Elimination of Herpes Simplex Virus-2 and Epstein-Barr Virus With Seraph 100 Microbind Affinity Blood Filter and Therapeutic Plasma Exchange: An Explorative Study in a Patient With Acute Liver Failure.

Herpes simplex virus (HSV)-2 is a rare cause of hepatitis that can lead to acute liver failure (ALF) and often death. The earlier the initiation of acyclovir treatment the better the survival. With regard to ALF, controlled randomized data support the use of therapeutic plasma exchange (TPE) both as bridge to recovery or transplantation-possibly by modulating the systemic inflammatory response and by replacing coagulation factors. Seraph 100 Microbind Affinity Blood Filter (Seraph; Ex Thera Medical, Martinez, CA), a novel extracorporeal adsorption device, removes living pathogens by binding to a heparin-coated surface was shown to efficiently clear HSV-2 particles in vitro. Here, we tested the combination of Seraph with TPE to reduce a massive HSV-2 viral load to reach a situation in that liver transplantation would be feasible. DESIGN: Explorative study. SETTING: Academic tertiary care transplant center. PATIENT: Single patient with HSV-2-induced ALF. INTERVENTIONS: TPE + Seraph 100 Microbind Affinity Blood Filter. MEASUREMENTS AND MAIN RESULTS: We report Seraph clearance data of HSV-2 and of Epstein-Barr virus (EBV) in vivo as well as total viral elimination by TPE. Genome copies/mL of HSV-2 and EBV in EDTA plasma were measured by polymerase chain reaction every 60 minutes over 6 hours after starting Seraph both systemically and post adsorber. Also, HSV-2 and EBV were quantified before and after TPE and in the removed apheresis plasma. We found a total elimination of 1.81 × e11 HSV-2 copies and 2.11 × e6 EBV copies with a single TPE (exchange volume of 5L; 1.5× calculated plasma volume). Whole blood clearance of HSV-2 in the first 6 hours of treatment was 6.64 mL/min (4.98-12.92 mL/min). Despite much lower baseline viremia, clearance of EBV was higher 36.62 mL/min (22.67-53.48 mL/min). CONCLUSIONS: TPE was able to remove circulating HSV-2 copies by 25% and EBV copies by 40% from the blood. On the other hand, clearance of HSV-2 by Seraph was clinically irrelevant, but Seraph seemed to be far more effective of removing EBV, implicating a possible use in EBV-associated pathologies, but this requires further study.

Mycobacterium helveticum sp. nov., a novel slowly growing mycobacterial species associated with granulomatous lesions in adult swine.

The occurrence of nontuberculous mycobacteria in different hosts and their implication as obligate or opportunistic pathogens remain mainly unclear. Mycobacteriosis in pigs is usually associated with members of the Mycobacterium avium complex and, in particular, with 'Mycobacterium avium subsp. hominissuis'. Here we describe a novel slow-growing mycobacterial species isolated from lymph nodes obtained from two sows housed in different Swiss farms. The animals presented chronic inappetence and mild diarrhoea. Gross pathology revealed focal caseous lymphadenopathy of the mesenteric lymph nodes. Complete genome sequencing of the two isolates from the two sows was performed. The genomes comprised 5.76 Mb and an average nucleotide identity score of 99.97 %. Whole genome sequence, mycolic acid and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the two isolates were not related to any previously described Mycobacterium species. The closest related species was Mycobacterium parmense, a slow-growing scotochromogenic mycobacterium first isolated from a cervical lymph node of a 3-year-old child. The name proposed for the new species is Mycobacterium helveticum sp. nov. and 16-83T (=DSM 109965T= LMG 2019-02457T) is the type strain.

Listeriosis Caused by Persistence of Listeria monocytogenes Serotype 4b Sequence Type 6 in Cheese Production Environment.

A nationwide outbreak of human listeriosis in Switzerland was traced to persisting environmental contamination of a cheese dairy with Listeria monocytogenes serotype 4b, sequence type 6, cluster type 7488. Whole-genome sequencing was used to match clinical isolates to a cheese sample and to samples from numerous sites within the production environment.

Siblings with typhoid fever: An investigation of intrafamilial transmission, clonality, and antibiotic susceptibility.

