Search

A hypervirulent Klebsiella pneumoniae SL218 (ST23-KL57), phylogenetically distinct from the classical hypervirulent SL23 (ST23-KL1) lineage, was transmitted between hospitalised patients in Denmark in 2021. The isolate carried a hybrid resistance and virulence plasmid containing bla NDM-1 and a plasmid containing bla OXA-48 (pOXA-48); the latter plasmid was horizontally transferred within-patient to Serratia marcescens. The convergence of drug resistance and virulence factors in single plasmids and in different lineages of K. pneumoniae is concerning and requires surveillance.

Syndromic testing for lower respiratory tract infections with BioFire® FilmArray® Pneumonia Panel Plus (BF) detects 27 pathogens with a turn-around-time of one hour. We compared the performance of BF with culture. Samples from 298 hospitalized patients with suspected pneumonia routinely sent for culture were also analyzed using BF. Retrospectively, patients were clinically categorized as having “pneumonia” or “no pneumonia.” BF and culture were compared by analytical performance, which was evaluated by pathogen concordance, and by clinical performance by comparing pathogen detections in patients with and without pneumonia. The BF results for viruses and atypical bacteria were not included in the performance analysis. In 298 patient samples, BF and culture detected 285 and 142 potential pathogens, respectively. Positive percent agreement (PPA) was 88% (125/142). In patients with community-acquired pneumonia (CAP), clinical sensitivity was 70% and 51%, and specificity was 43% and 71% for BF and culture, respectively. In patients with hospital-acquired pneumonia, the corresponding numbers were 55% and 23%, and 47% and 68%. There was no significant improvement of performance, when only high-quality sputum samples were considered. Efficacy of both BF and culture was low. Both tests are best used in CAP patients for whom the diagnosis has already been clinically established. Indiscriminate use may be clinically misleading and a cause of improper use of antibiotics.

Piperacillin/tazobactam (TZP) is a widely used penicillin/β-lactamase inhibitor combination with broad antimicrobial activity. Recently, Escherichia coli strains resistant to TZP but susceptible to third generation cephalosporins (TZP-R/3GC-S isolates) have been increasingly identified. Here, we investigated resistance mechanisms underlying the TZP-R/3GC-S phenotype in clinical E. coli isolates.A total of 29 TZP-R/3GC-S E. coli isolates were retrieved from urinary cultures and subjected to whole genome sequencing. Resistance to TZP was confirmed by minimum inhibitory concentration determination. β-lactamase activity in the presence and absence of tazobactam was determined to identify hyperproduction of β-lactamase and assess susceptibility to tazobactam inhibition. A previously unrecognized β-lactamase was identified and cloned to determine its resistance profile.Four different resistance mechanisms underlying the TZP-R/3-GC phenotype were identified: 1) In 18 out of 29 isolates (62%) β-lactamase production was increased and in 16 of these either strong alternative promoters or increased gene copy numbers of blaTEM-1 or blaSHV-1 were identified, 2) seven isolates (24%) produced blaOXA-1, 3) three isolates (10%) produced inhibitor-resistant TEM-β-lactamases, and 4) a single isolate (3%) harboured a blaCTX-M gene as the only β-lactamase present. This β-lactamase, CTX-M-255, only differs from CTX-M-27 by a G239S amino acid substitution. In contrast to CTX-M-27, CTX-M-255 conferred resistance to penicillin/β-lactamase inhibitor combinations but remained susceptible to cephalosporins.In conclusion, hyperproduction of blaTEM was the most prevalent mechanism of TZP-resistance underlying the TZP-R/3GC-S phenotype followed by production of blaOXA-1 and inhibitor-resistant TEM-β-lactamases. Furthermore, we identified a previously unrecognized CTX-M-β-lactamase, CTX-M-255 that was resistant to β-lactamase inhibitors.

BACKGROUND: Superinfection following viral infection is a known complication, which may lead to longer hospitalisation and worse outcome. Empirical antibiotic therapy may prevent bacterial superinfections, but may also lead to overuse, adverse effects and development of resistant pathogens. Knowledge about the incidence of superinfections in intensive care unit (ICU) patients with severe Coronavirus Disease 2019 (COVID-19) is limited.METHODS: We will conduct a nationwide cohort study comparing the incidence of superinfections in patients with severe COVID-19 admitted to the ICU compared with ICU patients with influenza A/B in Denmark. We will include approximately 1000 patients in each group from the time period of 1 October 2014 to 30 April 2019 and from 10 March 2020 to 1 March 2021 for patients with influenza and COVID-19, respectively. The primary outcome is any superinfection within 90 days of admission to the ICU. We will use logistic regression analysis comparing COVID-19 with influenza A/B after adjustment for relevant predefined confounders. Secondarily, we will use unadjusted and adjusted logistic regression analyses to assess six potential risk factors (sex, age, cancer [including haematological], immunosuppression and use of life support on day 1 in the ICU) for superinfections and compare outcomes in patients with COVID-19 with/without superinfections, and present descriptive data regarding the superinfections.CONCLUSION: This study will provide important knowledge about superinfections in ICU patients with severe COVID-19.

Enrichment culture (EC) remains gold standard for detecting MRSA colonisation, but molecular methods shorten turnaround time. The CE-marked automated Hologic Panther Fusion MRSA Assay (HPFM) is validated for nasal swabs. We compared HPFM with EC following an in-house PCR for detection of MRSA in nasal, pharyngeal, and perineal ESwabs. The same ESwabs were analysed using HPFM and inoculated in selective Tryptic Soy Broth (TSB) for overnight incubation. TSBs were screened by a PCR targeting nuc, femA, mecA, and mecC. Only samples with PCR results compatible with MRSA presence were inoculated onto 5% blood agar and chromogenic MRSA plates. HPFM detected MRSA in 103 of 132 EC positive samples indicating a sensitivity of 78.0% across sample types. When paired TSBs of 29 EC positive/HPFM negative samples were re-analysed by HPFM, MRSA was detected in 17/29 TSBs indicating that enrichment will increase the sensitivity of HPFM. HPFM analyses of cultured isolates from the remaining 12 EC positive/HPFM negative samples failed to detect orfX. HPFM reported the presence of MRSA in 22 samples where EC failed to identify MRSA. Fifteen of these ESwabs had been kept and direct culture without enrichment identified MRSA in seven samples. HPFM was useful for all sample sites. Compared to EC, the sensitivity of HPFM was limited because of lack of analytical sensitivity and failure to detect all MRSA variants. Failure of some MRSA-containing samples to enrich in cefoxitin-containing TSB indicates an unappreciated limitation of EC, which may lead to underestimation of the specificity of molecular assays.

ObjectiveHCV-genotype 4 infections are a major cause of liver diseases in the Middle East/Africa with certain subtypes associated with increased risk of direct-acting antiviral (DAA) treatment failures. We aimed at developing infectious genotype 4 cell culture systems to understand the evolutionary genetic landscapes of antiviral resistance, which can help preserve the future efficacy of DAA-based therapy.DesignHCV recombinants were tested in liver-derived cells. Long-term coculture with DAAs served to induce antiviral-resistance phenotypes. Next-generation sequencing (NGS) of the entire HCV-coding sequence identified mutation networks. Resistance-associated substitutions (RAS) were studied using reverse-genetics.ResultThe in-vivo infectious ED43(4a) clone was adapted in Huh7.5 cells, using substitutions identified in ED43(Core-NS5A)/JFH1-chimeric viruses combined with selected NS5B-changes. NGS, and linkage analysis, permitted identification of multiple genetic branches emerging during culture adaptation, one of which had 31 substitutions leading to robust replication/propagation. Treatment of culture-adapted ED43 with nine clinically relevant protease-DAA, NS5A-DAA and NS5B-DAA led to complex dynamics of drug-target-specific RAS with coselection of genome-wide substitutions. Approved DAA combinations were efficient against the original virus, but not against variants with RAS in corresponding drug targets. However, retreatment with glecaprevir/pibrentasvir remained efficient against NS5A inhibitor and sofosbuvir resistant variants. Recombinants with specific RAS at NS3-156, NS5A-28, 30, 31 and 93 and NS5B-282 were viable, but NS3-A156M and NS5A-L30Δ (deletion) led to attenuated phenotypes.ConclusionRapidly emerging complex evolutionary landscapes of mutations define the persistence of HCV-RASs conferring resistance levels leading to treatment failure in genotype 4. The high barrier to resistance of glecaprevir/pibrentasvir could prevent persistence and propagation of antiviral resistance.

