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BACKGROUND: Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches. METHODS: 96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher). RESULTS: According to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%). CONCLUSIONS: The ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.
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Antibacterianos , Compuestos de Azabiciclo , Aztreonam , Farmacorresistencia Bacteriana Múltiple , Bacterias Gramnegativas , Pruebas de Sensibilidad Microbiana , Aztreonam/farmacología , Compuestos de Azabiciclo/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Antibacterianos/farmacología , Humanos , Bacterias Gramnegativas/efectos de los fármacos , Combinación de Medicamentos , Pseudomonas aeruginosa/efectos de los fármacos , beta-Lactamasas/metabolismo , Enterobacteriaceae/efectos de los fármacos , Proteínas Bacterianas , Infecciones por Bacterias Gramnegativas/microbiologíaRESUMEN
BACKGROUND: Morganella spp are opportunistic pathogens involved in various infections. Intrinsic resistance to multiple antibiotics (including colistin) combined with the emergence of carbapenemase producers reduces the number of active antimicrobials. The aim of this study was to characterise genetic features related to the spread of carbapenem-resistant Morganella spp. METHODS: This comparative genomic study included extensively drug-resistant Morganella spp isolates collected between Jan 1, 2013, and March 1, 2021, by the French National Reference Center (NRC; n=68) and European antimicrobial resistance reference centres in seven European countries (n=104), as well as one isolate from Canada, two reference strains from the Pasteur Institute collection (Paris, France), and two colistin-susceptible isolates from Bicêtre Hospital (Kremlin-Bicêtre, France). The isolates were characterised by whole-genome sequencing, antimicrobial susceptibility testing, and biochemical tests. Complete genomes from GenBank (n=103) were also included for genomic analysis, including phylogeny and determination of core genomes and resistomes. Genetic distance between different species or subspecies was performed using average nucleotide identity (ANI). Intrinsic resistance mechanisms to polymyxins were investigated by combining genetic analysis with mass spectrometry on lipid A. FINDINGS: Distance analysis by ANI of 275 isolates identified three groups: Morganella psychrotolerans, Morganella morganii subspecies sibonii, and M morganii subspecies morganii, and a core genome maximum likelihood phylogenetic tree showed that the M morganii isolates can be separated into four subpopulations. On the basis of these findings and of phenotypic divergences between isolates, we propose a modified taxonomy for the Morganella genus including four species, Morganella psychrotolerans, Morganella sibonii, Morganella morganii, and a new species represented by a unique environmental isolate. We propose that M morganii include two subspecies: M morganii subspecies morganii (the most prevalent) and M morganii subspecies intermedius. This modified taxonomy was supported by a difference in intrinsic resistance to tetracycline and conservation of metabolic pathways such as trehalose assimilation, both only present in M sibonii. Carbapenemase producers were mostly identified among five high-risk clones of M morganii subspecies morganii. The most prevalent carbapenemase corresponded to NDM-1, followed by KPC-2, and OXA-48. A cefepime-zidebactam combination was the most potent antimicrobial against the 172 extensively drug-resistant Morganella spp isolates in our collection from different European countries, which includes metallo-ß-lactamase producers. Lipid A analysis showed that the intrinsic resistance to colistin was associated with the presence of L-ARA4N on lipid A. INTERPRETATION: This global characterisation of, to our knowledge, the widest collection of extensively drug-resistant Morganella spp highlights the need to clarify the taxonomy and decipher intrinsic resistance mechanisms, and paves the way for further genomic comparisons. FUNDING: None.