BACKGROUND: Typhoid fever usually manifests as an acute disease. However, asymptomatic carriage with Salmonella Typhi may occur. This study investigated a family setting of severe typhoid fever in Switzerland months after return from Bangladesh. METHOD: Standard microbiological procedures were performed. Testing for S. Typhi IgM antibodies was done using a novel immunochromographic lateral flow assay. Whole genome sequencing (WGS) followed by comparative core genome multilocus sequence typing (cgMLST) was performed on the S. Typhi isolates. RESULTS: Four months after returning from a visit to Bangladesh sibling 1 (9 months) was diagnosed with a S. Typhi meningitis and sibling 3 (8 years) was identified as asymptomatic S. Typhi carrier. Sibling 2 (2 years) was retrospectively diagnosed with typhoid fever by IgM serology at the time point of admission to the hospital. Parents were asymptomatic and culture-negative. WGS analysis of family S. Typhi isolates showed clonality and strongest homology with S. Typhi strains occurring in Bangladesh. The S. Typhi strain showed resistance against fluoroquinolones. A 4-week course of ceftriaxone resulted in full recovery of sibling 1. S. Typhi was eradicated from sibling 3 following azithromycin treatment for 14 days. CONCLUSION: S. Typhi, acquired from a visit to Bangladesh, was most likely transmitted within the family from one brother as asymptomatic shedder to his 9-month-old brother who manifested S. Typhi meningitis as a very rare and life-threatening presentation of typhoid fever. S. Typhi infection should be considered even in case of uncommon manifestations and irrespective of the interval between disease presentation and travel to an endemic area.

First Clinical Case of In Vivo Acquisition of DHA-1 Plasmid-Mediated AmpC in a Salmonella enterica subsp. enterica Isolate.

A pan-susceptible Salmonella enterica serovar Worthington isolate was detected in the stool of a man returning from Sri Lanka. Under ceftriaxone treatment, a third-generation cephalosporin (3GC)-resistant Salmonella Worthington was isolated after 8 days. Molecular analyses indicated that the two isolates were identical. However, the latter strain acquired a blaDHA-1-carrying IncFII plasmid probably from a Citrobacter amalonaticus isolate colonizing the gut. This is the first report of in vivo acquisition of plasmid-mediated resistance to 3GCs in S. enterica.

Vertical Transmission of Mycoplasma pneumoniae Infection.

Mycoplasma pneumoniae is a significant cause of pneumonia in school-aged children and young adults. We report a case of neonatal M. pneumoniae pneumonia in a preterm child manifesting in the first hours of life. Vertical transmission was demonstrated by the detection of M. pneumoniae in inflamed placental tissue indicating chorioamnionitis.

Increased Pathogen Identification in Vascular Graft Infections by the Combined Use of Tissue Cultures and 16S rRNA Gene Polymerase Chain Reaction.

Background: Vascular graft infections (VGI) are difficult to diagnose and treat, and despite redo surgery combined with antimicrobial treatment, outcomes are often poor. VGI diagnosis is based on a combination of clinical, radiological, laboratory and microbiological criteria. However, as many of the VGI patients are already under antimicrobial treatment at the time of redo surgery, microbiological identification is often difficult and bacterial cultures often remain negative rendering targeted treatment impossible. We aimed to assess the benefit of 16S rRNA gene polymerase chain reaction (broad-range PCR) for better microbiological identification in patients with VGI. Methods: We prospectively analyzed the clinical, microbiological, and treatment data of patients enrolled in the observational Vascular Graft Cohort Study (VASGRA), University Hospital Zurich, Switzerland. The routine diagnostic work-up involved microbiological cultures of minced tissue samples, and the use of molecular techniques in parallel. Patient-related and microbiological data were assessed in descriptive analyses, and we calculated sensitivity, specificity, negative and positive predictive value for broad-range 16S rRNA gene PCR versus culture (considered as gold standard). Results: We investigated 60 patients (median age 66 years (Interquartile range [IQR] 59-75)) with confirmed VGI between May 2013 and July 2017. The prevalence of antimicrobial pretreatment at the time of sampling was high [91%; median days of antibiotics 7 days (IQR 1-18)]. We investigated 226 microbiological specimens. Thereof, 176 (78%) were culture-negative and 50 (22%) were culture-positive. There was a concordance of 70% (158/226) between conventional culture and broad-range PCR (sensitivity 58% (95% CI 43-72); specificity 74% (67-80%)). Among the group of 176 culture-negative specimens, 46 specimens were broad-range PCR-positive resulting in identification of overall 69 species. Among the culture and/or broad-range PCR-positive specimens (n = 96), 74 (77%) were monomicrobial and 22 (23%) polymicrobial, whereas the rate of polymicrobial samples was higher in broad-range PCR-positive specimens (93%). Conclusions: Combined cultures and broad-range 16S rRNA gene PCR from periprosthetic tissue and/or explanted vascular grafts increased the diagnostic accuracy in VGI, particularly in patients already under antimicrobial treatment at the time of redo surgery. Ideally, antimicrobial treatment should be withheld until surgical sampling in order to optimize microbiological diagnostics.Clinical trials.gov identifier: NCT01821664.