Direct-acting antivirals (DAAs) have proven highly effective against chronic hepatitis C virus (HCV) infection. However, some patients experience treatment failure, associated with resistance-associated substitutions (RASs). Our aim was to investigate the complete viral coding sequence in hepatitis C patients treated with DAAs to identify RASs and the effects of treatment on the viral population. We selected 22 HCV patients with sustained virologic response (SVR) to match 21 treatment-failure patients in relation to HCV genotype, DAA regimen, liver cirrhosis and previous treatment experience. Viral-titre data were compared between the two patient groups, and HCV full-length open reading frame deep-sequencing was performed. The proportion of HCV NS5A-RASs at baseline was higher in treatment-failure (82%) than matched SVR patients (25%) (p =.0063). Also, treatment failure was associated with slower declines in viraemia titres. Viral population diversity did not differ at baseline between SVR and treatment-failure patients, but failure was associated with decreased diversity probably caused by selection for RAS. The NS5B-substitution 150V was associated with sofosbuvir treatment failure in genotype 3a. Further, mutations identified in NS2, NS3-helicase and NS5A-domain-III were associated with DAA treatment failure in genotype 1a patients. Six retreated HCV patients (35%) experienced 2nd treatment failure; RASs were present in 67% compared to 11% with SVR. In conclusion, baseline RASs to NS5A inhibitors, but not virus population diversity, and lower viral titre decline predicted HCV treatment failure. Mutations outside of the DAA targets can be associated with DAA treatment failure. Successful DAA retreatment in patients with treatment failure was hampered by previously selected RASs.

Objectives: Piperacillin-tazobactam (TZP) is a frequently prescribed antibiotic in hospital settings. Reports suggest in vivo efficacy of TZP, despite in vitro resistance of isolates susceptible to cephalosporins. Escherichia coli (E. coli) isolates hyperproducing TEM-1 β-lactamase possess this phenotype. This study investigated the influence of tazobactam (TAZ) concentration on piperacillin (PIP) inhibition of such isolates and compared the in vivo efficacy of TZP with cefotaxime (CTX) in an infection model. Methods: The PIP MICs for E. coli isolates, either hyperproducing TEM-1 because of promoter substitutions (n = 4) or because of gene amplification (n = 2) or producing an inhibitor-resistant TEM-35 (IRT) (n = 1), were determined using increasing concentrations of TAZ in a checkerboard setup. Furthermore, the efficacy of TZP and CTX against the isolates was investigated in a mouse peritonitis model using antibiotic exposures mimicking human conditions. Isolates producing either OXA-48 or CTX-M-15 β-lactamases were included as controls. Results: Using TAZ concentrations ≤ 64 mg/L, one isolate hyperproducing TEM-1 had a PIP MIC of 8 at TAZ 16 mg/L and two additional isolates at TAZ 64 mg/L. In the mouse peritonitis infection model, reduction of bacterial load in the peritoneum was larger for TZP than CTX only for the CTX-M-15-producing isolate. Larger reductions in bacterial load were observed after CTX treatment than TZP treatment for seven of the eight remaining test isolates. Conclusions: Piperacillin-tazobactam treatment of E. coli isolates hyperproducing TEM-1 was less effective than CTX treatment and may, for some isolates, be comparable with TZP treatment of isolates producing established resistance markers as IRT or OXA-48.

Introduction SARS-CoV-2 outbreaks at care homes are associated with a high morbidity and mortality. We aimed to study the molecular epidemiology of a major care home outbreak in Denmark. Methods After a staff member had been tested positive on 16 November 2020, a bundle approach programme was initiated including frequent surveillance screenings of residents and staff, isolation and cohorting procedures. This approach also involved limiting the number of visitors and enhancing the use of personal protective equipment, hand hygiene, and environmental cleaning. Naso/oropharyngeal swabs were tested for SARS-CoV-2 by polymerase chain reaction. Available positive samples were sequenced and phylogenetic relationships between the outbreak and local circulating strains were reconstructed. Results In all, 50% (56/114) of residents and 26% (49/190) of staff members became infected during the 46-day outbreak period. Altogether 16% of the infected residents died within 30 days after becoming infected. A total of 44% (46/105) of the samples with SARS-CoV-2 were sequenced. and phylogenetic analysis demonstrated a dominant outbreak lineage belonging to Global Lineage B.1.1.29 containing the mutation I233V in the S gene. The outbreak lineage was detected in the community 28 days before its introduction into the care home. Conclusions Introduction of SARS-CoV-2 to care homes is associated with severe outbreaks. Initiation of a bundle approach infection control programme in addition to measures ensuring enhanced herd immunity were successful in controlling the outbreak. Genome sequencing proved to be a powerful tool to describe the relatedness of the various clones and may help focusing outbreak interventions. Funding The study was funded in part by The Poul Due Jensen Foundation and The Danish Ministry of Higher Education and Science. The authors have no conflicts of interest to report. Trial registration not relevant.

Background & Aims Protease inhibitors (PIs) are of central importance in the treatment of patients with chronic hepatitis C virus (HCV) infection. HCV NS3 protease (NS3P) position 80 displays polymorphisms associated with resistance to the PI simeprevir for HCV genotype 1a. We investigated the effects of position-80-substitutions on fitness and PI-resistance for HCV genotypes 1-6, and analyzed evolutionary mechanisms underlying viral escape mediated by pre-existing Q80K. Methods The fitness of infectious NS3P recombinants of HCV genotypes 1-6, with engineered position-80-substitutions, was studied by comparison of viral spread kinetics in Huh-7.5 cells in culture. Median effective concentration (EC50) and fold resistance for PIs simeprevir, asunaprevir, paritaprevir, grazoprevir, glecaprevir and voxilaprevir were determined in short-term treatment assays. Viral escape was studied by long-term treatment of genotype 1a recombinants with simeprevir, grazoprevir, glecaprevir and voxilaprevir and of genotype 3a recombinants with glecaprevir and voxilaprevir, next generation sequencing, NS3P substitution linkage and haplotype analysis. Results Among tested PIs, only glecaprevir and voxilaprevir showed pan-genotypic activity against the original genotype 1-6 culture viruses. Variants with position-80-substitutions were all viable, but fitness depended on the specific substitution and the HCV isolate. Q80K conferred resistance to simeprevir across genotypes but had only minor effects on the activity of the remaining PIs. For genotype 1a, pre-existing Q80K mediated accelerated escape from simeprevir, grazoprevir and to a lesser extent glecaprevir, but not voxilaprevir. For genotype 3a, Q80K mediated accelerated escape from glecaprevir and voxilaprevir. Escape was mediated by rapid and genotype-, PI- and PI-concentration-dependent co-selection of clinically relevant resistance associated substitutions. Conclusions Position-80-substitutions had relatively low fitness cost and the potential to promote HCV escape from clinically relevant PIs in vitro, despite having a minor impact on results in classical short-term resistance assays. Lay summary Among all clinically relevant hepatitis C virus protease inhibitors, voxilaprevir and glecaprevir showed the highest and most uniform activity against cell culture infectious hepatitis C virus with genotype 1-6 proteases. Naturally occurring amino acid changes at protease position 80 had low fitness cost and influenced sensitivity to simeprevir, but not to other protease inhibitors in short-term treatment assays. Nevertheless, the pre-existing change Q80K had the potential to promote viral escape from protease inhibitors during long-term treatment by rapid co-selection of additional resistance changes, detected by next generation sequencing.

