RESUMO
IVDR regulation represents a major challenge for diagnostic microbiology laboratories. IVDR complicates a broad range of aspects and poses a risk given the high diversity of pathogens (including rare but highly virulent microbes) and the large variety of samples submitted for analysis. The regular emergence of new pathogens (including Echovirus E-11, Adenovirus 41, Monkeypox virus, Alongshan virus, and Enterovirus D68, as recent examples in Europe in the post SARS-CoV-2 era) is another factor that makes IVDR regulation risky, because its detrimental effect on production of in-house tests will negatively impact knowledge and expertise in the development of new diagnostic tests. Moreover, such regulations negatively impact the availability of diagnostic tests, especially for neglected pathogens, and has a detrimental effect on the overall costs of the tests. The increased regulatory burden of IVDR may thereby pose an important risk for public health. Taken together, it will have a negative impact on the financial balance of diagnostic microbiology laboratories (especially small ones). The already-high standards of quality management of all ISO-accredited and Swissmedic-authorized laboratories render IVDR law of little value, at least in Switzerland, while tremendously increasing the regulatory burden and associated costs. Eventually, patients will need to pay for diagnostic assays outside of the framework of their insurance in order to obtain a proper diagnostic assessment, which may result in social inequity. Thus, based on the risk assessment outlined above, the coordinated commission for clinical microbiology proposes adjusting the IvDO ordinance by (i) introducing an obligation to be ISO 15189 accredited and (ii) not implementing the IvDO 2028 milestone.
RESUMO
OBJECTIVES: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it. METHODS: For baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared. RESULTS: At baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2-25]), reproducibility between technical replicates (range = [55%-86%]), and measurement error (range = [147 parts per million (ppm)-588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values <0.001, paired Wilcoxon test); for six out of ten devices, which detected a low number of marker masses, the number of detected marker masses increased (p-values <0.001, paired Wilcoxon test). DISCUSSION: We have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.
Assuntos
Bactérias , Laboratórios , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Reprodutibilidade dos Testes , Fluxo de TrabalhoRESUMO
Facing multidrug resistant (MDR) bacterial pathogens is one of the most important challenges for our society. The spread of highly virulent and resistant pathogens can be described using molecular typing technologies; in particular, whole genome sequencing (WGS) data can be used for molecular typing purposes with high resolution. WGS data analysis can explain the spatiotemporal patterns of pathogen transmission. However, the transmission between compartments (human, animal, food, environment) is very complex. Interoperable and curated metadata are a key requirement for fully understanding this complexity. In addition, high quality sequence data are a key element between centres using WGS data for diagnostic and epidemiological applications. We aim to describe steps to improve WGS data analysis and to implement a molecular surveillance platform allowing integration of high resolution WGS typing data and epidemiological data.
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Bactérias/patogenicidade , Genoma Bacteriano/genética , Epidemiologia Molecular , Tipagem Molecular , Sequenciamento Completo do Genoma/métodos , Fluxo de Trabalho , Bactérias/genética , Surtos de Doenças , Resistência Microbiana a Medicamentos , Humanos , Epidemiologia Molecular/organização & administração , Tipagem Molecular/métodos , Vigilância da População/métodos , Suíça , Sequenciamento Completo do Genoma/normasRESUMO
The aims of the present study were to characterize the mechanisms of resistance to fluoroquinolones, macrolides, and imipenem in Haemophilus influenzae, to assess the extent of the AcrAB-TolC-mediated resistance, and to define a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae by using whole-genome sequencing. Four amino acid substitutions in GyrA (at Ser84 and Asp88), ParC (at Ser84), and ParE (at Asp420) were found to be closely associated to the MICs. We did not find any amino acid substitution surrounding the three highly conserved amino acid motifs in PBP3 related to imipenem resistance. All the isolates possessed the ermB gene. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the MIC of imipenem by twofold for FQR-6 and fourfold for GE47 and GE88 strains. For erythromycin, the MICs were decreased by twofold. We found that the six FQR isolates were clustered in two groups. The number of different loci within FQR-1_FQR-3_FQR-5 cluster was 6, while FQR-2 and FQR-4 differed for 21 loci. FQR-1_FQR-3_FQR-5 and FQR-2_FQR-4 clusters were distant among each other and compared to 19 genomes downloaded from NCBI, to 8 strains heteroresistant to imipenem, and to 4 strains monoresistant to ciprofloxacin isolated in Denmark. We confirmed that specific amino acid substitutions in GyrA, ParC, and ParE are implicated in quinolone resistance. Additionally, the degree of resistance is related to the number of these amino acid substitutions. We provide robust evidence that drug efflux is one of the substantial mechanisms of imipenem and erythromycin resistance in H. influenzae.