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Antibacterianos , Proteínas Bacterianas , Infecciones por Enterobacteriaceae , Genoma Bacteriano , Pruebas de Sensibilidad Microbiana , Morganella , Filogenia , beta-Lactamasas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Antibacterianos/farmacología , Genoma Bacteriano/genética , Humanos , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/epidemiología , Morganella/genética , Genómica , Secuenciación Completa del Genoma , Europa (Continente)/epidemiología , Carbapenémicos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Colistina/farmacologíaRESUMEN
SCOPE: Pseudomonas aeruginosa, a ubiquitous opportunistic pathogen considered one of the paradigms of antimicrobial resistance, is among the main causes of hospital-acquired and chronic infections associated with significant morbidity and mortality. This growing threat results from the extraordinary capacity of P. aeruginosa to develop antimicrobial resistance through chromosomal mutations, the increasing prevalence of transferable resistance determinants (such as the carbapenemases and the extended-spectrum ß-lactamases), and the global expansion of epidemic lineages. The general objective of this initiative is to provide a comprehensive update of P. aeruginosa resistance mechanisms, especially for the extensively drug-resistant (XDR)/difficult-to-treat resistance (DTR) international high-risk epidemic lineages, and how the recently approved ß-lactams and ß-lactam/ß-lactamase inhibitor combinations may affect resistance mechanisms and the definition of susceptibility profiles. METHODS: To address this challenge, the European Study Group for Antimicrobial Resistance Surveillance (ESGARS) from the European Society of Clinical Microbiology and Infectious Diseases launched the 'Improving Surveillance of Antibiotic-Resistant Pseudomonas aeruginosa in Europe (ISARPAE)' initiative in 2022, supported by the Joint programming initiative on antimicrobial resistance network call and included a panel of over 40 researchers from 18 European Countries. Thus, a ESGARS-ISARPAE position paper was designed and the final version agreed after four rounds of revision and discussion by all panel members. QUESTIONS ADDRESSED IN THE POSITION PAPER: To provide an update on (a) the emerging resistance mechanisms to classical and novel anti-pseudomonal agents, with a particular focus on ß-lactams, (b) the susceptibility profiles associated with the most relevant ß-lactam resistance mechanisms, (c) the impact of the novel agents and resistance mechanisms on the definitions of resistance profiles, and (d) the globally expanding XDR/DTR high-risk lineages and their association with transferable resistance mechanisms. IMPLICATION: The evidence presented herein can be used for coordinated epidemiological surveillance and decision making at the European and global level.
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Antibacterianos , Infecciones por Pseudomonas , Humanos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , beta-Lactamasas/genética , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/epidemiología , Infecciones por Pseudomonas/microbiología , Pseudomonas , Pseudomonas aeruginosa/genética , Inhibidores de beta-Lactamasas/uso terapéutico , beta-Lactamas/farmacología , beta-Lactamas/uso terapéutico , Pruebas de Sensibilidad MicrobianaRESUMEN
Accurate susceptibility result of temocillin (TMO) is important for treating infections caused by multidrug-resistant Enterobacterales. This multicenter study aimed to investigate the performance of routine temocillin testing assays against Enterobacterales challenging strains. Forty-seven selected clinical isolates were blindly analyzed by 12 Belgian laboratories using VITEK® 2 (n = 5) and BD Phoenix™ (n = 3) automated systems, ETEST® gradient strip (n = 3), and disk (3 brands) diffusion method (DD; n = 6) for temocillin susceptibility using standardized methodology. Results were interpreted using EUCAST 2023 criteria and compared to the broth microdilution (BMD; Sensititre™ panel) method used as gold standard. Methods' reproducibility was assessed by testing 3 reference strains in triplicate. A total of 702 organism-drug results were obtained against 33 TMO-susceptible and 14 TMO-resistant isolates. Excluding Proteae species (P. mirabilis and M. morganii), the essential agreement rates were excellent (91.5-100%) for all MIC-based methods. The highest category agreement was achieved by ETEST® (97.5%) followed by VITEK® 2 (93.2%), disk diffusion (91.6%), and BD Phoenix™ (88.5%). BD Phoenix™ and paper disk diffusion overcalled resistance (11.5% and 6.8% of major discrepancies, respectively), while ROSCO tablets diffusion and VITEK® 2 generated higher very major discrepancies (7.1% and 4.2% respectively). Inter-assay reproducibility was unsatisfactory using recommended E. coli ATCC 25922 strain but was excellent with E. coli ATCC 35218 and K. pneumoniae ATCC 700603 strains. This interlaboratory study suggests that routine testing methods provide accurate and reproducible TMO categorization results except for Proteae species.