Enrichment culture (EC) remains gold standard for detecting MRSA colonisation, but molecular methods shorten turnaround time. The CE-marked automated Hologic Panther Fusion MRSA Assay (HPFM) is validated for nasal swabs. We compared HPFM with EC following an in-house PCR for detection of MRSA in nasal, pharyngeal, and perineal ESwabs. The same ESwabs were analysed using HPFM and inoculated in selective Tryptic Soy Broth (TSB) for overnight incubation. TSBs were screened by a PCR targeting nuc, femA, mecA, and mecC. Only samples with PCR results compatible with MRSA presence were inoculated onto 5% blood agar and chromogenic MRSA plates. HPFM detected MRSA in 103 of 132 EC positive samples indicating a sensitivity of 78.0% across sample types. When paired TSBs of 29 EC positive/HPFM negative samples were re-analysed by HPFM, MRSA was detected in 17/29 TSBs indicating that enrichment will increase the sensitivity of HPFM. HPFM analyses of cultured isolates from the remaining 12 EC positive/HPFM negative samples failed to detect orfX. HPFM reported the presence of MRSA in 22 samples where EC failed to identify MRSA. Fifteen of these ESwabs had been kept and direct culture without enrichment identified MRSA in seven samples. HPFM was useful for all sample sites. Compared to EC, the sensitivity of HPFM was limited because of lack of analytical sensitivity and failure to detect all MRSA variants. Failure of some MRSA-containing samples to enrich in cefoxitin-containing TSB indicates an unappreciated limitation of EC, which may lead to underestimation of the specificity of molecular assays.

Background During 2018–19, an increase of vanB vancomycin-resistant Enterococcus faecium (VREfm) was observed in the Capital Region of Denmark. vanA/vanB PCR performed directly on rectal swabs is accurate in detection of vanA; however, the positive predictive value for vanB-positive samples is low because of the presence of vanB in non-enterococcal gut commensals. Objectives We investigated the epidemiology and clonal relatedness of vanB VREfm from the period 2015–19 and describe the application of a clone-specific vanB VREfm PCR assay for rapid and accurate detection of vanB VREfm in rectal screening samples. Methods vanB VREfm were investigated using epidemiological data and WGS data. The SeqSphere+ software was used to analyse MLST and cgMLST, and de novo assemblies were annotated to determine insertion sites for the vanB transposon (Tn1549). A clone-specific vanB VREfm PCR assay was designed to detect the sequence bridging Tn1549 and the E. faecium chromosome (araA2) in the dominant cluster. Results Two hundred and seventy-five vanB VREfm isolates were identified, of which 76% were identified in 2019. A dominant cluster (Cluster 1, n = 204, 74%), six minor clusters and 15 singletons were identified. All Cluster 1 isolates and six non-Cluster 1 isolates had Tn1549 integrated into araA2. In 2019, the PCR assay would have detected 92% of all rectal screening samples containing vanB VREfm. Conclusions vanB VREfm increased due to the introduction and nosocomial transmission of the successful Cluster 1. The clone-specific PCR assay detected vanB VREfm outbreak isolates in rectal screening samples rapidly and accurately.

A defined bacterial mixture could be a safer alternative to faecal microbiota transplantation (FMT).To compare the efficacy of a 12-strain mixture termed rectal bacteriotherapy with either FMT or vancomycin for recurrent Clostridioides difficile infection (CDI) in an open-label 3-arm randomised controlled trial.We screened all individuals positive for C difficile from May 2017 to March 2019. Persons with laboratory-confirmed recurrent CDI were included. Before FMT and rectal bacteriotherapy, we pre-treated with vancomycin for 7-14 days. Rectal bacteriotherapy was applied by enema on three consecutive days and FMT by enema once with possible repetition for two to three infusions within 14 days. The vancomycin group was treated for 14 days with additional five weeks of tapering for multiple recurrences. The primary outcome was clinical cure within 90 days. A secondary outcome was 180-day all-cause mortality.Participants in the FMT group (n = 34) were cured more often than participants receiving vancomycin (n = 31), 76% vs 45% (OR 3.9 (1.4-11.4), P 0.01) or rectal bacteriotherapy (n = 31), 76% vs 52% (OR 3.0 (1.1-8.8), P = 0.04). Rectal bacteriotherapy and vancomycin performed similarly (P = 0.61). The mortality rate was 6% in the FMT group, 13% in the bacteriotherapy group and 23% in the vancomycin group. FMT tended to reduce mortality compared with vancomycin, OR 0.2 (0.04-1.12), P = 0.07.Rectal bacteriotherapy appears as effective as vancomycin but less effective than 1-3 FMTs. FMT by enema with 1-3 infusions is superior to vancomycin for treating recurrent C difficile infections and might reduce mortality.

Background HIV-RNA is the most important parameter for monitoring antiviral treatment in individuals infected with HIV-1. Knowledge of the performance of different tests for the quantification of HIV-1 RNA is therefore important for clinical care. Objectives To compare the analytical performance of the Aptima HIV-1 Quant Dx Assay (Aptima) and the COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 (CAPCTMv2) for the quantification of HIV-1 RNA in plasma samples. Study design The performance of the two tests was compared on 216 clinical plasma samples, on dilutions series in seven replicates of five clinical samples of known subtype and on ten replicates of the Acrometrix High and Low Positive Control. Results Bland-Altman analysis of 130 samples that quantified in both tests did not show indications of gross mis-quantification of either test. A tendency of the Aptima assay to quantify higher at high viral load compared to the CAPCTMv2 was observed in Bland-Altman analysis, by Deming regression (Slope 1.13) and in dilution series of clinical samples. Precision evaluated using the Acrometrix Positive Controls was similar for the High Control (CV: 1.2% vs. 1.3%; Aptima assay vs. CAPCTMv2 test, respectively), but differed for the Low control (CV: 17.9% vs. 7.1%; Aptima assay vs. CAPCTMv2 test, respectively). However, this did not impact clinical categorization of clinical samples at neither the 50 cp/mL nor 200 cp/mL level. Conclusion The Aptima assay and the CAPCTMv2 test are highly correlated and are useful for monitoring HIV-infected individuals.

The surveillance and correct subtyping of hepatitis C virus strains require available and up-to-date publicly available reference genomes. Here, we present the complete open reading frame sequence of a hepatitis C virus genotype 6 strain of an unknown subtype that was discovered during routine subtyping of patients in the clinic.

Pivmecillinam (amdinocillin pivoxil) is the recommended first‐choice antibiotic used to treat urinary tract infections (UTIs) in Denmark. The frequency of mutation to mecillinam (MEC) resistance is described as high in vitro; however, treatment of UTI has a good clinical response and prevalence of mecillinam resistance in Escherichia coli remains low despite many years of use. We describe occurrence of in vivo mecillinam resistance in a clinical isolate of ESBL‐producing E. coli following pivmecillinam treatment. The identified phenotypic differences in the mecillinam resistant isolate compared with the original mecillinam susceptible isolate were a full‐length LPS with O‐antigen (O25), mecillinam resistance and a lower MIC for ceftazidime. Regarding genotype, the resistant isolate differed with a mutation in bla CTX‐M‐15 to bla CTX‐M‐127, loss of a part of a plasmid and a genomic island, respectively, and insertion of a transposase in wbbL, causing the rough phenotype. The observed mecillinam resistance is expected to be caused by the mutation in bla CTX‐M‐15 with additional contribute from the serotype shift. We continue to recommend the use of pivmecillinam as first‐line treatment for UTI., In this study, we investigated the genetics behind mecillinam resistance which occurred in an extended‐spectrum β‐lactamase‐producing Escherichia coli during pivmecillinam treatment of a patient. The genomic comparison of the two isolates points toward a mutational change of blaCTX‐M‐15 into blaCTX‐M‐127 along with a serotype shift from rough to full‐length O25 as the mechanisms contributing to the change in mecillinam susceptibility.