Global outbreak of severe Mycobacterium chimaera disease after cardiac surgery: a molecular epidemiological study.

BACKGROUND: Since 2013, over 100 cases of Mycobacterium chimaera prosthetic valve endocarditis and disseminated disease were notified in Europe and the USA, linked to contaminated heater-cooler units (HCUs) used during cardiac surgery. We did a molecular epidemiological investigation to establish the source of these patients' disease. METHODS: We included 24 M chimaera isolates from 21 cardiac surgery-related patients in Switzerland, Germany, the Netherlands, and the UK, 218 M chimaera isolates from various types of HCUs in hospitals, from LivaNova (formerly Sorin; London, UK) and Maquet (Rastatt, Germany) brand HCU production sites, and unrelated environmental sources and patients, as well as eight Mycobacterium intracellulare isolates. Isolates were analysed by next-generation whole-genome sequencing using Illumina and Pacific Biosciences technologies, and compared with published M chimaera genomes. FINDINGS: Phylogenetic analysis based on whole-genome sequencing of 250 isolates revealed two major M chimaera groups. Cardiac surgery-related patient isolates were all classified into group 1, in which all, except one, formed a distinct subgroup. This subgroup also comprised isolates from 11 cardiac surgery-related patients reported from the USA, most isolates from LivaNova HCUs, and one from their production site. Isolates from other HCUs and unrelated patients were more widely distributed in the phylogenetic tree. INTERPRETATION: HCU contamination with M chimaera at the LivaNova factory seems a likely source for cardiothoracic surgery-related severe M chimaera infections diagnosed in Switzerland, Germany, the Netherlands, the UK, the USA, and Australia. Protective measures and heightened clinician awareness are essential to guarantee patient safety. FUNDING: Partly funded by the EU Horizon 2020 programme, its FP7 programme, the German Center for Infection Research (DZIF), the Swiss National Science Foundation, the Swiss Federal Office of Public Health, and National Institute of Health Research Oxford Health Protection Research Units on Healthcare Associated Infection and Antimicrobial Resistance.

Evaluation of the AID carbapenemase line probe assay for rapid detection and identification of carbapenemase genes in Gram-negative bacilli.

Objectives: This study evaluated the AID carbapenemase line probe assay (LPA) for the detection and identification of carbapenem resistance genes in Enterobacteriaceae and other Gram-negative bacilli (GNB) using bacterial cultures and DNA extracts directly from patient urine samples. Methods: The AID carbapenemase LPA detects 13 different carbapenemase genes. Test probe accuracy was verified for using clinical Enterobacteriaceae isolates harbouring bla KPC , bla VIM , bla NDM , bla GIM , bla AIM , bla SPM , bla IMP and bla OXA-48 and a well-characterized set of Escherichia coli DH5α strains transformed with the vector plasmid pUC57- kan harbouring bla BIC , bla SIM , bla DIM , bla IMI-3 , bla IMI-1 and bla NMC-A . Sensitivity and specificity was determined by testing 151 clinical GNB strains previously characterized for the production of carbapenemase activity and carbapenemase genes. Direct detection of carbapenemase genes using the LPA was determined using 299 clinical urine specimens. Analytical sensitivity for detection in urine was determined by testing serial dilutions of bla KPC and bla NDM in clinical Klebsiella pneumoniae strains. Results: All carbapenemase gene probes showed 100% accuracy without cross-reactions. Sensitivity and specificity of the LPA using clinical isolates was 100% for each. Analytical sensitivity for detection of bla KPC and bla NDM in urine was 10 1 -10 2 cfu. The LPA detected carbapenemase genes in 20 urines, which were confirmed in 12 samples by conventional multiplex PCR. Remarkably, 0 of the 20 urines grew carbapenemase-suspicious GNB applying EUCAST recommendations. Conclusions: The AID carbapenemase LPA is an accurate, sensitive and easy-to-use test for the detection and identification of carbapenemase genes, which can readily be implemented in any diagnostic laboratory.