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Fluoroquinolonas/farmacologia , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Imipenem/farmacologia , Macrolídeos/farmacologia , Adulto , Idoso , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feminino , Genoma Bacteriano , Genômica/métodos , Haemophilus influenzae/classificação , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Mutação , Sorogrupo , Sequenciamento Completo do GenomaRESUMO
BACKGROUND The degree of bacterial contamination of stethoscopes can vary significantly following a physical examination. OBJECTIVE To conduct a prospective study to investigate the impact of various environmental and patient characteristics on stethoscope contamination. METHODS Following a standardized examination, the levels of bacterial contamination of 4 regions of the physicians' hands and 2 sections of the stethoscopes, and the presence of different pathogenic bacteria, were assessed. Predictors of heavy stethoscope contamination were identified through multivariate logistic regression. RESULTS In total, 392 surfaces were sampled following examination of 56 patients. The microorganisms most frequently recovered from hands and stethoscopes were Enterococcus spp. (29% and 20%, respectively) and Enterobacteriaceae (16% and 7%, respectively). Staphylococcus aureus (either methicillin susceptible or resistant), extended-spectrum ß-lactamase-producing Enterobacteriaceae, and Acinetobacter baumannii were recovered from 4%-9% of the samples from either hands or stethoscopes. There was a correlation between the likelihood of recovering these pathogens from the stethoscopes vs from the physicians' hands (ρ=0.79; P=.04). The level of patient's skin contamination was an independent predictor of contamination of the stethoscope diaphragm (adjusted odds ratio [aOR], 1.001; P=.007) and tube (aOR, 1.001; P=.003). Male sex (aOR, 28.24; P=.01) and reception of a bed bath (aOR, 7.52; P=.048) were also independently associated with heavy tube contamination. CONCLUSIONS Stethoscope contamination following a single physical examination is not negligible and is associated with the level of contamination of the patient's skin. Prevention of pathogen dissemination is needed. Infect Control Hosp Epidemiol 2016;37:673-679.
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Contaminação de Equipamentos , Exame Físico/efeitos adversos , Estetoscópios/microbiologia , Acinetobacter baumannii , Idoso , Carga Bacteriana , Enterobacteriaceae , Enterococcus , Contaminação de Equipamentos/estatística & dados numéricos , Feminino , Mãos/microbiologia , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina , Pessoa de Meia-Idade , Estudos Prospectivos , Gestão de Riscos , Staphylococcus aureusRESUMO
BACKGROUND: Identification of unexpected taxa in 16S rRNA surveys of low-density microbiota, diluted mock communities and cultures demonstrated that a variable fraction of sequence reads originated from exogenous DNA. The sources of these contaminants are reagents used in DNA extraction, PCR, and next-generation sequencing library preparation, and human (skin, oral and respiratory) microbiota from the investigators. RESULTS: For in silico removal of reagent contaminants, a pipeline was used which combines the relative abundance of operational taxonomic units (OTUs) in V3-4 16S rRNA gene amplicon datasets with bacterial DNA quantification based on qPCR targeting of the V3 segment of the 16S rRNA gene. Serially diluted cultures of Escherichia coli and Staphylococcus aureus were used for 16S rDNA profiling, and DNA from each of these species was used as a qPCR standard. OTUs assigned to Escherichia or Staphylococcus were virtually unaffected by the decontamination procedure, whereas OTUs from Pseudomonas, which is a major reagent contaminant, were completely or nearly completely removed. The decontamination procedure also attenuated the trend of increase in OTU richness in serially diluted cultures. CONCLUSIONS: Removal of contaminant sequences derived from reagents based on use of qPCR data may improve taxonomic representation in samples with low DNA concentration. Using the described pipeline, OTUs derived from cross-contamination of negative extraction controls were not recognized as contaminants and not removed from the sample dataset.