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Antibacterianos , Escherichia coli , Penicilinas , Humanos , Antibacterianos/farmacología , Reproducibilidad de los Resultados , Pruebas de Sensibilidad Microbiana , Klebsiella pneumoniaeRESUMEN
Pseudomonas aeruginosa (Pa) remains among clinically-significant Gram-negative species. The carbapenems are often the last resort for treating infections due to multidrug resistant isolates such as Pa. The carbapenems' efficacy is increasingly compromised by the emergence and the rapid spread of Pa carrying carbapenemases which represent a serious threat to public health. This study aimed to establish the resistance profile and to identify carbapenemase genes in isolates with imipenem resistant phenotypes. Among 134 Pa isolates collected both in the community (46) and hospital (88) from January 2021 to December 2021 in Morocco, 18 (8 were from the community and 10 from the hospital settings) were carbapenem resistant. The identification of these strains has been confirmed using matrix assisted laser desorption ionization-time of flight (MALDI-TOF). The antibiotic susceptibility testing against 16 antibiotics was carried out and interpreted according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing (2021). The worrying antibiotics resistance profiles, which spread to cefiderocol for two isolates, were obtained for all isolates, which were eXtensive Drug Resistance showing highly resistant to all antibiotic categories tested, even to ceftolozane-tazobactam. Colistin (100% susceptible) and cefiderocol (88.88%) were the most active agents against carbapenem-resistant Pa (CRPa). Phenotypic detection by NP-CARBA and NG-CARBA tests of metalloßlactamase (MßL) production was confirmed by PCR amplification and sequencing. Three CRPa isolates coharboring blaVIM-2-blaNDM-1 (two isolates) and blaVIM-2-blaIMP-8 (one isolate) genes were detected. In this study, we describe the coexistence of these MßL genes and the cefiderocol resistance in CRPa strains in Morocco. The alarming antibiotic resistance patterns of all these CRPa isolates and their resistance genes emphasize the importance of antimicrobial susceptibility testing in the choice of antibiotics for treating Pa infections.
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Pseudomonas aeruginosa , beta-Lactamasas , Pseudomonas aeruginosa/genética , Marruecos , beta-Lactamasas/genética , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Carbapenémicos/farmacología , Hospitales , Resistencia a Medicamentos , Pruebas de Sensibilidad Microbiana , CefiderocolRESUMEN
Rapid and recurrent breakthroughs of new SARS-CoV-2 strains (variants) have prompted public health authorities worldwide to set up surveillance networks to monitor the circulation of variants of concern. The use of next-generation sequencing technologies has raised the need for quality control assessment as required in clinical laboratories. The present study is the first to propose a validation guide for SARS-CoV-2 typing using three different NGS methods fulfilling ISO15189 standards. These include the assessment of the risk, specificity, accuracy, reproducibility, and repeatability of the methods. Among the three methods used, two are amplicon-based involving reverse transcription polymerase chain reaction (Artic v3 and Midnight v1) on Oxford Nanopore Technologies while the third one is amplicon-based using reverse complement polymerase chain reaction (Nimagen) on Illumina technology. We found that all methods met the quality requirement (e.g., 100% concordant typing results for accuracy, reproducibility, and repeatability) for SARS-CoV-2 typing in clinical setting. Additionally, the typing results emerging from each of the three sequencing methods were compared using three widely known nomenclatures (WHO, Pangolineage, and Nextclade). They were also compared regarding single nucleotide variations. The outcomes showed that Artic v3 and Nimagen should be privileged for outbreak investigation as they provide higher quality results for samples that do not meet inclusion criteria for analysis in a clinical setting. This study is a first step towards validation of laboratory developed NGS tests in the context of the new European regulation for medical devices and in vitro diagnostics.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reproducibilidad de los Resultados , AcreditaciónRESUMEN
Three soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against the CMY-2 ß-lactamase behaved as inhibitors. The structure of the complex VHH cAbCMY-2(254)/CMY-2 showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes into the catalytic site. The ß-lactamase inhibition pattern followed a mixed profile with a predominant noncompetitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binders. Our study identified a binding site that can be targeted by a new class of ß-lactamase inhibitors designed on the sequence of the paratope. Furthermore, the use of mono- or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enables the development of the first generation of enzyme-linked immunosorbent assay (ELISA) for the detection of CMY-2 produced by CMY-2-expressing bacteria, irrespective of resistotype.