Reemergence of Mycobacterium chimaera in Heater-Cooler Units despite Intensified Cleaning and Disinfection Protocol.

Invasive Mycobacterium chimaera infections after open-heart surgery have been reported internationally. These devastating infections result from aerosols generated by contaminated heater-cooler units used with extracorporeal circulation during surgery. Despite intensified cleaning and disinfection, surveillance samples from factory-new units acquired during 2014 grew nontuberculous mycobacteria after a median of 174 days.

The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis.

Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell.

Diagnostic Molecular Mycobacteriology in Regions With Low Tuberculosis Endemicity: Combining Real-time PCR Assays for Detection of Multiple Mycobacterial Pathogens With Line Probe Assays for Identification of Resistance Mutations.

Molecular assays have not yet been able to replace time-consuming culture-based methods in clinical mycobacteriology. Using 6875 clinical samples and a study period of 35months we evaluated the use of PCR-based assays to establish a diagnostic workflow with a fast time-to-result of 1-2days, for 1. detection of Mycobacterium tuberculosis complex (MTB), 2. detection and identification of nontuberculous mycobacteria (NTM), and 3. identification of drug susceptible MTB. MTB molecular-based detection and culture gave concordant results for 97.7% of the specimens. NTM PCR-based detection and culture gave concordant results for 97.0% of the specimens. Defining specimens on the basis of combined laboratory data as true positives or negatives with discrepant results resolved by clinical chart reviews, we calculated sensitivity, specificity, PPV and NPV for PCR-based MTB detection as 84.7%, 100%, 100%, and 98.7%; the corresponding values for culture-based MTB detection were 86.3%, 100%, 100%, and 98.8%. PCR-based detection of NTM had a sensitivity of 84.7% compared to 78.0% of that of culture-based NTM detection. Molecular drug susceptibility testing (DST) by line-probe assay was found to predict phenotypic DST results in MTB with excellent accuracy. Our findings suggest a diagnostic algorithm to largely replace lengthy culture-based techniques by rapid molecular-based methods.

Similar efficacy of broad-range ITS PCR and conventional fungal culture for diagnosing fungal infections in non-immunocompromised patients.

BACKGROUND: Broad-range fungal inter spacer region (ITS) polymerase chain reaction (PCR) has been evaluated for the detection and identification of fungi in clinical specimens from severely immunocompromised patients, but not in non-selected patients. Thus, the aim of this study was to compare the diagnostic performance of ITS PCR with that of fungal culture and to investigate its clinical impact on the diagnosis of fungal infections in non-immunocompromised patients. The corresponding patients' data were retrieved by detailed medical chart reviews. RESULTS: Results from 251 specimens showed a high concordance of 89.6 % for ITS PCR and fungal culture. The analytical sensitivity and specificity of ITS PCR considering culture as gold standard were 87.7 and 90.3 %, respectively, the positive and negative predictive value (PPV and NPV) were 76 and 95.5 %, respectively. Assessing the clinical probability of a fungal infection based on detailed chart reviews, PCR had a clinical sensitivity of 88.9 %, a specificity of 86.3 %, a PPV of 64.0 % and a NPV of 96.6 %. The overall performance of fungal broad-range PCR was similar to that of culture. CONCLUSIONS: Our data show that, in non-selected and non-immunocompromised patients, the performance of ITS PCR is similar to that of culture for detecting fungal infections, not the least because sensitivity of culture in patients under antifungal treatment is surprisingly high. Compared to culture, PCR has the advantage of a rapid time-to-result (approximately two working days), proper identification of rare pathogens, prompt initiation of a species-targeted antifungal treatment, and prospects for automation.

Evaluation of the Bruker MALDI Biotyper for Identification of Fastidious Gram-Negative Rods.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has entered clinical laboratories, facilitating identification of bacteria. Here, we evaluated the MALDI Biotyper (Bruker Daltonics) for the identification of fastidious Gram-negative rods (GNR). Three sample preparation methods, direct colony transfer, direct transfer plus on-target formic acid preparation, and ethanol-formic acid extraction, were analyzed for 151 clinical isolates. Direct colony transfer applied with the manufacturer's interpretation criteria resulted in overall species and genus identification rates of 43.0% and 32.5%, respectively; 23.2% of the isolates were not identified, and two misidentifications (1.3%) were observed. The species identification rates increased to 46.4% and 53.7% for direct transfer plus formic acid preparation and ethanol-formic acid extraction, respectively. In addition, we evaluated score value cutoff alterations. The identification rates hardly increased by reducing the genus cutoff, while reducing the 2.0 species cutoff to 1.9 and to 1.8 increased the identification rates to up to 66.2% without increasing the rate of misidentifications. This study shows that fastidious GNR can reliably be identified using the MALDI Biotyper. However, the identification rates do not reach those of nonfastidious GNR such as the Enterobacteriaceae. In addition, two approaches optimizing the identification of fastidious GNR by the MALDI Biotyper were demonstrated: formic acid-based on-target sample treatment and reductions in cutoff scores to increase the species identification rates.

Acquired Resistance to Bedaquiline and Delamanid in Therapy for Tuberculosis.

Treatment of multidrug-resistant Mycobacterium tuberculosis is a challenge. This letter describes the emergence of resistance to new therapies, bedaquiline and delamanid.

Evaluation of the Rapidec Carba NP Test for Detection of Carbapenemases in Enterobacteriaceae.

This study evaluated the performance of the Rapidec Carba NP test, which was introduced recently into the market for the detection of carbapenemase production in a broad spectrum of ß-lactamase-producing Enterobacteriaceae clinical isolates. In total, 252 clinical Enterobacteriaceae isolates that had been genetically characterized with respect to carbapenemase, extended-spectrum ß-lactamase (ESBL), and AmpC genes were analyzed; 51/252 isolates (20.2%) were genetically confirmed to be carbapenemase producers, whereas 201/252 isolates (79.8%) were genetically negative for the presence of carbapenemase genes. The Rapidec Carba NP test was applied according to the manufacturer's instructions, and results were read after 30 and 120 min of incubation. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the Rapidec Carba NP test were 90.2%, 100%, 100%, and 97.6%, respectively, when the manufacturer's instructions were followed. Four of 5 false-negative results occurred with OXA-48-like enzymes. After an incubation time of 30 min, the sensitivity was 49%. The sensitivity increased to 100% when the recommended bacterial inoculum was doubled and the test was read strictly after 120 min of incubation. The Rapidec Carba NP test is a useful tool for the reliable confirmation of carbapenemase-producing Enterobacteriaceae isolates. The test should be read strictly after 120 min of incubation and the inoculum should be larger than recommended by the manufacturer.

Determination of MIC distribution and epidemiological cutoff values for bedaquiline and delamanid in Mycobacterium tuberculosis using the MGIT 960 system equipped with TB eXiST.

Bedaquiline (Sirturo) and delamanid (Deltyba) have recently been approved by the regulatory authorities for treatment of multidrug-resistant tuberculosis (MDR-TB). Antimicrobial susceptibility testing is not established for either substance. On the basis of the use of the MGIT 960 system equipped with EpiCenter/TB eXiST, we determined a mean bedaquiline MIC for wild-type strains of 0.65 mg/liter (median, 0.4 mg/liter) and an epidemiological cutoff (ECOFF) of 1.6 mg/liter; for delamanid, a mean wild-type drug MIC of 0.013 mg/liter (median, 0.01 mg/liter) and an ECOFF of 0.04 mg/liter were determined.