Backgroundbla TEM-1 encodes a narrow-spectrum β-lactamase that is inhibited by β-lactamase inhibitors and commonly present in Escherichia coli. Hyperproduction of blaTEM-1 may cause resistance to penicillin/β-lactamase inhibitor (P/BLI) combinations.ObjectivesTo characterize EC78, an E. coli bloodstream isolate, resistant to P/BLI combinations, which contains extensive amplification of blaTEM-1 within the chromosome.MethodsEC78 was sequenced using Illumina and Oxford Nanopore Technology (ONT) methodology. Configuration of blaTEM-1 amplification was probed using PCR. Expression of blaTEM-1 mRNA was determined using quantitative PCR and β-lactamase activity was determined spectrophotometrically in a nitrocefin conversion assay. Growth rate was assessed to determine fitness and stability of the gene amplification was assessed by passage in the absence of antibiotics.ResultsIllumina sequencing of EC78 identified blaTEM-1B as the only acquired β-lactamase preceded by the WT P3 promoter and present at a copy number of 182.6 with blaTEM-1B bracketed by IS26 elements. The chromosomal location of the IS26-blaTEM-1B amplification was confirmed by ONT sequencing. Hyperproduction of blaTEM-1 was confirmed by increased transcription of blaTEM-1 and β-lactamase activity and associated with a significant fitness cost; however, the array was maintained at a relatively high copy number for 150 generations. PCR screening for blaTEM amplification of isolates resistant to P/BLI combinations identified an additional strain containing an IS26-associated amplification of a blaTEM gene.ConclusionsIS26-associated amplification of blaTEM can cause resistance to P/BLI combinations. This adaptive mechanism of resistance may be overlooked if simple methods of genotypic prediction (e.g. gene presence/absence) are used to predict antimicrobial susceptibility from sequencing data.

Large conjugative plasmids are important drivers of bacterial evolution and contribute significantly to the dissemination of antibiotic resistance. Although plasmid borne multidrug resistance is recognized as one of the main challenges in modern medicine, the adaptive forces shaping the evolution of these plasmids within pathogenic hosts are poorly understood. Here we study plasmid-host adaptations following transfer of a 73 kb conjugative multidrug resistance plasmid to naïve clinical isolates of Klebsiella pneumoniae and Escherichia coli We use experimental evolution, mathematical modelling and population sequencing to show that the long-term persistence and molecular integrity of the plasmid is highly influenced by multiple factors within a 25 kb plasmid region constituting a host-dependent burden. In the E. coli hosts investigated here, improved plasmid stability readily evolves via IS26 mediated deletions of costly regions from the plasmid backbone, effectively expanding the host-range of the plasmid. Although these adaptations were also beneficial to plasmid persistence in a naïve K. pneumoniae host, they were never observed in this species, indicating that differential evolvability can limit opportunities of plasmid adaptation. While insertion sequences are well known to supply plasmids with adaptive traits, our findings suggest that they also play an important role in plasmid evolution by maintaining the plasticity necessary to alleviate plasmid-host constrains. Further, the observed evolutionary strategy consistently followed by all evolved E. coli lineages exposes a trade-off between horizontal and vertical transmission that may ultimately limit the dissemination potential of clinical multidrug resistance plasmids in these hosts.

CMY-2 is the most common plasmid-mediated AmpC β-lactamase in Escherichia coli isolates of human and animal origin. The aim of this study was to elucidate the epidemiology of CMY-2-producing E. coli in Denmark. Strain and plasmid relatedness was studied in 93 CMY-2-producing clinical and commensal E. coli isolates collected from 2006 to 2012 from humans, retail poultry meat, broilers, and dogs. Multilocus sequence typing (MLST), antimicrobial susceptibility testing, and conjugation were performed in conjunction with plasmid replicon typing, plasmid multilocus sequence typing (pMLST), restriction fragment length polymorphism (RFLP), and sequencing of selected bla CMY-2 -harboring plasmids. MLST revealed high strain diversity, with few E. coli lineages occurring in multiple host species and sample types. bla CMY-2 was detected on plasmids in 83 (89%) isolates. Most (75%) of the plasmids were conjugative and did not (96%) cotransfer resistance to antimicrobials other than cephalosporins. The main replicon types identified were IncI1-Iγ (55%) and IncK (39%). Isolates from different host species mainly carried distinct plasmid subtypes. Seven of the 18 human isolates harbored IncI1-Iγ/sequence type 2 (ST2), IncI1-Iγ/ST12, or IncK plasmids highly similar to those found among animal isolates, even though highly related human and animal plasmids differed by nonsynonymous single nucleotide polymorphisms (SNPs) or insertion sequence elements. This study clearly demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this β-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids that are generally distributed according to host-specific patterns. These baseline data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources. IMPORTANCE CMY-2 is the most common plasmid-mediated AmpC β-lactamase in Escherichia coli . This β-lactamase is poorly inhibited by clavulanic acid and confers resistance to cephamycins, third-generation cephalosporins, and aztreonam. Furthermore, resistance to carbapenems has been reported in E. coli as a result of production of plasmid-encoded CMY-2 β-lactamase in combination with decreased outer membrane permeability. The gene encoding CMY-2 generally resides on transferable plasmids belonging to different incompatibility groups. The prevalence of CMY-2-mediated cephalosporin resistance in E. coli varies significantly depending on the geographical region and host. This study demonstrates that the epidemiology of CMY-2 can be understood only by thorough plasmid characterization. To date, the spread of this β-lactam resistance determinant in Denmark is mainly associated with IncK and IncI1-Iγ plasmids, which are generally distributed according to host-specific patterns. These data will be useful to assess the consequences of the increasing human exposure to CMY-2-producing E. coli via animal sources.

Rapid and sensitive detection of macrolide resistance in Mycoplasma genitalium is required for the guidance of adequate antimicrobial treatment. Previous studies have confirmed that single-base mutations at position 2058 or 2059 in domain V of the 23S rRNA gene of M. genitalium result in high-level macrolide resistance. Sequencing of PCR products remains the gold standard for the identification of mutations conferring resistance to macrolides but is laborious and time-consuming. The aim of the present study was to develop a 5′ nuclease genotyping assay to detect single nucleotide polymorphisms in the 23S rRNA gene of Mycoplasma genitalium that are associated with macrolide resistance by combining PCR with hydrolysis probes and subsequent endpoint genotyping analysis. The 5′ nuclease genotyping assay was used as a referral test to be used on M. genitalium -positive samples and was validated on 259 positive samples, of which 253 (97.7%) were successfully sequenced. With the newly developed assay, 237/259 (91.5%) investigated M. genitalium -positive samples were genotyped. The positive and the negative predictive values were 100% when evaluated on successfully genotyped samples. The newly developed assay discriminated macrolide-resistant M. genitalium in clinical specimens possessing A2058G, A2058C, A2058T, and A2059G mutations with a sensitivity of 94.4% (95% confidence interval [CI], 90.7% to 98.2%) and a specificity of 92.7% (95% CI, 87.8% to 97.6%) when evaluated on successfully sequenced samples. The assay can correctly guide antimicrobial treatment of M. genitalium infections.

Protease inhibitors (PIs) are important components of treatment regimens for patients with chronic hepatitis C virus (HCV) infection. However, emergence and persistence of antiviral resistance could reduce their efficacy. Thus, defining resistance determinants is highly relevant for efforts to control HCV. Here, we investigated patterns of PI resistance–associated substitutions (RASs) for the major HCV genotypes and viral determinants for persistence of key RASs. We identified protease position 156 as a RAS hotspot for genotype 1-4, but not 5 and 6, escape variants by resistance profiling using PIs grazoprevir and paritaprevir in infectious cell culture systems. However, except for genotype 3, engineered 156-RASs were not maintained. For genotypes 1 and 2, persistence of 156-RASs depended on genome-wide substitution networks, co-selected under continued PI treatment and identified by next-generation sequencing with substitution linkage and haplotype reconstruction. Persistence of A156T for genotype 1 relied on compensatory substitutions increasing replication and assembly. For genotype 2, initial selection of A156V facilitated transition to 156L, persisting without compensatory substitutions. The developed genotype 1, 2, and 3 variants with persistent 156-RASs had exceptionally high fitness and resistance to grazoprevir, paritaprevir, glecaprevir, and voxilaprevir. A156T dominated in genotype 1 glecaprevir and voxilaprevir escape variants, and pre-existing A156T facilitated genotype 1 escape from clinically relevant combination treatments with grazoprevir/elbasvir and glecaprevir/pibrentasvir. In genotype 1 infected patients with treatment failure and 156-RASs, we observed genome-wide selection of substitutions under treatment. Conclusion: Comprehensive PI resistance profiling for HCV genotypes 1-6 revealed 156-RASs as key determinants of high-level resistance across clinically relevant PIs. We obtained in vitro proof of concept for persistence of highly fit genotype 1-3 156-variants, which might pose a threat to clinically relevant combination treatments.