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Anticuerpos de Dominio Único , Animales , Conejos , Medicina de Precisión , beta-Lactamasas/genética , beta-Lactamasas/química , Inhibidores de beta-Lactamasas , Penicilinas , Anticuerpos , EpítoposRESUMEN
OXA-48-producing Enterobacterales have now widely disseminated throughout the world. Several variants have now been reported, differing by just a few amino-acid substitutions or deletions, mostly in the region of the loop ß5-ß6. As OXA-48 hydrolyzes carbapenems but lacks significant expanded-spectrum cephalosporin (ESC) hydrolytic activity, ESCs were suggested as a therapeutic option. Here, we have characterized OXA-517, a natural variant of OXA-48- with an Arg214Lys substitution and a deletion of Ile215 and Glu216 in the ß5-ß6 loop, capable of hydrolyzing at the same time ESC and carbapenems. MICs values of E. coli expressing blaOXA-517 gene revealed reduced susceptibility to carbapenems (similarly to OXA-48) and resistance to ESCs. Steady-state kinetic parameters revealed high catalytic efficiencies for ESCs and carbapenems. The blaOXA-517 gene was located on a ca. 31-kb plasmid identical to the prototypical IncL blaOXA-48-carrying plasmid except for an IS1R-mediated deletion of 30.7-kb in the tra operon. The crystal structure of OXA-517, determined to 1.86 Å resolution, revealed an expanded active site compared to that of OXA-48, which allows for accommodation of the bulky ceftazidime substrate. Our work illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutation/deletion in the ß5-ß6 loop to extend its hydrolysis profile to encompass most ß-lactam substrates.
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Carbapenémicos , Cefalosporinas , Carbapenémicos/farmacología , Escherichia coli/genética , beta-Lactamasas/genética , beta-Lactamasas/química , Ceftazidima , Monobactamas , Antibacterianos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Infections with carbapenem-resistant (CR) Gram-negative (GN) pathogens have increased in many countries worldwide, leaving only few therapeutic options. Cefiderocol (CFDC) is approved in Europe for the treatment of aerobic GN infections in adults with limited treatment options. This study evaluated the in vitro activity of cefiderocol and comparators against multidrug-resistant (MDR) bacteria including meropenem-resistant (MR) or pandrug-resistant (PR) GN clinical isolates from France and Belgium. The minimum inhibitory concentrations (MICs) of CFDC were determined by broth microdilution, using iron-depleted cation-adjusted Mueller-Hinton broth, and were compared to those of 10 last-line antibiotics. The MICs were interpreted according to EUCAST and CLSI breakpoints, and in the absence of species-specific breakpoints, non-species-related pharmacokinetic/pharmacodynamic breakpoints were used. Among the 476 isolates tested, 322 were carbapenemase producers (CP), 58 non-CP-CRs, 52 intrinsically CR, 41 expanded-spectrum cephalosporin resistant and 5 were multi-susceptible. Susceptibility to CFDC was high using EUCAST breakpoints 81%, 99% and 84%, and was even higher using CLSI breakpoints to 93%, 100% and 88% for Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii, respectively. Susceptibility to cefiderocol using non-species-related breakpoints for Stenotrophomonas maltophilia, Achromobacter xylosoxydans and Burkholderia cepacia, was 100%, 100% and 92.3%, respectively. The susceptibility rates were lower with the NDM producers, with values of 48% and 30% using EUCAST breakpoints and 81% and 50% using CLSI breakpoints for Enterobacterales and Acinetobacter spp, respectively. CFDC demonstrated high in vitro susceptibility rates against a wide range of MDR GN pathogens, including MR and PR isolates.
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From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (107, 105 and 103 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.
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COVID-19 , SARS-CoV-2 , Bélgica/epidemiología , COVID-19/diagnóstico , COVID-19/epidemiología , Humanos , Pandemias , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/genéticaRESUMEN
Hypervirulent Klebsiella pneumoniae (hvKp) raised concern worldwide. We studied 22 hvKp clinical invasive isolates referred to the Belgian national reference laboratory between 2014 and 2020. Sixty-four percent of the isolates expressed K2 capsular serotype and belonged to 7 different MLST lineages, while 32% expressed K1 (all belonging to ST23) and were associated with liver abscesses. Primary extra-hepatic infections were reported in 36% and sepsis for 95% of the patients with 30% of deaths. Improved clinical and microbiological diagnostics are required as hvKp may represent an underestimated cause of community-acquired invasive infections in Belgium.
