for TT, they were 1.61 (0.58 to 4.86). Regarding 30-day mortality, the adjusted OR (95% CI) values were 0.55 (0.25 to 1.18) for ET and 0.59 (0.19 to 1.71) for TT. The results were consistent in all subgroups studied, in a stratified analysis according to quartiles of PS, in PS-matched cases, and in the GC. BLBLIs, if active in vitro, appear to be as effective as carbapenems for ET and TT of BSI due to ESLB-E regardless of the source and specific species. These data may help to avoid the overuse of carbapenems. (This study has been registered at ClinicalTrials.gov under registration no. NCT01764490.)

In Vivo Selection of a Missense Mutation in adeR and Conversion of the Novel blaOXA-164 Gene into blaOXA-58 in Carbapenem-Resistant Acinetobacter baumannii Isolates from a Hospitalized Patient

ABSTRACT The mechanism of stepwise acquired multidrug resistance in Acinetobacter baumannii isolates from a hospitalized patient was investigated. Thirteen consecutive multidrug-resistant isolates were recovered from the same patient over a 2-month period. The Vitek 2 system identified the isolates as meropenem-sensitive Acinetobacter lwoffii; however, molecular identification showed that the isolates were A. baumannii. Etest revealed that the isolates were meropenem resistant. The presence of oxacillinase (OXA)-type enzymes were investigated by sequencing. The clonal relatedness of isolates was assessed by pulsed-field gel electrophoresis (PFGE). Expression of the genes encoding the efflux pumps AdeB and AdeJ was performed by semiquantitative real-time reverse transcription-PCR (qRT-PCR). The adeRS two-component system was sequenced. All isolates had identical PFGE fingerprints, suggesting clonal identity. The first six isolates were positive for the novel blaOXA-164 gene. The following seven isolates, recovered after treatment with a combination of meropenem, amikacin, ciprofloxacin, and co-trimoxazole showed an increase of >7-fold in adeB mRNA transcripts and a missense mutation in blaOXA-164, converting it to blaOXA-58. Sequencing revealed a novel mutation in adeR. These data illustrate how A. baumannii can adapt during antimicrobial therapy, leading to increased antimicrobial resistance.

A multicentre cohort study on colonization and infection with ESBL-producing Enterobacteriaceae in high-risk patients with haematological malignancies

BACKGROUND Bloodstream infections (BSIs) caused by enterobacteria remain a leading cause of mortality in patients with chemotherapy-induced neutropenia. The rate and type of colonization and infection with ESBL-producing Enterobacteriaceae (ESBL-E) and their mode of transmission in German cancer centres is largely unknown. METHODS We performed a prospective, observational study at five German university-based haematology departments. Participating sites screened for intestinal ESBL-E colonization within 72 h of admission, every 10 ± 2 days thereafter and before discharge. Three of the five centres performed contact isolation for patients colonized or infected with ESBL-E. Molecular characterization of resistance mechanisms and epidemiological typing of isolates by repetitive extragenic palindromic PCR (rep-PCR) and PFGE was performed to assess strain transmission between patients. RESULTS Between October 2011 and December 2012, 719 hospitalizations of 497 haematological high-risk patients comprising 20,143 patient-days were analysed. Mean duration of in-hospital stay was 36.6 days (range: 2-159 days). ESBL-E were identified from screening samples (82.8% Escherichia coli and 14.6% Klebsiella pneumoniae) in 55/497 patients (11.1%; range by centre: 5.8%-23.1%). PFGE and rep-PCR revealed only a single case of potential cross-transmission among two patients colonized with K. pneumoniae. Six episodes of BSI with ESBL-E were observed. Multivariate analysis revealed previous colonization with ESBL-E as the most important risk factor for BSI with ESBL-E (OR 52.00; 95% CI 5.71-473.89). CONCLUSIONS Even though BSI with ESBL-E is still rare in this high-risk population, colonization rates are substantial and vary considerably between centres. In-hospital transmission of ESBL-E as assessed by molecular typing was the exception.

Incidence of Cyp51 A Key Mutations in Aspergillus fumigatus-A Study on Primary Clinical Samples of Immunocompromised Patients in the Period of 1995-2013

As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA) in immunocompromised patients is rarely based on positive culture yield, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat), L98H and M220 alterations). We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL), biopsies, cerebrospinal fluid (CSF)) of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46) directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.

