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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.

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.

From October 2022 through January 2023, nine patients with NDM-5/OXA-48-carbapenemase-producing Enterobacter hormaechei ST79 were detected in Denmark and subsequently one patient in Iceland. There were no nosocomial links between patients, but they had all been treated with dicloxacillin capsules. An NDM-5/OXA-48-carbapenemase-producing E. hormaechei ST79, identical to patient isolates, was cultured from the surface of dicloxacillin capsules in Denmark, strongly implicating them as the source of the outbreak. Special attention is required to detect the outbreak strain in the microbiology laboratory. From October 2022 through January 2023, nine patients with NDM-5/OXA-48-carbapenemase-producing Enterobacter hormaechei ST79 were detected in Denmark and subsequently one patient in Iceland. There were no nosocomial links between patients, but they had all been treated with dicloxacillin capsules. An NDM-5/OXA-48-carbapenemase-producing E. hormaechei ST79, identical to patient isolates, was cultured from the surface of dicloxacillin capsules in Denmark, strongly implicating them as the source of the outbreak. Special attention is required to detect the outbreak strain in the microbiology laboratory.

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.

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.

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.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has been associated with a high risk of adverse outcomes in solid organ transplant (SOT) recipients in the pre-vaccination era. In this retrospective cohort study, we examined the incidence and severity of COVID-19 in kidney and liver transplant recipients in Denmark in the post-vaccination era, from December 27, 2020, to December 27, 2021. We included 1428 SOT recipients with 143 cases of first-positive SARS-CoV-2 PCR test. The cumulative incidence of first-positive SARS-CoV-2 PCR test 1 year after initiation of vaccination was 10.4% (95% CI: 8.8–12.0), and the incidence was higher in kidney than in liver transplant recipients (11.6% [95% CI: 9.4–13.8] vs. 7.4% [95% CI: 5.1–9.8], p =.009). After the first-positive SARS-CoV-2 PCR test, the hospitalization rate was 31.5% (95% CI: 23.9–39.1), and 30-day all-cause mortality was 3.7% (95% CI: 0.5–6.8). Hospitalization was lower in vaccinated than in unvaccinated SOT recipients (26.4% [95% CI: 18.1–34.6] vs. 48.5% [95% CI: 31.4–65.5], p =.011), as was mortality (1.8% [95% CI: 0.0–4.3] vs. 9.1% [95% CI: 0.0–18.9], p =.047). In conclusion, SOT recipients remain at high risk of adverse outcomes after SARS-CoV-2 infections, with a lower risk observed in vaccinated than in unvaccinated SOT recipients.

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.

The COVID-19 pandemic has led to an unprecedented demand for real-time surveillance data in order to inform critical decision makers regarding the management of the pandemic. The aim of this review was to describe how the Danish national microbiology database, MiBa, served as a cornerstone for providing data to the real-time surveillance system by linkage to other nationwide health registries. The surveillance system was established on an existing IT health infrastructure and a close network between clinical microbiologists, information technology experts, and public health officials. In 2020, testing capacity for SARS-CoV-2 was ramped up from none to over 10,000 weekly PCR tests per 100,000 population. The crude incidence data mirrored this increase in testing. Real-time access to denominator data and patient registries enabled adjustments for fluctuations testing activity, providing robust data on crude SARS-CoV-2 incidence during the changing diagnostic and management strategies. The use of the same data for different purposes, for example, final laboratory reports, information to the public, contact tracing, public health, and science, has been a critical asset for the pandemic response. It has also raised issues concerning data protection and critical capacity of the underlying technical systems and key resources. However, even with these limitations, the setup has enabled decision makers to adopt timely interventions. The experiences from COVID-19 may motivate a transformation from traditional indicator-based public health surveillance to an all-encompassing information system based on access to a comprehensive set of data sources, including diagnostic and reference microbiology.

Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for large-scale detection of total antibodies (Ab), immunoglobulin G (IgG), and IgM against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays., Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for large-scale detection of total antibodies (Ab), immunoglobulin G (IgG), and IgM against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays. This study was a Danish national collaboration and evaluated 15 commercial and one in-house anti-SARS-CoV-2 assays in 16 laboratories. Sensitivity was evaluated using 150 samples from individuals with asymptomatic, mild, or moderate COVID-19, nonhospitalized or hospitalized, confirmed by nucleic acid amplification tests (NAAT); samples were collected 13 to 73 days either from symptom onset or from positive NAAT (patients without symptoms). Specificity and cross-reactivity were evaluated in samples collected prior to the SARS-CoV-2 epidemic from >586 blood donors and patients with autoimmune diseases, cytomegalovirus or Epstein-Barr virus infections, and acute viral infections. A specificity of ≥99% was achieved by all total-Ab and IgG assays except one, DiaSorin Liaison XL IgG (97.2%). Sensitivities in descending order were Wantai ELISA total Ab (96.7%), CUH-NOVO in-house ELISA total Ab (96.0%), Ortho Vitros total Ab (95.3%), YHLO iFlash IgG (94.0%), Ortho Vitros IgG (93.3%), Siemens Atellica total Ab (93.2%), Roche Elecsys total Ab (92.7%), Abbott Architect IgG (90.0%), Abbott Alinity IgG (median 88.0%), DiaSorin Liaison XL IgG (median 84.6%), Siemens Vista total Ab (81.0%), Euroimmun/ELISA IgG (78.0%), and Snibe Maglumi IgG (median 78.0%). However, confidence intervals overlapped for several assays. The IgM results were variable, with the Wantai IgM ELISA showing the highest sensitivity (82.7%) and specificity (99%). The rate of seropositivity increased with time from symptom onset and symptom severity.

Background Diagnostic real time reverse transcription PCR (rRT-PCR) is usually done using nucleic acid (NA) purified from the sample. In the SARS-CoV-2 pandemic reagents and utensils for NA purification has been in short supply. This has generated interest in methods that eliminate the need for NA purification. Objectives To investigate if addition of detergent to rRT-PCR master mix (MM) enabled in-well direct lysis and detection of SARS-CoV-2 in clinical eSwab specimens. Study design IGEPAL-CA-630 (IGEPAL) was added to SARS-CoV-2 MM to 0.3 % final concentration and crude sample was added directly to the PCR well containing MM. Cycle of positivity (Cp) and categorical agreement was compared in samples tested in standard rRT-PCR after NA purification and in in-well lysis, direct rRT-PCR. Results In-well lysis direct rRT-PCR detected SARS-CoV-2 in 27/30 previously SARS-CoV-2+ samples with an average bias of 3.26 cycles (95 %CI: 0.08–6.43 cycles). All 30 previously test negative samples remained negative when tested in in-well lysis, direct PCR. Conclusions Supplementation of detergent to MM was shown to be useful for the detection of SARS CoV-2 in eSwab specimens (COPAN) by direct rRT-PCR without prior NA purification.

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 & 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.

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.

We describe clonal shifts in vanA Enterococcus faecium isolates from clinical samples obtained from patients in Denmark from 2015 to the first quarter (Q1) of 2019. During Q1 2019, the vancomycin-variable enterococci (VVE) ST1421-CT1134 vanA E. faecium became the most dominant vanA E. faecium clone and has spread to all five regions in Denmark. Among 174 E. faecium isolates with vanA, vanB or vanA/vanB genes in Q1 2019, 44% belonged to this type. We describe the clonal shift for vanA Enterococcus faecium during the last 4 years and the national spread of a vancomycin-variable vanA E. faecium ST1421-CT1134 clone in Denmark. The aim is to highlight the importance of using molecular methods for detecting vancomycin-variable enterococci (VVE), and to alert other countries about this emerging nosocomial clone.

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.

Viral gastroenteritis causes high morbidity worldwide. In this study, stool samples from 179 children aged 0–6 years attending Danish day care centers were investigated for gastrointestinal viruses. Each child was observed for one year with submission of samples and questionnaires every two months. Adenovirus, norovirus, rotavirus, and sapovirus were detected in samples using real-time PCR. A total of 229 (33%) of the 688 samples collected tested positive for at least one virus. At the first sampling point, adenovirus was shed by 6%, norovirus genotype I by 3% and genotype II by 12%, rotavirus A by 9%, and sapovirus by 21% of the 142 children included in the risk factor analyses. Increasing age was identified as a protective factor against testing positive for gastrointestinal virus, whereas nausea during the previous two months was positively associated with testing positive. Odds of shedding adenovirus were 9.6 times higher among children treated with antibiotics within the previous two months than among children who were not. Gastrointestinal viruses were shed year-round and high viral loads were observed in samples from both symptomatic and asymptomatic children, suggesting children in day care as a reservoir and a possible source of spreading of viruses into the community.

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.

Objectives Mycoplasma genitalium (MG) causes urethritis and cervicitis, potentially causing reproductive complications. Resistance in MG to first-line (azithromycin) and second-line (moxifloxacin) treatment has increased. We examined the clinical and analytical performance of the new Conformite Europeene (CE)/ in vitro diagnostics (IVD) Aptima Mycoplasma genitalium assay (CE/IVD AMG; Hologic); the prevalence of MG, Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG); and MG resistance to azithromycin and moxifloxacin in Denmark, Norway and Sweden in 2016. Methods From February 2016 to February 2017, urogenital and extragenital (only in Denmark) specimens from consecutive attendees at three sexually transmitted disease clinics were tested with the CE/IVD AMG, the research-use-only MG Alt TMA-1 assay (Hologic), Aptima Combo 2 (CT/NG) assay and a laboratory-developed TaqMan real-time mgp B quantitative real-time PCR (qPCR). Resistance-associated mutations were determined by sequencing. Strains of MG and other mycoplasma species in different concentrations were also tested. Results In total 5269 patients were included. The prevalence of MG was 7.2% (382/5269; 4.9–9.8% in the countries). The sensitivity of the CE/IVD AMG, MG Alt TMA-1 and mgp B qPCR ranged 99.13–100%, 99.13–100% and 73.24–81.60%, respectively, in the countries. The specificity ranged 99.57–99.96%, 100% and 99.69–100%, respectively. The prevalence of resistance-associated mutations for azithromycin and moxifloxacin was 41.4% (120/290; 17.7–56.6%) and 6.6% (18/274; 4.1–10.2%), respectively. Multidrug resistance was found in all countries (2.7%; 1.1–4.2%). Conclusions Both transcription-mediated amplification (TMA)-based MG assays had a highly superior sensitivity compared to the mgp B qPCR. The prevalence of MG and azithromycin resistance was high. Validated and quality-assured molecular tests for MG, routine resistance testing of MG-positive samples and antimicrobial resistance surveillance are crucial.

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: The current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5 B proteins. OBJECTIVE: To develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5 B RASs without prior knowledge of sample genotype. STUDY DESIGN: In total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (∼ 9 kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs. RESULTS: The method successfully amplified and sequenced 94% (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22%) samples at a 15% threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4%) of 95 successfully sequenced samples. CONCLUSIONS: The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype.

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.

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.

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.