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Infecciones Comunitarias Adquiridas , Infecciones por Klebsiella , Bélgica/epidemiología , Infecciones Comunitarias Adquiridas/epidemiología , Infecciones Comunitarias Adquiridas/microbiología , Humanos , Infecciones por Klebsiella/epidemiología , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae , Tipificación de Secuencias Multilocus , Virulencia , Factores de Virulencia/genéticaRESUMEN
OBJECTIVES: In order to elaborate a new national challenge panel of resistant Gram-negative bacilli and Gram-positive cocci strains for the validation of routine antimicrobial susceptibility testing (AST) methods, an interlaboratory evaluation was organised. METHODS: The results of 12 well-characterised multidrug-resistant strains tested by nine laboratories using local disk diffusion (DD) and automated AST (AUST) methods were compared with the reference broth microdilution method. RESULTS: Overall categorical agreement ranged from 70% to 100% both for DD and AUST and was >90% for all but one strain for all antibiotics. CONCLUSION: Our multicentre AST study showed good reproducibility and the panel can be used as national resistant reference strains for routine AST validation.
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Antiinfecciosos , Bacterias Gramnegativas , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana , Reproducibilidad de los ResultadosRESUMEN
Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-ß-lactamases (MBLs), which inactivate virtually all ß-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a Klebsiella pneumoniae clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime compared to VIM-1. These results were confirmed by steady-state kinetic measurements, where VIM-52 yielded a lower activity toward ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221 to 241), which borders the active site. As Arg 224 and Ser 228 both play an important and interrelated role in enzymatic activity, stability, and substrate specificity for the MBLs, targeted mutagenesis at both positions was performed and further confirmed their crucial role for substrate specificity.
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Antibacterianos , Klebsiella pneumoniae , Antibacterianos/farmacología , Ceftazidima/farmacología , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/genéticaRESUMEN
The current reliable recommended test for coronavirus disease 2019 (COVID-19) diagnosis is quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Rapid antigen test devices could be useful as they are less expensive, faster without the need of specialized laboratories to perform the test. We report the performances of two rapid immunochromatographic antigen testing devices compared with RT-qPCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal samples. We carried out a lateral-flow tests study on 401 nasopharyngeal swab samples from nonduplicated suspected COVID-19 subjects. An equal volume of universal transport medium tubes-containing samples (dilution ratio = 1:15) were added to the manufacturer's extraction buffer solution (dilution ratio = 1:2) and analyzed on BioSpeedia COVID19Speed-Antigen Test and on Abbott Panbio™ COVID-19 Ag Rapid Test, devices. Qualitative results were compared to those obtained by the RT-qPCR (Allplex™ SARS-CoV-2 Assay Seegene). Based on our data, the overall sensitivity for BioSpeedia and Panbio devices was estimated at 65.5% and 75.0%, respectively. The sensitivity was greater for cycle threshold values less than 25 achieving 90.4 and 96.8 for BioSpeedia and Panbio devices, respectively. A perfect specificity of 100.0% was observed for both devices.
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Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/aislamiento & purificación , Antígenos Virales/análisis , Pruebas Diagnósticas de Rutina , Humanos , Nasofaringe/virología , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Sensibilidad y EspecificidadRESUMEN
PURPOSE: Broth microdilution (BMD) stays as the reference testing method for determination of antimicrobial susceptibility testing (AST) to colistin and is considered essential for patient management and for monitoring of colistin resistance. This multicenter study aimed to evaluate the performance of automated systems for colistin AST among Enterobacterales as an alternative for BMD since the majority of laboratories use automated systems as first-line method. METHODS: Twenty colistin resistant (COL-R) including 10 MCR producers and 10 colistin-susceptible (COL-S) Enterobacterales isolates were blindly tested for colistin susceptibility with the routine automated AST systems used by 8 laboratories (3 with BD Phoenix, 3 with Vitek2 and 2 with MicroScan). Additionally, 3 reference strains (E. coli ATCC 25922, E. coli NCTC 13846, and one COL-R mcr-negative K. pneumoniae M/14750) were tested in triplicate by each laboratory. RESULTS AND CONCLUSION: Results were compared with BMD performed at the reference laboratory. BD Phoenix and MicroScan automated AST systems provide accurate and reproducible categorical results for the testing of colistin in Enterobacterales. However, Vitek2 system showed poor performance for the detection of COL-R isolates especially those with MICs close to the susceptibility breakpoint (categorical agreement of 88% and precision categorical agreement of 81%).