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Bactérias/classificação , Biologia Computacional/métodos , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , Bactérias/genética , Carga Bacteriana , Análise por Conglomerados , Simulação por Computador , Contaminação por DNA , DNA Bacteriano/genética , DNA Ribossômico/genética , Humanos , Microbiota , Análise de Sequência de DNARESUMO
BACKGROUND: Streptococcus mutans biofilms are considered as primary causative agents of dental caries. Photodynamic antimicrobial chemotherapy (PACT) has been recently proposed as a strategy for inactivating dental biofilms. This study aimed to investigate the effect of blue light-activated curcumin on S. mutans viability and to explore its potential as a new anti-caries therapeutic agent. The effect of different concentrations and incubation times of photo-activated curcumin on the survival of S. mutans in planktonic and biofilm models of growth was assessed by flow cytometry. METHODS: Streptococcus mutans in planktonic suspensions or biofilms formed on hydroxyapatite disks were incubated for 5 or 10min with curcumin prior to blue light activation. Bacteria were labeled with SYTO 9 and propidium iodide before viability was assessed by flow cytometry. Results were statistically analyzed using one-way ANOVA and Tukey multiple comparison intervals (α=0.05). RESULTS: For planktonic cultures, 0.2µM of light-activated curcumin significantly reduced S. mutans viability (p<0.05). For biofilm cultures, light-activated curcumin at concentration of 40-60µM only suppressed viability by 50% (p<0.05). Independently of the mode of growth, incubation time has no significant effect on PACT efficiency. CONCLUSION: This study indicates that blue light-activated curcumin can efficiently inactivate planktonic cultures of S. mutans whereas biofilms were more resistant to treatment. Flow cytometry allowed the detection of bacteria with damaged membranes that were unable to replicate and grow after cell sorting. Further studies seem warranted to optimize the efficacy of light-activated curcumin against S. mutans biofilms.
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Curcumina/efeitos da radiação , Citometria de Fluxo/métodos , Estimulação Luminosa/métodos , Fotoquimioterapia/métodos , Streptococcus mutans/citologia , Streptococcus mutans/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cor , Luz , Fármacos Fotossensibilizantes/efeitos da radiação , Doses de Radiação , Streptococcus mutans/fisiologiaRESUMO
OBJECTIVE: Determine the prevalence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-PE) contamination of food and colonization of food handlers in a hospital kitchen and compare retrieved ESBL-PE strains with patient isolates. DESIGN: Cross-sectional study. SETTING: A 2,200-bed tertiary care university hospital in Switzerland. PARTICIPANTS: Food handlers. METHODS: Raw and prepared food samples were obtained from the hospital kitchen, with a comparator group from local supermarkets. Fecal samples collected from food handlers and selectively pre-enriched homogenized food samples were inoculated onto selective chromogenic media. Phenotypic confirmation of ESBL production was performed using the double disk method. Representative ESBL-PE were characterized using polymerase chain reaction (PCR) and sequencing for blaCTX-M, blaSHV, and blaTEM genes, and Escherichia coli strains were typed using phylotyping, repetitive element palindromic PCR, and multilocus sequence typing. Meat samples were screened for antibiotic residues using liquid chromatography time-of-flight mass spectrometry. RESULTS: Sixty (92%) of the raw chicken samples were ESBL-PE positive, including 30 (86%) of the hospital samples and all supermarket samples. No egg, beef, rabbit, or cooked chicken samples were ESBL-PE positive. No antibiotic residues were detected. Six (6.5%) of 93 food handlers were ESBL-PE carriers. ESBL-PE strains from chicken meat more commonly possessed blaCTX-M-1 and blaCTX-M-2, whereas blaCTX-M-14 and blaCTX-M-15 were predominant among strains of human origin. There was partial overlap in the sequence type of E. coli strains of chicken and human origin. No E. coli ST131 strains or blaCTX-M-15 genes were isolated from meat. CONCLUSIONS: Although there is significant ESBL-PE contamination of delivered chicken meat, current preventive strategies minimize risks to food handlers, hospital staff, and patients.
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Enterobacteriaceae/isolamento & purificação , Microbiologia de Alimentos , Serviço Hospitalar de Nutrição , beta-Lactamases/biossíntese , Estudos Transversais , Resíduos de Drogas/análise , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Genes Bacterianos , Hospitais Universitários , Humanos , Reação em Cadeia da Polimerase , Medição de Risco , SuíçaRESUMO
OBJECTIVE: To obtain an unbiased estimate of the excess hospital length of stay (LOS) and cost attributable to extended-spectrum ß-lactamase (ESBL) positivity in bloodstream infections (BSIs) due to Enterobacteriaceae. DESIGN: Retrospective cohort study. SETTING: A 2,200-bed academic medical center in Geneva, Switzerland. PATIENTS: Patients admitted during 2009. METHODS: We used multistate modeling and Cox proportional hazards models to determine the excess LOS and adjusted end-of-LOS hazard ratio (HR) for ESBL-positive and ESBL-negative BSI. We estimated economic burden as the product of excess LOS and average bed-day cost. Patient-level accounting data provided a complementary analysis of economic burden. A predictive model was fitted to national surveillance data. RESULTS: Thirty ESBL-positive and 96 ESBL-negative BSI cases were included. The excess LOS attributable to ESBL-positive and ESBL-negative BSI was 9.4 (95% confidence interval [CI], 0.4-18.4) and 2.6 (95% CI, 0.7-5.9) days, respectively. ESBL positivity was therefore associated with 6.8 excess days and CHF 9,473 per BSI. The adjusted end-of-LOS HRs for ESBL-positive and ESBL-negative BSI were 0.62 (95% CI, 0.43-0.89) and 0.90 (95% CI, 0.74-1.10), respectively. After reimbursement, the average financial loss per acute care episode in ESBL-positive BSI, ESBL-negative BSI, and control cohorts was CHF 48,674, 48,131, and 13,532, respectively. Our predictive model estimated that the nationwide cost of third-generation cephalosporin resistance would increase from CHF 2,084,000 in 2010 to CHF 3,526,000 in 2015. CONCLUSIONS: This is the first hospital-wide analysis of excess LOS attributable to ESBL positivity determined using multistate modeling to avoid time-dependent bias. These results may inform health-economic evaluations of interventions targeting ESBL control.
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Bacteriemia/economia , Infecção Hospitalar/economia , Infecções por Enterobacteriaceae/economia , Enterobacteriaceae/enzimologia , Tempo de Internação/economia , beta-Lactamases/biossíntese , Idoso , Bacteriemia/microbiologia , Intervalos de Confiança , Efeitos Psicossociais da Doença , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/epidemiologia , Infecções por Enterobacteriaceae/microbiologia , Feminino , Previsões , Hospitais Universitários , Humanos , Tempo de Internação/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Distribuição por Sexo , SuíçaRESUMO
Molecular biology has not yet fully reached its ambitious goals in clinical bacteriology. Notwithstanding the tremendous technical challenges, the detection of nucleic acids directly from the blood of septic patients has not been shown cost-effective or even clinically relevant. Yet the potential for rapid molecular detection of circulating DNA (DNAemia) coupled to an educated antimicrobial drug adaptation has been repetitively advocated as a predicted breakthrough. Why do we still remain in such uncertainty?
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Custos de Cuidados de Saúde , Modelos Teóricos , Reação em Cadeia da Polimerase/economia , Sepse/economia , Sepse/mortalidade , HumanosRESUMO
Bacterial identification relies primarily on culture-based methodologies requiring 24 h for isolation and an additional 24 to 48 h for species identification. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to the problem of bacterial species identification. We evaluated two MALDI-TOF MS systems with 720 consecutively isolated bacterial colonies under routine clinical laboratory conditions. Isolates were analyzed in parallel on both devices, using the manufacturers' default recommendations. We compared MS with conventional biochemical test system identifications. Discordant results were resolved with "gold standard" 16S rRNA gene sequencing. The first MS system (Bruker) gave high-confidence identifications for 680 isolates, of which 674 (99.1%) were correct; the second MS system (Shimadzu) gave high-confidence identifications for 639 isolates, of which 635 (99.4%) were correct. Had MS been used for initial testing and biochemical identification used only in the absence of high-confidence MS identifications, the laboratory would have saved approximately US$5 per isolate in marginal costs and reduced average turnaround time by more than an 8-h shift, with no loss in accuracy. Our data suggest that implementation of MS as a first test strategy for one-step species identification would improve timeliness and reduce isolate identification costs in clinical bacteriology laboratories now.
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Bactérias/classificação , Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Bactérias/química , Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Técnicas de Tipagem Bacteriana/métodos , Humanos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economia , Fatores de TempoRESUMO
Microarray comparative genomic hybridisation (aCGH) provides an estimate of the relative abundance of genomic DNA (gDNA) taken from comparator and reference organisms by hybridisation to a microarray containing probes that represent sequences from the reference organism. The experimental method is used in a number of biological applications, including the detection of human chromosomal aberrations, and in comparative genomic analysis of bacterial strains, but optimisation of the analysis is desirable in each problem domain.We present a method for analysis of bacterial aCGH data that encodes spatial information from the reference genome in a hidden Markov model. This technique is the first such method to be validated in comparisons of sequenced bacteria that diverge at the strain and at the genus level: Pectobacterium atrosepticum SCRI1043 (Pba1043) and Dickeya dadantii 3937 (Dda3937); and Lactococcus lactis subsp. lactis IL1403 and L. lactis subsp. cremoris MG1363. In all cases our method is found to outperform common and widely used aCGH analysis methods that do not incorporate spatial information. This analysis is applied to comparisons between commercially important plant pathogenic soft-rotting enterobacteria (SRE) Pba1043, P. atrosepticum SCRI1039, P. carotovorum 193, and Dda3937.Our analysis indicates that it should not be assumed that hybridisation strength is a reliable proxy for sequence identity in aCGH experiments, and robustly extends the applicability of aCGH to bacterial comparisons at the genus level. Our results in the SRE further provide evidence for a dynamic, plastic 'accessory' genome, revealing major genomic islands encoding gene products that provide insight into, and may play a direct role in determining, variation amongst the SRE in terms of their environmental survival, host range and aetiology, such as phytotoxin synthesis, multidrug resistance, and nitrogen fixation.
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Hibridização Genômica Comparativa/métodos , Enterobacteriaceae/genética , Genoma Bacteriano , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Doenças das Plantas/microbiologia , Análise por Conglomerados , Simulação por Computador , Transferência Genética Horizontal , Variação Genética , Ilhas Genômicas , Cadeias de Markov , Modelos Estatísticos , Distribuição Normal , Reprodutibilidade dos TestesRESUMO
A high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) carriage at hospital readmission among previous MRSA carriers warrants screening and preemptive isolation precautions. The replacement of culture on chromogenic agar with rapid quantitative polymerase chain reaction for readmission screening reduces the number of unnecessary preemptive isolation-days by 54% (from 6.88 to 3.14 isolation-days) and related costs by 45% (from US dollars 113.2 to US dollars 62.1) for patients who test negative for MRSA.
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Portador Sadio/diagnóstico , Programas de Rastreamento/economia , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Isolamento de Pacientes , Reação em Cadeia da Polimerase/métodos , Infecções Estafilocócicas/diagnóstico , Técnicas Bacteriológicas/economia , Técnicas Bacteriológicas/métodos , Portador Sadio/microbiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/prevenção & controle , Humanos , Staphylococcus aureus Resistente à Meticilina/genética , Isolamento de Pacientes/economia , Reação em Cadeia da Polimerase/economia , Infecções Estafilocócicas/microbiologiaRESUMO
Novel high-throughput DNA sequencing technologies allow researchers to characterize a bacterial genome during a single experiment and at a moderate cost. However, the increase in sequencing throughput that is allowed by using such platforms is obtained at the expense of individual sequence read length, which must be assembled into longer contigs to be exploitable. This study focuses on the Illumina sequencing platform that produces millions of very short sequences that are 35 bases in length. We propose a de novo assembler software that is dedicated to process such data. Based on a classical overlap graph representation and on the detection of potentially spurious reads, our software generates a set of accurate contigs of several kilobases that cover most of the bacterial genome. The assembly results were validated by comparing data sets that were obtained experimentally for Staphylococcus aureus strain MW2 and Helicobacter acinonychis strain Sheeba with that of their published genomes acquired by conventional sequencing of 1.5- to 3.0-kb fragments. We also provide indications that the broad coverage achieved by high-throughput sequencing might allow for the detection of clonal polymorphisms in the set of DNA molecules being sequenced.