Background Primary screening for VRE with PCR directed against vanA allowed identification of vanA+ samples from which VRE could not be isolated when selective culture methods were used. From such a sample a vancomycin-susceptible, vanA+ Enterococcus faecium, Efm-V1511, was isolated, when vancomycin selection was not used during culture. Similar isolates with variable susceptibility to vancomycin were obtained in the following months. Objectives To characterize Efm-V1511 and investigate the causes of variable susceptibility to vancomycin. Methods All strains were sequenced using Illumina technology. Plasmids containing vanA were reconstructed by scaffolding to known plasmids or plasmids were sequenced using Oxford Nanopore MinION. Derived structures were verified by PCR and sequencing. Furthermore, selected vanA+ vancomycin-susceptible isolates were passaged in the presence of vancomycin and vancomycin-resistant variants obtained were sequenced. Results Efm-V1511 belonged to ST1421 and contained a 49 696 bp plasmid pHVH-V1511 carrying a Tn1546-derived genetic element. Within this element vanX was truncated by a 252 bp 3' deletion explaining the susceptibility of Efm-V1511. Between March 2016 and April 2017, 48 isolates containing pHVH-V1511 were identified. All were ST1421. In isolates resistant to vancomycin, resistance could be attributed to changes in ddl disrupting gene function sometimes accompanied by changes in vanS, increased pHVH-V1511 copy number or the existence of an additional vanA-containing plasmid encoding a functional vanX. Conclusions E. faecium carrying pHVH-V1511 is capable of nosocomial transmission and may develop clinical resistance to vancomycin. Strains may not be detected using standard culture methods for VRE.

Clostridium difficile infection (CDI) is a common complication to antibiotic use. Saccharomyces boulardii has shown effect as a prophylactic agent. We aimed to evaluate the efficacy of S. boulardii in preventing CDI in unselected hospitalized patients treated with antibiotics. We conducted a 1 year controlled prospective intervention study aiming to prescribe Sacchaflor (S. boulardii 5 × 109, Pharmaforce ApS) twice daily to hospitalized patients treated with antibiotics. Comparable departments from three other hospitals in our region were included as controls. All occurrences of CDI in patients receiving antibiotics were reported and compared to a baseline period defined as 2 years prior to intervention. Results were analyzed using run chart tests for non-random variation in CDI rates. In addition, odds ratios for CDI were calculated. S. boulardii compliance reached 44% at the intervention hospital, and 1389 patients were treated with Sacchaflor. Monthly CDI rates dropped from a median of 3.6% in the baseline period to 1.5% in the intervention period. S. boulardii treatment was associated with a reduced risk of CDI at the intervention hospital: OR = 0.06 (95% CI 0.02–0.16). At two control hospitals, CDI rates did not change. At one control hospital, the median CDI rate dropped from 3.5 to 2.4%, possibly reflecting the effects of simultaneous multifaceted intervention against CDI at that hospital. The results from this controlled prospective interventional study indicate that S. boulardii is effective for the prevention of CDI in an unselected cohort of mainly elderly patients from departments of internal medicine.

For future decisions on respiratory syncytial virus (RSV)-vaccination strategies and implementation into national immunisation-programmes, we used national registry data (hospitalisation, microbiology and vital statistics) to determine the age-specific incidence and direct medical costs of annual RSV-associated admissions in children

Background Quantification of HBV DNA is used for initiating and monitoring antiviral treatment. Analytical test performance consequently impacts treatment decisions. Objectives To compare the analytical performance of the Aptima HBV Quant Assay (Aptima) and the COBAS Ampliprep/COBAS TaqMan HBV Test v2.0 (CAPCTMv2) for the quantification of HBV DNA in plasma samples. Study design The performance of the two tests was compared on 129 prospective plasma samples, and on 63 archived plasma samples of which 53 were genotyped. Linearity of the two assays was assessed on dilutions series of three clinical samples (Genotype B, C, and D). Results Bland-Altman analysis of 120 clinical samples, which quantified in both tests, showed an average quantification bias (Aptima – CAPCTMv2) of −0.19 Log IU/mL (SD: 0.33 Log IU/mL). A single sample quantified more than three standard deviations higher in Aptima than in CAPCTMv2. Only minor differences were observed between genotype A (N = 4; average difference −0.01 Log IU/mL), B (N = 8; −0.13 Log IU/mL), C (N = 8; −0.31 Log IU/mL), D (N = 25; −0.22 Log IU/mL), and E (N = 7; −0.03 Log IU/mL). Deming regression showed that the two tests were excellently correlated (slope of the regression line 1.03; 95% CI: 0.998-1.068). Linearity of the tests was evaluated on dilution series and showed an excellent correlation of the two tests. Both tests were precise with %CV less than 3% for HBV DNA ≥3 Log IU/mL. Conclusions The Aptima and CAPCTMv2 tests are highly correlated, and both tests are useful for monitoring patients chronically infected with HBV.

Background: We aimed to evaluate the relevance of dual target real-time polymerase chain (PCR) assays targeting the rtxA and cpn60 genes of the paediatric pathogen Kingella kingae. We also studied for the first time the clinical and epidemiological features of K. kingae infections in a Danish population. Method: Children with K. kingae-positive cultures were identified from 11,477 children and 86 children younger than 16 years old from whom blood cultures and joint fluid cultures were obtained between January 2010 and November 2016. Results were then compared to microbiological results obtained from 29 joint fluids (28 children) tested by dual target K. kingae real-time PCR from September 2014 to November 2016. Epidemiological data of all children with microbiologically confirmed K. kingae infections were collected. Results: From 2010 to 2016, we diagnosed 17 children with microbiological-proven K. kingae infections. During this period, blood cultures from five children and joint fluid cultures from a single child yielded K. kingae. Dual target K. kingae real-time PCR allowed us to increase the diagnostic yield of K. kingae infections by detecting the organism in 12 of 29 (41.4%) specimens. Notably, the 12 real-time PCR-positive specimens were rtxA-positive whereas only 10 (83.3%) were cpn60-positive. PCR-positive children were significantly younger than PCR-negative children (p-value: .01). A significant seasonal variation was found for patients with proven K. kingae infection (p-value: Conclusion: Dual target-specific real-time PCR markedly improved the detection of K. kingae in clinical specimens when compared to culture methods.

Background Chronic hepatitis C virus (HCV) infection can be effectively treated with directly acting antiviral (DAA) therapy. Measurement of HCV RNA is used to evaluate patient compliance and virological response during and after treatment. Objectives To compare the analytical performance of the Aptima HCV Quant Dx Assay (Aptima) and the COBAS Ampliprep/COBAS TaqMan HCV Test v2.0 (CAPCTMv2) for the quantification of HCV RNA in plasma samples, and compare the clinical utility of the two tests in patients undergoing treatment with DAA therapy. Study design Analytical performance was evaluated on two sets of plasma samples: 125 genotyped samples and 172 samples referred for quantification of HCV RNA. Furthermore, performance was evaluated using dilutions series of four samples containing HCV genotype 1a, 2b, 3a, and 4a, respectively. Clinical utility was evaluated on 118 plasma samples obtained from 13 patients undergoing treatment with DAAs. Results Deming regression of results from 187 plasma samples with HCV RNA > 2 Log IU/mL indicated that the Aptima assay quantified higher than the CAPCTMv2 test for HCV RNA > 4.9 Log IU/mL. The linearity of the Aptima assay was excellent across dilution series of four HCV genotypes (slope of the regression line: 1.00-1.02). The Aptima assay detected significantly more replicates below targeted 2 Log IU/mL than the CAPCTMv2 test, and yielded clearly interpretable results when used to analyze samples from patients treated with DAAs. Conclusions The analytical performance of the Aptima assay makes it well suited for monitoring patients with chronic HCV infection undergoing antiviral treatment.