Colonization with third-generation cephalosporin-resistant Enterobacteriaceae on hospital admission: prevalence and risk factors

OBJECTIVES The objectives of this study were to prospectively assess the rectal carriage rate of third-generation cephalosporin-resistant Enterobacteriaceae (3GCREB) in non-ICU patients on hospital admission and to investigate resistance mechanisms and risk factors for carriage. METHODS Adult patients were screened for 3GCREB carriage at six German tertiary care hospitals in 2014 using rectal swabs or stool samples. 3GCREB isolates were characterized by phenotypic and molecular methods. Each patient answered a questionnaire about potential risk factors for colonization with MDR organisms (MDROs). Univariable and multivariable risk factor analyses were performed to identify factors associated with 3GCREB carriage. RESULTS Of 4376 patients, 416 (9.5%) were 3GCREB carriers. Escherichia coli was the predominant species (79.1%). ESBLs of the CTX-M-1 group (67.3%) and the CTX-M-9 group (16.8%) were the most frequent β-lactamases. Five patients (0.11%) were colonized with carbapenemase-producing Enterobacteriaceae. The following risk factors were significantly associated with 3GCREB colonization in the multivariable analysis (P < 0.05): centre; previous MDRO colonization (OR = 2.12); antibiotic use within the previous 6 months (OR = 2.09); travel outside Europe (OR = 2.24); stay in a long-term care facility (OR = 1.33); and treatment of gastroesophageal reflux disease (GERD) (OR = 1.22). CONCLUSIONS To our knowledge, this is the largest admission prevalence study of 3GCREB in Europe. The observed prevalence of 9.5% 3GCREB carriage was higher than previously reported and differed significantly among centres. In addition to previously identified risk factors, the treatment of GERD proved to be an independent risk factor for 3GCREB colonization.

Ertapenem for the treatment of bloodstream infections due to ESBL-producing Enterobacteriaceae: a multinational pre-registered cohort study

OBJECTIVES Data about the efficacy of ertapenem for the treatment of bloodstream infections (BSI) due to ESBL-producing Enterobacteriaceae (ESBL-E) are limited. We compared the clinical efficacy of ertapenem and other carbapenems in monomicrobial BSI due to ESBL-E. METHODS A multinational retrospective cohort study (INCREMENT project) was performed (ClinicalTrials.gov identifier: NCT01764490). Patients given monotherapy with ertapenem or other carbapenems were compared. Empirical and targeted therapies were analysed. Propensity scores were used to control for confounding; sensitivity analyses were performed in subgroups. The outcome variables were cure/improvement rate at day 14 and all-cause 30 day mortality. RESULTS The empirical therapy cohort (ETC) and the targeted therapy cohort (TTC) included 195 and 509 patients, respectively. Cure/improvement rates were 90.6% with ertapenem and 75.5% with other carbapenems (P = 0.06) in the ETC and 89.8% and 82.6% (P = 0.02) in the TTC, respectively; 30 day mortality rates were 3.1% and 23.3% (P = 0.01) in the ETC and 9.3% and 17.1% (P = 0.01) in the TTC, respectively. Adjusted ORs (95% CI) for cure/improvement with empirical and targeted ertapenem were 1.87 (0.24-20.08; P = 0.58) and 1.04 (0.44-2.50; P = 0.92), respectively. For the propensity-matched cohorts it was 1.18 (0.43-3.29; P = 0.74). Regarding 30 day mortality, the adjusted HR (95% CI) for targeted ertapenem was 0.93 (0.43-2.03; P = 0.86) and for the propensity-matched cohorts it was 1.05 (0.46-2.44; P = 0.90). Sensitivity analyses were consistent except for patients with severe sepsis/septic shock, which showed a non-significant trend favouring other carbapenems. CONCLUSIONS Ertapenem appears as effective as other carbapenems for empirical and targeted therapy of BSI due to ESBL-E, but further studies are needed for patients with severe sepsis/septic shock.

Making moulds meet. Information retrieval as a basis for understanding Pseudallescheria and Scedosporium.

G. S. de Hoog, V. Robert, M. Lackner, M. J. G. T. Vehreschild, J. J. Vehreschild, F. Symoens, E. Gottlich-Fligg, D. Garcia-Hermoso, A. Harun, W. Meyer, S. C. A. Chen, A. Hamprecht, G. Fischer, W. Buzina, O. A. Cornely, J. Guarro, J. Cano and R. Horre* CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, The Netherlands, Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China, Division of Hygiene and Medical Microbiology, Innsbruck Medical University, Innsbruck, Austria, 1st Department of Internal Medicine, University of Cologne, Cologne, Germany, Scientific Institute of Public Health, Brussels, Belgium, BZH Deutsches Beratungszentrum fur Hygiene des Universitatsklinikums Freiburg, Freiburg, Germany, Institut Pasteur, Unite de Mycologie Moleculaire, Centre National de Reference Mycologie et Antifongiques, Paris, France, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia, Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, Sydney Medical School Westmead Hospital, Sydney, The University of Sydney, Australia, Landesgesundheitsamt Baden-Wurttemberg, Stuttgart, Germany, Institute of Hygiene, Microbiology and Environmental Medicine, Medical University Graz, Graz, Austria, Clinical Trials Centre Cologne, ZKS Koln, University of Cologne, Cologne, Germany, Center for Integrated Oncology CIO Koln-Bonn, University of Cologne, Cologne, Germany, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany, Facultad de Medicina, Universitat Rovira i Virgili, Reus, Spain, Institute for Medical Microbiology, Immunology and Parasitology, University of Bonn, Bonn, Germany and Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany