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1.
J Antimicrob Chemother ; 79(4): 810-814, 2024 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-38366372

RESUMO

OBJECTIVES: An Escherichia coli isolate, WGS1363, showed resistance to piperacillin/tazobactam but susceptibility to cephalosporins and contained a previously unrecognized ß-lactamase, CTX-M-255, as the only acquired ß-lactamase. CTX-M-255 was identical to CTX-M-27 except for a G239S substitution. Here, we characterize the hydrolytic spectrum of CTX-M-255 and a previously reported ß-lactamase, CTX-M-178, also containing a G239S substitution and compare it to their respective parental enzymes, CTX-M-27 and CTX-M-15. METHODS: All ß-lactamase genes were expressed in E. coli TOP10 and MICs to representative ß-lactam-antibiotics were determined. Furthermore, blaCTX-M-15,  blaCTX-M-27, blaCTX-M-178 and blaCTX-M-255 with C-terminal His-tag fusions were affinity purified for enzyme kinetic assays determining Michaelis-Menten kinetic parameters against representative ß-lactam-antibiotics and IC50s of clavulanate, sulbactam, tazobactam and avibactam. RESULTS: TOP10-transformants expressing blaCTX-M-178 and blaCTX-M-255 showed resistance to penicillin/ß-lactamase combinations and susceptibility to cephalothin and cefotaxime in contrast to transformants expressing blaCTX-M-15 and blaCTX-M-27. Determination of enzyme kinetic parameters showed that CTX-M-178 and CTX-M-255 both lacked hydrolytic activity against cephalosporins and showed impaired hydrolytic efficiency against penicillin antibiotics compared to their parental enzymes. Both enzymes appeared more active against piperacillin compared to benzylpenicillin and ampicillin. Compared to their parental enzymes, IC50s of ß-lactamase-inhibitors were increased more than 1000-fold for CTX-M-178 and CTX-M-255. CONCLUSIONS: CTX-M-178 and CTX-M-255, both containing a G239S substitution, conferred resistance to piperacillin/tazobactam and may be characterized as inhibitor-resistant CTX-M ß-lactamases. Inhibitor resistance was accompanied by loss of activity against cephalosporins and monobactams. These findings add to the necessary knowledge base for predicting antibiotic susceptibility from genotypic data.


Assuntos
Antibacterianos , Inibidores de beta-Lactamases , Inibidores de beta-Lactamases/farmacologia , Antibacterianos/farmacologia , Escherichia coli , beta-Lactamases/genética , Penicilinas/farmacologia , Cefalosporinas/farmacologia , Tazobactam/farmacologia , Piperacilina/farmacologia , Monobactamas , Combinação Piperacilina e Tazobactam , Testes de Sensibilidade Microbiana
2.
Euro Surveill ; 28(17)2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37103784

RESUMO

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.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Humanos , Klebsiella pneumoniae/genética , Serratia marcescens/genética , beta-Lactamases/genética , Proteínas de Bactérias/genética , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/epidemiologia , Plasmídeos/genética , Dinamarca/epidemiologia
3.
Euro Surveill ; 28(9)2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36862098

RESUMO

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.


Assuntos
Dicloxacilina , Surtos de Doenças , Humanos , Islândia/epidemiologia , Dinamarca/epidemiologia
4.
Gut ; 71(3): 627-642, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-33833066

RESUMO

OBJECTIVE: HCV-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. DESIGN: HCV 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. RESULT: The 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. CONCLUSION: Rapidly 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.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/genética , Hepacivirus/efeitos dos fármacos , Hepacivirus/fisiologia , Hepatócitos/virologia , Mutação/genética , Benzimidazóis/farmacologia , Técnicas de Cultura de Células , Combinação de Medicamentos , Genótipo , Hepacivirus/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Pirrolidinas/farmacologia , Quinoxalinas/farmacologia , Sofosbuvir/farmacologia , Sulfonamidas/farmacologia
5.
Am J Transplant ; 22(11): 2637-2650, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35801693

RESUMO

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.


Assuntos
COVID-19 , Transplante de Rim , Transplante de Órgãos , Humanos , COVID-19/epidemiologia , SARS-CoV-2 , Incidência , Estudos Retrospectivos , Transplante de Rim/efeitos adversos , Transplante de Órgãos/efeitos adversos , Transplantados , Vacinação , Fígado , Dinamarca/epidemiologia
6.
J Clin Microbiol ; 59(5)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33574119

RESUMO

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.


Assuntos
Anticorpos Antivirais/isolamento & purificação , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Imunoensaio , Infecções por Citomegalovirus , Ensaio de Imunoadsorção Enzimática , Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Imunoglobulina G/isolamento & purificação , Imunoglobulina M/isolamento & purificação , Laboratórios , SARS-CoV-2 , Sensibilidade e Especificidade
7.
J Antimicrob Chemother ; 76(9): 2260-2267, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34151364

RESUMO

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.


Assuntos
Infecção Hospitalar , Enterococcus faecium , Infecções por Bactérias Gram-Positivas , Enterococos Resistentes à Vancomicina , Proteínas de Bactérias/genética , Células Clonais , Dinamarca/epidemiologia , Enterococcus faecium/genética , Infecções por Bactérias Gram-Positivas/epidemiologia , Humanos , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase , Enterococos Resistentes à Vancomicina/genética
8.
J Viral Hepat ; 28(2): 302-316, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33131178

RESUMO

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.


Assuntos
Antivirais , Hepatite C Crônica , Antivirais/farmacologia , Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Quimioterapia Combinada , Genótipo , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Humanos , Retratamento , Falha de Tratamento , Proteínas não Estruturais Virais/genética
9.
Eur J Clin Microbiol Infect Dis ; 40(10): 2177-2183, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33977413

RESUMO

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.


Assuntos
Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Nariz/microbiologia , Períneo/microbiologia , Faringe/microbiologia , Infecções Estafilocócicas/microbiologia , Humanos , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Reação em Cadeia da Polimerase Multiplex , Infecções Estafilocócicas/diagnóstico
10.
Acta Anaesthesiol Scand ; 65(9): 1345-1350, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34086975

RESUMO

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.


Assuntos
COVID-19 , Influenza Humana , Superinfecção , Estudos de Coortes , Dinamarca/epidemiologia , Humanos , Influenza Humana/complicações , Influenza Humana/epidemiologia , Unidades de Terapia Intensiva , SARS-CoV-2 , Superinfecção/epidemiologia
11.
Hepatology ; 70(3): 771-787, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30964552

RESUMO

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.


Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Hepatite C Crônica/tratamento farmacológico , Inibidores de Proteases/uso terapêutico , 2-Naftilamina , Ácidos Aminoisobutíricos , Anilidas/uso terapêutico , Benzimidazóis/uso terapêutico , Carbamatos/uso terapêutico , Ciclopropanos , Dinamarca , Quimioterapia Combinada , Feminino , Genótipo , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C Crônica/diagnóstico , Humanos , Lactamas Macrocíclicas , Leucina/análogos & derivados , Masculino , Prognóstico , Prolina/análogos & derivados , Inibidores de Proteases/farmacologia , Pirrolidinas , Quinoxalinas/uso terapêutico , Sulfonamidas/uso terapêutico , Uracila/análogos & derivados , Uracila/uso terapêutico , Valina
12.
J Hepatol ; 70(3): 388-397, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30395912

RESUMO

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.


Assuntos
Antivirais , Farmacorresistência Viral/genética , Hepacivirus , Hepatite C Crônica , Proteínas não Estruturais Virais , Antivirais/classificação , Antivirais/farmacologia , Ligação Genética , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Humanos , Polimorfismo Genético , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética
13.
J Antimicrob Chemother ; 74(11): 3179-3183, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31411684

RESUMO

BACKGROUND: bla TEM-1 encodes a narrow-spectrum ß-lactamase that is inhibited by ß-lactamase inhibitors and commonly present in Escherichia coli. Hyperproduction of blaTEM-1 may cause resistance to penicillin/ß-lactamase inhibitor (P/BLI) combinations. OBJECTIVES: To characterize EC78, an E. coli bloodstream isolate, resistant to P/BLI combinations, which contains extensive amplification of blaTEM-1 within the chromosome. METHODS: EC78 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 ß-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. RESULTS: Illumina sequencing of EC78 identified blaTEM-1B as the only acquired ß-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 ß-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. CONCLUSIONS: IS26-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.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Combinação Piperacilina e Tazobactam/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genética , Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Escherichia coli/enzimologia , Humanos , Testes de Sensibilidade Microbiana , Análise de Sequência de DNA
14.
Euro Surveill ; 24(34)2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31456560

RESUMO

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.


Assuntos
Antibacterianos/farmacologia , Enterococcus faecium/genética , Enterococcus faecium/isolamento & purificação , Infecções por Bactérias Gram-Positivas/microbiologia , Enterococos Resistentes à Vancomicina/genética , Enterococos Resistentes à Vancomicina/isolamento & purificação , Vancomicina/farmacologia , Proteínas de Bactérias/genética , Carbono-Oxigênio Ligases , DNA Bacteriano/genética , Dinamarca/epidemiologia , Eletroforese em Gel de Campo Pulsado , Enterococcus faecium/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase , Prevalência , Vigilância de Evento Sentinela , Análise de Sequência de DNA , Enterococos Resistentes à Vancomicina/efeitos dos fármacos
15.
Gastroenterology ; 152(5): 1031-1041.e2, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28063955

RESUMO

BACKGROUND & AIMS: Nosocomial infections with Clostridium difficile present a considerable problem despite numerous attempts by health care workers to reduce risk of transmission. Asymptomatic carriers of C difficile can spread their infection to other patients. We investigated the effects of asymptomatic carriers on nosocomial C difficile infections. METHODS: We performed a population-based prospective cohort study at 2 university hospitals in Denmark, screening all patients for toxigenic C difficile in the intestine upon admittance, from October 1, 2012, to January 31, 2013. Screening results were blinded to patients, staff, and researchers. Patients were followed during their hospital stay by daily registration of wards and patient rooms. The primary outcomes were rate of C difficile infection in exposed and unexposed patients and factors associated with transmission. RESULTS: C difficile infection was detected in 2.6% of patients not exposed to carriers and in 4.6% of patients exposed to asymptomatic carriers at the ward level (odds ratio for infection if exposed to carrier, 1.79; 95% confidence interval, 1.16-2.76). Amount of exposure correlated with risk of C difficile infection, from 2.2% in the lowest quartile to 4.2% in the highest quartile of exposed patients (P = .026). Combining the load of exposure to carriers and length of stay seemed to have an additive effect on the risk of contracting C difficile. CONCLUSIONS: In a population-based prospective cohort study in Denmark, we found that asymptomatic carriers of toxigenic C difficile in hospitals increase risk of infection in other patients.


Assuntos
Infecções Assintomáticas/epidemiologia , Portador Sadio/epidemiologia , Infecção Hospitalar/epidemiologia , Enterocolite Pseudomembranosa/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Portador Sadio/diagnóstico , Clostridioides difficile/genética , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/epidemiologia , Estudos de Coortes , Dinamarca/epidemiologia , Feminino , Hospitalização , Hospitais Universitários , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Razão de Chances , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Adulto Jovem
16.
J Antimicrob Chemother ; 73(11): 2936-2940, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30113682

RESUMO

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.


Assuntos
Proteínas de Bactérias/genética , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Deleção de Genes , D-Ala-D-Ala Carboxipeptidase Tipo Serina/genética , Resistência a Vancomicina/genética , Vancomicina/farmacologia , Antibacterianos/farmacologia , Infecção Hospitalar/microbiologia , Infecção Hospitalar/transmissão , Humanos , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
17.
Eur J Clin Microbiol Infect Dis ; 37(8): 1431-1439, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29725956

RESUMO

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.


Assuntos
Antibiose , Infecções por Clostridium/prevenção & controle , Infecção Hospitalar/prevenção & controle , Probióticos/uso terapêutico , Saccharomyces boulardii/fisiologia , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/efeitos adversos , Antibacterianos/uso terapêutico , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/etiologia , Infecção Hospitalar/etiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Profilaxia Pré-Exposição , Probióticos/administração & dosagem , Resultado do Tratamento
18.
Scand J Gastroenterol ; 53(7): 849-856, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29720023

RESUMO

OBJECTIVES: We describe factors associated with and barriers to initiation of Direct Acting Antiviral (DAA) treatment in patients with chronic hepatitis C, who fulfill national fibrosis treatment guidelines in Denmark. MATERIALS AND METHODS: In this nationwide cohort study, we included patients with chronic hepatitis C from The Danish Database for Hepatitis B and C (DANHEP) who fulfilled fibrosis treatment criteria. Factors associated with treatment initiation and treatment failure were determined by logistic regression analyses. Medical records were reviewed from patients who fulfilled fibrosis treatment criteria, but did not initiate DAA treatment to determine the cause. RESULTS: In 344 (49%) of 700 patients, who fulfilled treatment criteria, factors associated with DAA treatment initiation were transmission by other routes than injecting drug use odds ratio (OR) 2.13 (CI: 1.38-3.28), previous treatment failure OR 2.58 (CI: 1.84-3.61) and ALT above upper limit of normal OR 1.60 (CI: 1.18-2.17). The most frequent reasons for not starting treatment among 356 (51%) patients were non-adherence to medical appointments (n = 107/30%) and ongoing substance use (n = 61/17%). Treatment failure with viral relapse occurred in 19 (5.5%) patients, who were more likely to have failed previous treatment OR 4.53 (CI: 1.59-12.91). CONCLUSIONS: In this nationwide cohort study, we found non-adherence to medical appointments and active substance use to be major obstacles for DAA treatment initiation. Our findings highlight the need for interventions that can overcome these barriers and increase the number of patients who can initiate and benefit from curative DAA treatment.


Assuntos
Antivirais/uso terapêutico , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/epidemiologia , Cooperação do Paciente , Adulto , Estudos de Coortes , Dinamarca/epidemiologia , Esquema de Medicação , Feminino , Hepacivirus/genética , Hepatite C Crônica/complicações , Humanos , Cirrose Hepática/epidemiologia , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Guias de Prática Clínica como Assunto , Fatores de Risco , Resposta Viral Sustentada , Falha de Tratamento
19.
Euro Surveill ; 23(3)2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29386093

RESUMO

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 < 5 years-old for the period of 2010-2015. We identified ca 2,500 RSV-associated hospitalisations annually amounting to total direct medical-costs of ca EUR 4.1 million per year. The incidence of RSV-associated hospitalisations peaked in infants 1-2 months of age followed by infants 2-3 months of age, and infants < 1 month of age, respectively. Infant boys were at higher risk of severe RSV infection as compared to infant girls: male-to-female ratio peaked with 1.4 at four months of age and gradually levelled out with increasing age to 1.0 at 4 years of age. Five RSV-associated deaths were identified. Our findings demonstrate that in a western country as Denmark, RSV constitutes a considerable burden on childhood health. Furthermore, the best approach to reduce the high incidence of RSV-associated hospitalisations in young infants < 3 months of age may be maternal vaccination due to general challenges in achieving sufficient and protective immune responses in young infants.


Assuntos
Custos de Cuidados de Saúde , Hospitalização/estatística & dados numéricos , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Estações do Ano , Vacinação/estatística & dados numéricos , Pré-Escolar , Dinamarca/epidemiologia , Feminino , Hospitalização/economia , Humanos , Lactente , Recém-Nascido , Masculino , Vigilância da População , Sistema de Registros , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/economia , Infecções por Vírus Respiratório Sincicial/virologia , Estudos Retrospectivos , Distribuição por Sexo , Vacinação/economia
20.
Mol Biol Evol ; 33(11): 2860-2873, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27501945

RESUMO

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.


Assuntos
Adaptação Biológica/genética , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Conjugação Genética , Elementos de DNA Transponíveis , Resistência Microbiana a Medicamentos , Resistência a Múltiplos Medicamentos , Escherichia coli/genética , Evolução Molecular , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Plasmídeos/metabolismo
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