The genus Kluyvera comprises potential pathogens that can cause many infections. This study reports a Kluyvera intermedia strain (FOSA7093) from a pancreatic cyst specimen from a long-term hospitalised patient. Whole-genome sequencing (WGS) of the K. intermedia isolate was performed and the strain was reported as sensitive to Danish-registered antibiotics although it had a fosA-like gene in the genome. There were nine contigs that aligned to a plasmid, and these contigs contained several heavy metal resistance gene homologues. Furthermore, a prophage was discovered in the genome. WGS represents an efficient tool for monitoring Kluyvera spp. and its role as a reservoir of multidrug resistance. Therefore, this susceptible K. intermedia genome has many characteristics that allow comparison of resistant K. intermedia that might be discovered in the future.

Objectives To investigate the clinical relevance and molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Klebsiella species in animals. Methods Antimicrobial susceptibilities and presence of ESBLs were examined among Klebsiella spp. (n = 1519) from clinical samples (>1200 senders from Germany and other European countries) mainly from companion animals and horses from October 2008 to March 2010. Multilocus sequence typing (MLST) and PFGE were performed including human isolates for comparative purposes. Results The overall ESBL rate was 8% for Klebsiella pneumoniae subsp. pneumoniae. Most K. pneumoniae subsp. pneumoniae ESBL producers were isolated from soft tissue infections (29.3%) and urinary tract infections (14.9%). The major ESBL type was CTX-M-15 (85.4%), located on different plasmid scaffolds (HI2, I1, FIA, FIB, FII, A/C, R and N). Other ESBL genes, such as bla(CTX-M-1) (5.6%), bla(CTX-M-3), bla(CTX-M-9), bla(SHV-2) and bla(SHV-12) (1.1% each), were also detected. Additional resistances, e.g. to fluoroquinolones (89.9%), were frequently present. ST15-CTX-M-15, a clonal group that recently emerged in humans, accounted for 75.8% of the strains analysed by MLST and there was evidence for nosocomial events in five veterinary clinics. Human ST15-CTX-M-15 strains shared PFGE clusters with animal isolates, suggesting the dissemination of this clonal group between both populations. Conclusions Our data indicate a wide spread of ST15-CTX-M-15 K. pneumoniae subsp. pneumoniae, which should be considered as a zoonotic agent of high clinical relevance for humans and animals. Further research should be undertaken to unravel both microevolutionary and biological aspects probably contributing to this global success.

The primary aim of the Danish enterovirus (EV) surveillance system is to document absence of poliovirus infection. The conflict in Syria has left many children unvaccinated and movement from areas with polio cases to Europe calls for increased awareness to detect and respond to virus-transmission in a timely manner. We evaluate the national EV laboratory surveillance, to generate recommendations for system strengthening. The system was analysed for completeness of viral typing analysis and clinical information and timeliness of specimen collection, laboratory results and reporting of clinical information. Of 23,720 specimens screened, 2,202 (9.3%) were EV-positive. Submission of cerebrospinal fluid and faecal specimens from primary diagnostic laboratories was 79.5% complete (845/1,063), and varied by laboratory and patient age. EV genotypes were determined in 68.5% (979/1,430) of laboratory-confirmed cases, clinical information was available for 63.1% (903/1,430). Primary diagnostic results were available after a median of 1.4 days, typing results after 17 days, detailed clinical information after 33 days. The large number of samples typed demonstrated continued monitoring of EV-circulation in Denmark. The system could be strengthened by increasing the collection of supplementary faecal specimens, improving communication with primary diagnostic laboratories, adapting the laboratory typing methodology and collecting clinical information with electronic forms.

Background Escherichia coli is the most common cause of urinary tract infection (UTI). The pathogenic isolates are becoming increasingly resistant to antibiotics; with a worldwide dissemination of resistant sequence types (ST). We characterized three different uropathogenic E. coli populations, from non-hospitalized patients to describe the genetic kinship between resistant and susceptible isolates. We studied the populations by use of multi-locus sequence typing (MLST) and abbreviated-multi locus variable number of tandem repeat analysis (a-MLVA). Urine samples submitted for testing, by general practitioners, were identified at Dept. of Clinical Microbiology at Hvidovre Hospital, Denmark, from Oct. 2011 to July 2012. We included 94 fully susceptible, 94 resistant (non-ESBL) and 98 Extended Spectrum Beta-lactamases- (ESBL)-producing E. coli isolates. Results The ESBL population was dominated vastly by ST131 (51 %), ST38 (9 %) and ST69 (6 %). In the resistant group ST69 (18 %), ST73 (11 %) and ST131 (15 %) were the largest clusters. In the susceptible population more STs and a-MLVA codes were identified compared to the other groups and ST73 and ST95 were found as the only clusters with 16 % and 6 %, respectively. Ninety-eight per cent of the ESBL-producing E. coli isolates were CTX-M-producers. Conclusion ST131 dominated the population of community-associated uropathogenic ESBL-producing E. coli, but was less frequent among non-ESBL-producing E. coli. The fully susceptible E. coli population was a much more diverse group than the resistant and ESBL-producing E. coli populations. Overall, these findings suggest that dominant ESBL-producing lineages are derived from UPEC lineages already established in the general UPEC population. Electronic supplementary material The online version of this article (doi:10.1186/s12866-016-0681-z) contains supplementary material, which is available to authorized users.

Background: Escherichia coli is the most common cause of urinary tract infection (UTI). The pathogenic isolates are becoming increasingly resistant to antibiotics; with a worldwide dissemination of resistant sequence types (ST). We characterized three different uropathogenic E. coli populations, from non-hospitalized patients to describe the genetic kinship between resistant and susceptible isolates. We studied the populations by use of multi-locus sequence typing (MLST) and abbreviated-multi locus variable number of tandem repeat analysis (a-MLVA). Urine samples submitted for testing, by general practitioners, were identified at Dept. of Clinical Microbiology at Hvidovre Hospital, Denmark, from Oct. 2011 to July 2012. We included 94 fully susceptible, 94 resistant (non-ESBL) and 98 Extended Spectrum Beta-lactamases- (ESBL)-producing E. coli isolates.Results: The ESBL population was dominated vastly by ST131 (51 %), ST38 (9 %) and ST69 (6 %). In the resistant group ST69 (18 %), ST73 (11 %) and ST131 (15 %) were the largest clusters. In the susceptible population more STs and a-MLVA codes were identified compared to the other groups and ST73 and ST95 were found as the only clusters with 16 % and 6 %, respectively. Ninety-eight per cent of the ESBL-producing E. coli isolates were CTX-M-producers.Conclusion: ST131 dominated the population of community-associated uropathogenic ESBL-producing E. coli, but was less frequent among non-ESBL-producing E. coli. The fully susceptible E. coli population was a much more diverse group than the resistant and ESBL-producing E. coli populations. Overall, these findings suggest that dominant ESBL-producing lineages are derived from UPEC lineages already established in the general UPEC population.

Background: Around a quarter of individuals infected with hepatitis C virus (HCV) are spontaneously able to clear the virus. Correlates of spontaneous HCV clearance are not well established and the aim of this study was to characterize factors associated with spontaneous HCV clearance in a human immunodeficiency virus (HIV)-co-infected cohort. Methods: We analyzed 327 anti-HCV-positive HIV-1-infected patients using multivariate logistic regression. HCV clearance was defined as the presence of anti-HCV with undetectable HCV RNA from at least 2 measurements more than 6 months apart. Results: We included 327 HIV-1-infected individuals, predominantly of Caucasian race; 112 (34%) were females, 258 (79%) were injecting drug users (IDU), 25 (8%) were men who have sex with men (MSM), and 20 (6%) were hepatitis B surface antigen (HBsAg)-positive. Seventy-six (23%; 95% confidence interval (CI) 18-28) had cleared their HCV infection and 251 (77%; 95% CI 72-82) had a chronic infection. The clearance rate in HBsAg-positive individuals was 65%. Being female, HBsAg-positive, or belonging to HIV exposure groups IDU and MSM predicted higher HCV clearance rates (adjusted odds ratio (aOR) 1.8, 95% CI 1-3.2; aOR 7.6, 95% CI 2.7-21; aOR 5.2, 1.2-23.5; and aOR 10.2, 95% CI 1.8-58, respectively). Race, acquired immunodeficiency syndrome (AIDS), and antiretroviral therapy were not associated with HCV clearance. Conclusions: The HCV clearance rate in this HIV-1 cohort was 23%. MSM and IDUs may have higher clearance rates due to their repeated exposure to low-dose HCV, leading to immune memory. Our data suggest an interaction of hepatitis B virus and HCV that influences the outcome of acute HCV infection.

Summary. Twenty-five per cent of individuals infected with hepatitis C virus (HCV) are able to clear HCV spontaneously. Differences in host genetics are believed to affect the outcome of HCV infection. We analysed an exonic, a promoter and an intronic single nucleotide polymorphism (SNP) of the interferon-λ3 coding interleukin (IL)-28B gene to study the relationship between IL28B SNPs and outcome of HCV infection. Among 206 HIV-1-infected Europeans with evidence of HCV infection, 47 (23%) individuals had cleared HCV and 159 (77%) had developed chronic infection. The exonic rs8103142 CT, the promoter rs12979860 CT and the intronic rs11881222 AG genotypes were associated with a decreased HCV clearance rate with adjusted odds ratios (aOR) of 0.3 (95% CI, 0.1–0.7), 0.4 (95% CI, 0.2–0.8) and 0.4 (95% CI, 0.2–0.8), respectively. The haplotype block TCG CTA was associated with a decreased HCV clearance rate (aOR 0.4, 95% CI, 0.2–0.8). Further, we found significant differences in HCV RNA levels among individuals chronically infected with HCV genotype 1 for rs8103142 and rs12979860 (P ≤ 0.05). Chronically infected individuals with HCV genotype 3 and with the favourable haplotype block CTA CTA had higher median HCV RNA levels than individuals with unfavourable haplotype blocks (P ≤ 0.05). Our findings suggest that IL28B may account for some differences in HCV outcome but that other factors including the viral genotype, host genetics and the host–virus interaction are likely to influence the outcome of HCV infection.

Global surveillance for a novel rhinovirus genotype indicated its association with community outbreaks and pediatric respiratory disease in Africa, Asia, Australia, Europe, and North America. Molecular dating indicates that these viruses have been circulating for at least 250 years.

BackgroundLittle is known about the underlying causes of differences in immunological response to antiretroviral therapy during multidrug-resistant (MDR) HIV type-1 (HIV-1) infection. This study aimed to identify virological factors associated with immunological response during therapy failure.MethodsIndividuals with MDR HIV-1 receiving therapy for ≥3 months were included. CD4+T-cell count slopes and pol and clonal env sequences were determined. Genetic analyses were performed using distance-based and maximum likelihood methods. Synonymous mutations rates of env were used to estimate viral replication.ResultsOf 1,000 patients treated between 1995 and 2003, 72 individuals fulfilled the definition for triple-class failure, but 25 were non-compliant, 21 were successfully resuppressed and 3 had died or quit therapy. Of the 23 that fulfilled study criteria, 16 had samples available for analysis. In a longitudinal mixed-effects model, plasma HIV-1 RNA only tended to predict immunological response ( P=0.06), whereas minor protease inhibitor (PI) and nucleoside reverse transcriptase (NRTI) mutations at baseline correlated significantly with CD4+T-cell count slopes ( r=-0.56, P=0.04 and r=-0.64, P=0.008, respectively). Interestingly, synonymous mutations of env correlated inversely with CD4+T-cell count slopes ( r=-0.60; P=0.01) and individuals with codons under positive selection had significantly better CD4+T-cell responses than individuals without (0.42 versus -5.34; P=0.02).ConclusionsOur results suggest that minor PI mutations and NRTI mutations present early during therapy failure are predictive of the CD4+T-cell count slopes. Synonymous mutation rates of the env gene suggested that underlying differences in fitness could cause this association.

The purpose of the study was to evaluate how use of antibiotics precedes the presence of ESBL-producing E.coli in general practice. The authors performed a triple-case-control study where three case groups were individually compared to a single control group of uninfected individuals. Urine samples were prospectively collected and retrospective statistical analyses were done. This study included 98 cases with urinary tract infection (UTI) caused by ESBL-producing E. coli, 174 with antibiotic-resistant (non-ESBL) E. coli, 177 with susceptible E. coli and 200 with culture negative urine samples. Case groups had significantly higher use of antibiotics than the control group within 30 days before infection (p < 0.0001). The ESBL group had significantly more hospital admissions than the other case groups (p < 0.05). Hospital admission was an independent risk factor for community onset UTI by ESBL-producing E. coli. Exposure to antibiotics was a risk factor for UTI with E. coli, while prior antibiotic usage was not an indisputable predictor for infection with ESBL-producing E.coli in general practice.

Typing of meticillin resistant Staphylococcus aureus (MRSA) by whole genome sequencing (WGS) is performed routinely in Copenhagen since January 2013. We describe the relatedness, based on WGS data and epidemiological data, of 341 MRSA isolates. These comprised all MRSA (n?=?300) identified in Copenhagen in the first five months of 2013. Moreover, because MRSA of staphylococcal protein A (spa)-type 304 (t304), sequence type (ST) 6 had been associated with a continuous neonatal ward outbreak in Copenhagen starting in 2011, 41 t304 isolates collected in the city between 2010 and 2012 were also included. Isolates from 2013 found to be of t304, ST6 (n=14) were compared to the 41 earlier isolates. In the study, isolates of clonal complex (CC) 22 were examined in detail, as this CC has been shown to include the hospital-acquired epidemic MRSA (EMRSA-15) clone. Finally, all MRSA ST80 were also further analysed, as representatives of an important community-acquired MRSA in Europe. Overall the analysis identified 85 spa-types and 35 STs from 17 CCs. WGS confirmed the relatedness of epidemiologically linked t304 neonatal outbreak isolates. Several non-outbreak related patients had isolates closely related to the neonatal isolates suggesting unrecognised community chains of transmission and insufficient epidemiological data. Only four CC22 isolates were related to EMRSA-15. No community spread was observed among the 13 ST80 isolates. WGS successfully replaced conventional typing and added information to epidemiological surveillance. Creation of a MRSA database allows clustering of isolates based on single nucleotide polymorphism (SNP) calling and has improved our understanding of MRSA transmission.

Hydroxy chalcones, for example, Licochalcone A, has for several years been known to be antibacterial. The low aqueous solubility and the medium antibacterial potency have limited the usefulness of the compounds. We describe the bioisosteric replacement of the essential 4'-hydroxy group in the hydroxy chalcones with bioisosters of varied degrees of acidity resulting in both more potent and more soluble compounds. The more acidic 4'-hydroxy analogues (e.g., 3'-fluoro- or 3',5'-difluoro-) gave almost inactive compounds whereas exchanging the hydroxy group with a carboxy group resulted in a potent compound with a high aqueous solubility. Further optimisation and SAR-analysis resulted in soluble and potent carboxy chalcones [e.g., 3,5-dibromo- and 3,5-di(trifluoromethyl)-].