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Antibacterianos/farmacología , Automatización de Laboratorios , Colistina/farmacología , Pruebas de Sensibilidad Microbiana , Bélgica , Pruebas Diagnósticas de Rutina , Farmacorresistencia Bacteriana/efectos de los fármacos , Enterobacteriaceae/efectos de los fármacos , HumanosRESUMEN
Objectives: This study evaluated the performance of the Novodiag® CarbaR+ an automated qualitative nucleic acid-based diagnostic assay detecting the blaVIM, blaNDM, blaIMP, blaOXA-48, blaKPC, blaOXA-23, blaOXA-58, blaOXA-24, and ISAba1 associated blaOXA-51 carbapenemase genes and colistin resistance mcr-1 gene from clinical isolates or directly from rectal swabs. Materials and Methods: CarbaR+ was evaluated on 201 clinical isolates and on 100 rectal swabs (80 selected swabs from patients that were evaluated by culture method and/or Xpert Carba-R assay and 20 spiked samples). PCR-sequencing on colonies was considered as the gold standard. Results: The CarbaR+ detected all the variants of the targeted resistance genes (39 blaVIM-, 30 blaNDM-, 20 blaIMP-, 19 blaOXA-48-, 15 blaKPC-, 19 blaOXA-23-, 13 blaOXA-58-, 4 blaOXA-24-, 8 ISAba1-blaOXA-51-, and 3 mcr-1-like genes) with sensitivity and specificity of 98.2% and 99.7%, respectively. On the 80 rectal swabs, 71 CarbaR+ results were fully concordant with the results on selective culture media (66 positive samples with 1 to 3 carbapenemases and 5 negative samples). In eight rectal swabs, CarbaR+ identified additional carbapenemase genes. One false negative result with an Escherichia coli producing-OXA-181 was observed and one CarbaR+ result for OXA-48 was in agreement with Xpert Carba-R assay, without growth on culture media. A concordance of 100% was observed on spiked samples. Conclusions: Novodiag CarbaR+ is a random-access fully automated system that achieves excellent performances for the detection of carbapenemase and/or colistin resistance determinants either from cultured clinical isolates or directly from rectal swabs in 80 minutes.
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Carbapenémicos/farmacología , Colistina/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Humanos , Pruebas de Sensibilidad Microbiana , Sensibilidad y Especificidad , beta-Lactamasas/genéticaRESUMEN
The emergence of the SARS-CoV-2 virus and the exponential growth of COVID-19 cases have created a major crisis for public health systems. The critical identification of contagious asymptomatic carriers requires the isolation of viral nucleic acids, reverse transcription, and amplification by PCR. However, the shortage of specific proprietary reagents or the lack of automated platforms have seriously hampered diagnostic throughput in many countries. Here, we provide a procedure for SARS-CoV-2 detection for diagnostic purposes from clinical samples in the setting of a basic research molecular biology lab. The procedure details the necessary steps for daily analysis of up to 500 clinical samples with a team composed of 12 experienced researchers. The protocol has been designed to rely on widely available reagents and devices, to cope with heterogeneous clinical specimens, to guarantee nucleic acid extraction from very scarce biological material, and to minimize the rate of false-negative results.
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Antimicrobial resistance (AMR) is one of the most prominent public health threats. AMR genes localized on plasmids can be easily transferred between bacterial isolates by horizontal gene transfer, thereby contributing to the spread of AMR. Next-generation sequencing (NGS) technologies are ideal for the detection of AMR genes; however, reliable reconstruction of plasmids is still a challenge due to large repetitive regions. This study proposes a workflow to reconstruct plasmids with NGS data in view of AMR gene localization, i.e., chromosomal or on a plasmid. Whole-genome and plasmid DNA extraction methods were compared, as were assemblies consisting of short reads (Illumina MiSeq), long reads (Oxford Nanopore Technologies) and a combination of both (hybrid). Furthermore, the added value of conjugation of a plasmid to a known host was evaluated. As a case study, an isolate harboring a large, low-copy mcr-1-carrying plasmid (>200 kb) was used. Hybrid assemblies of NGS data obtained from whole-genome DNA extractions of the original isolates resulted in the most complete reconstruction of plasmids. The optimal workflow was successfully applied to multidrug-resistant Salmonella Kentucky isolates, where the transfer of an ESBL-gene-containing fragment from a plasmid to the chromosome was detected. This study highlights a strategy including wet and dry lab parameters that allows accurate plasmid reconstruction, which will contribute to an improved monitoring of circulating plasmids and the assessment of their risk of transfer.
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OBJECTIVE: To assess the prevalence of influenza and respiratory syncytial virus (RSV) in adults hospitalized for a respiratory infection in the winter months and to evaluate the impact of a viral diagnosis on empirical antimicrobial management (antibiotics and antivirals). DESIGN: Observational cohort study. SETTING: Acute-care university hospital. PATIENTS: The study included 963 adult patients hospitalized over a 4-year surveillance period. METHODS: Annual surveillance timelines were defined according to epidemiological criteria related to the circulation of RSV and influenza viruses in the general population. Patients were screened following a severe acute respiratory infection (SARI) case definition at the emergency department and were enrolled for molecular assay targeting influenza/RSV viruses after oral informed consent. Epidemiological and clinical data were recorded prospectively, microbiological investigations, antimicrobial management, and outcome data were reviewed retrospectively. RESULTS: An influenza or RSV virus was documented in 316 of 963 patients (33%). Optimization of antimicrobial management (AM) was achieved in 162 of 265 patients (61%) with a positive viral diagnosis and no bacterial infection at admission (AM treatment not initiated, n = 111; discontinued, n = 51). In contrast, only 128 of 462 patients (28%) with negative microbiological investigations did not have AM treatment initiated (n = 116) or had such treatment discontinued (n = 12). Early, targeted antiviral treatment was prescribed in 235 of 253 patients (93%) confirmed with influenza. Epidemiological, clinical, and outcome data were similar in both groups. CONCLUSION: Epidemiological surveillance associated with influenza/RSV molecular diagnosis in adults hospitalized for severe winter respiratory infections dramatically enhanced antimicrobial management.
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Programas de Optimización del Uso de los Antimicrobianos , Gripe Humana , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Infecciones del Sistema Respiratorio , Adulto , Hospitalización , Hospitales , Humanos , Lactante , Gripe Humana/diagnóstico , Gripe Humana/tratamiento farmacológico , Gripe Humana/epidemiología , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones del Sistema Respiratorio/epidemiología , Estudios RetrospectivosRESUMEN
In Enterobacterales, the most common carbapenemases are Ambler's class A (KPC-like), class B (NDM-, VIM- or IMP-like) or class D (OXA-48-like) enzymes. This study describes the characterization of twenty-four OXA-23 or OXA-58 producing-Proteus mirabilis isolates recovered from human and veterinary samples from France and Belgium. Twenty-two P. mirabilis isolates producing either OXA-23 (n = 21) or OXA-58 (n = 1), collected between 2013 and 2018, as well as 2 reference strains isolated in 1996 and 2015 were fully sequenced. Phylogenetic analysis revealed that 22 of the 24 isolates, including the isolate from 1996, belonged to a single lineage that has disseminated in humans and animals over a long period of time. The blaOXA-23 gene was located on the chromosome and was part of a composite transposon, Tn6703, bracketed by two copies of IS15∆II. Sequencing using Pacbio long read technology of OXA-23-producing P. mirabilis VAC allowed the assembly of a 55.5-kb structure encompassing the blaOXA-23 gene in that isolate. By contrast to the blaOXA-23 genes, the blaOXA-58 gene of P. mirabilis CNR20130297 was identified on a 6-kb plasmid. The acquisition of the blaOXA-58 gene on this plasmid involved XerC-XerD recombinases. Our results suggest that a major clone of OXA-23-producing P. mirabilis is circulating in France and Belgium since 1996.