Several functions have been assigned to the extensive glycosylation of HIV-1 envelope glycoprotein gp120, especially immune escape mechanisms, but the intramolecular interactions between gp120 and its carbohydrate complement are not well understood. To analyse this phenomenon we established a new microwell deglycosylation assay for determining N-linked glycan accessibility after binding of gp120-specific agents. Orientation-specific exposition of gp120 in ELISA microplates was achieved by catching with either anti-C5 antibody D7324 or anti-V3 antibody NEA-9205. We found that soluble CD4 inhibited the deglycosylation of gp120 only when gp120 was caught by D7324 and not by NEA9205. In contrast, antibodies from HIV-infected individuals inhibited the deglycosylation best when gp120 was caught by NEA9205. These results demonstrated that both the CD4-binding site and the epitopes recognised by antibodies from HIV-infected individuals have N-glycans in the close vicinity. However, the difference in gp120 orientation indicates that antibodies in HIV-infected individuals, at least partly, bind to epitopes different from the CD4-binding site. Finally, we determined the structural class of the glycan of one V1 glycosylation site of prototype HIV-1 LAI gp120, which remained unsolved from previous studies, and found that it belonged to the complex type of glycans.

Objectives There are limited data available on the epidemiology and prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the human population that encode the recently described mecA homologue, mecC. To address this knowledge gap we undertook a prospective prevalence study in England to determine the prevalence of mecC among MRSA isolates. Patients and methods Three hundred and thirty-five sequential MRSA isolates from individual patients were collected from each of six clinical microbiology laboratories in England during 2011–12. These were tested by PCR or genome sequencing to differentiate those encoding mecA and mecC. mecC-positive isolates were further characterized by multilocus sequence typing, spa typing, antimicrobial susceptibility profile and detection of PBP2a using commercially available kits. Results Nine out of the 2010 MRSA isolates tested were mecC positive, indicating a prevalence among MRSA in England of 0.45% (95% CI 0.24%–0.85%). The remainder were mecA positive. Eight out of these nine mecC MRSA isolates belonged to clonal complex 130, the other being sequence type 425. Resistance to non-β-lactam antibiotics was rare among these mecC MRSA isolates and all were phenotypically identified as MRSA using oxacillin and cefoxitin according to BSAC disc diffusion methodology. However, all nine mecC isolates gave a negative result using three different commercial PBP2a detection assays. Conclusions mecC MRSA are currently rare among MRSA isolated from humans in England and this study provides an important baseline prevalence rate to monitor future changes, which may be important given the increasing prevalence of mecC MRSA reported in Denmark.

Background Quantification of Hepatitis C Virus (HCV)-RNA is important for the clinical management of patients undergoing antiviral therapy. Objectives To compare the quantification of clinical plasma samples by the Roche COBAS AmpliPrep/COBAS TaqMan HCV test v2.0 and the artus HCV QS-RGQ test. Study design HCV-RNA viral load in 155 plasma samples from HCV-seropositive individuals was determined using the COBAS test and retrospectively with the artus . Furthermore, a dilution series of an Acrometrix standard was tested with both tests in replicates of five to assess differences in limit of detection and precision. Results Two clinical samples showed inhibition using the artus test and were excluded from analysis. Of the clinical samples, 20 tested negative in both tests, 7 tested positive in the COBAS test and negative in the artus test, and 126 samples were quantified by both tests. The mean overall difference between tests ( artus –COBAS) was 0.27logIU/mL. The mean difference of quantification varied little across genotype 1a, 1b, 2b and 3a (range: +0.15 to +0.35logIU/mL). Both tests were precise (%CV at 1000IU/mL 1.1 and 1.8 for the COBAS and artus test, respectively). Conclusions The limit of detection appeared lower in the COBAS test than the artus test when analyzed from a limited number of replicates. Both tests were precise with the artus test quantifying higher than the COBAS test on average. It is therefore recommended to monitor individual patients with the same test throughout treatment.

The V3 region of the human immunodeficiency virus type 1 envelope protein gp120 constitutes a potential neutralization target, but the oligosaccharide of one conserved N-glycosylation site in this region protects it from neutralizing antibodies. Here, we determined whether N-linked glycans of other gp120 domains were also involved in protection of V3 neutralization epitopes. Two molecular clones of HIV-1, one lacking three N-linked glycans of the V1 region (HIV-1(3N/V1)) and another lacking three N-linked glycans of the C2 region (HIV-1(3N/C2)), were created and characterized. gp120 from both mutated viral clones had higher electrophoretic mobilities than gp120 from wild-type virus, confirming loss of N-linked glycans. Wild-type virus and both mutant clones replicated equally well in established T cell lines and all three viruses were able to utilize CXCR4 but not CCR5 as a coreceptor. The induced mutations increased gp120 affinity for CXCR4 but caused no corresponding increase in viral ability to replicate in T cell lines. HIV-1(3N/V1) was neutralized at about 25 times lower concentrations of an antibody to the V3 region than were wild-type virus and HIV-1(3N/C2). Soluble, monomeric gp120 from HIV-1(3N/V1) and wild type virus had identical avidity for the V3 antibody, indicating that the V1 glycans were able to shield V3 only in oligomeric but not monomeric gp120. In conclusion, one or more N-linked glycans of gp120 V1 is engaged in protection of the V3 region from potential neutralizing antibodies, and this effect is dependent on the oligomeric organization of gp120/gp41.

We previously identified a group of 10 long-term nonprogressors (LTNP) with HIV-1 infection. In this study, we have sequenced the envelope gene (C2-V3-C3) from the 10 LTNPs and from a control group of 9 people with rapidly progressing infection (RPI). The 19 individuals' CCR5 genotype and virus phenotype (i.e., syncytium-inducing/non-syncytium-inducing [SI/NSI]) were obtained from a previous study. A phylogenetic tree was constructed containing the 19 envelope sequences together with 42 local control env sequences obtained from other studies. Analysis of the phylogenetic tree did not reveal any relation between the envelope gene (C2-V3-C3) from LTNPs versus RPIs. When data from the CCR5 genotype and the virus phenotype were assembled in the phylogenetic tree, no significant clustering was observed. From alignment of the protein sequences, we found a possible N-glycan in position aa294 in env that was conserved in only 1 of 10 LTNPs; however, it was conserved in 6 of 9 RPIs. Our study could not demonstrate any association between LTNPs and the sequenced envelope gene segment (C2-V3-C3). This lack of association could be due to the relatively small sample size of the data set. Nor did we find any relation between the CCR5 genotype or the SI/NSI phenotype with the sequenced envelope genes from the 19 participants. The possible N-glycan position we have described is an interesting observation that may require further investigation.

We have previously shown that an N-glycosylation site of N306 of HIV-1 gp120 is not necessary for the HIV-1 infectivity but protects HIV-1 from neutralising antibodies. In contrast Nakayama et al. [FEBS Lett. (1998) 426, 367–372], using a virus with an identical V3 region, suggested that elimination of this particular glycan reduced the ability of T-tropic HIV to bind to CXCR4 and hence its ability to infect T cell lines. We therefore re-examined the ability of a mutant virus, lacking the N306 glycan, to replicate in various types of cells and found no change in co-receptor usage for mutant virus. The ability of mutant virus to replicate or to induce syncytia in infected cells was similar to that of wild type virus. These results corroborate our original observation, confirming that the induced mutation in the N306 glycosylation site neither impairs nor improves the ability of mutant virus to replicate in permissive cells.

An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected to elicit antibodies preferentially neutralizing mutant variants of HIV-BRU lacking the N306 glycan. Therefore, two guinea pigs were immunized with monomeric wild-type HIV-BRU gp120 possessing the N306 glycan and immune sera were tested for neutralization against target viruses HIV-BRU, -A308, and -A308T321. HIV-A308 and HIV-A308T321 lack the N306 glycan; HIV-A308T321 contains an additional mutation at the tip of V3 rendering it resistant to MAb binding at this epitope. Both immune sera preferentially neutralized the two mutant virus variants lacking the N306 glycan, with a 10- to 20-fold increase in neutralization titer compared with the wild-type HIV-BRU. Thus, immunization with monomeric HIV-BRU gp120 elicited antibodies preferentially neutralizing HIV variants lacking the N306 glycan. In addition to antibodies directed against the tip of V3, other antibodies directed against epitopes shielded by the N306 glycan on the envelope oligomer were elicited by the immunization, as demonstrated by the ability of the immune sera to neutralize HIV-A308T321. One such epitope was overlapping the NEA-9284 epitope located at the amino-terminal flank of the V3 loop. Our results demonstrate that monomeric gp120 contains immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation.