RESUMO
The clinical microbiology laboratory is capable of identifying microorganisms in clinical specimens faster and more accurately than ever before. At face value, this should enable patient care providers to make better-informed decisions and target antimicrobial therapies to deliver individualized care. Ironically, more complete and specific reporting of microorganisms isolated from specimens may result in overtreatment based on the presence of a pathogen, even in the absence of clear signs of clinical infection. This conundrum calls into question the role of the laboratory in contributing to care through selective or "exception" reporting whereby some results are selectively withheld when there is a low probability that laboratory findings correlate with the clinical infection. In a recent article published in the Journal of Clinical Microbiology, Bloomfield et al. (J Clin Microbiol 62:e00342-24, 2024, https://doi.org/10.1128/jcm.00342-24) examine the impact and safety of an exception reporting strategy applied to wound swab specimens. Canonical pathogens associated with skin and soft tissue infections including S. aureus and beta-hemolytic streptococci are withheld from the laboratory report if certain patient criteria are met that would put them at low risk of adverse outcomes if untreated, or if treated with guideline-recommended empiric therapy. Their central finding was an approximately 50% reduction in post-laboratory report antibiotic initiation without adverse events or increased 30-day admission rate (indicative of infection-related complications, e.g., disseminated disease). While effectively achieving their goal, the premise of exception reporting and other modified reporting strategies raises questions about the potential risk of underreporting and how to ensure that the message is being interpreted, and acted upon, by care providers as was intended by the laboratory.
Assuntos
Técnicas Microbiológicas , Humanos , Técnicas Microbiológicas/normas , Infecções dos Tecidos Moles/microbiologia , Infecções dos Tecidos Moles/tratamento farmacológico , Gestão de Antimicrobianos , Antibacterianos/uso terapêuticoRESUMO
In the execution of its legislated responsibilities, the United States Food and Drug Administration commonly refers to standard test methods detailed in the United States Pharmacopeia (USP). Microbiological test methods (contained in general chapters) are listed in chapters <51> to <80> with details regarded as enforceable where referenced as a test method. USP <61> "Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests" is a globally harmonized chapter that has been successfully employed for the enumeration of microorganisms recoverable from nonsterile finished drug products. The content of USP <61> is not always scientifically principled nor emphatically understood by all pharmaceutical microbiologists. Consequently, misunderstanding and misapplication of USP <61> may result in analyses and assessments of microbiological quality that are flawed or erroneous. In this article, clarification is provided to assist the pharmaceutical microbiologist in the appropriate and intended use of USP <61>, including provision of details not always commonly known or understood.
Assuntos
Contaminação de Medicamentos , Farmacopeias como Assunto , Farmacopeias como Assunto/normas , Contaminação de Medicamentos/prevenção & controle , Estados Unidos , United States Food and Drug Administration/normas , Técnicas Microbiológicas/normas , Técnicas Microbiológicas/métodos , Contagem de Colônia Microbiana/normas , Preparações Farmacêuticas/normas , Preparações Farmacêuticas/análiseRESUMO
Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.
Assuntos
Ensaios de Triagem em Larga Escala , Técnicas Microbiológicas , Técnicas de Diagnóstico Molecular , Hibridização de Ácido Nucleico , RNA , Sondas de DNA , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/normas , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , RNA/análise , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Viroses/diagnóstico , Infecções Bacterianas/diagnóstico , Linhagem Celular Tumoral , HumanosRESUMO
Campylobacter spp. are considered the most frequent cause of acute gastroenteritis worldwide. However, outside high-income countries, its burden is poorly understood. Limited published data suggest that Campylobacter prevalence in low- and middle-income countries is high, but their reservoirs and age distribution are different. Culturing Campylobacter is expensive due to laboratory equipment and supplies needed to grow the bacterium (e.g., selective culture media, microaerophilic atmosphere, and a 42°C incubator). These requirements limit the diagnostic capacity of clinical laboratories in many resource-poor regions, leading to significant underdiagnosis and underreporting of isolation of the pathogen. CAMPYAIR, a newly developed selective differential medium, permits Campylobacter isolation without the need for microaerophilic incubation. The medium is supplemented with antibiotics to allow Campylobacter isolation in complex matrices such as human feces. The present study aims to evaluate the ability of the medium to recover Campylobacter from routine clinical samples. A total of 191 human stool samples were used to compare the ability of CAMPYAIR (aerobic incubation) and a commercial Campylobacter medium (CASA, microaerophilic incubation) to recover Campylobacter. All Campylobacter isolates were then identified by MALDI-TOF MS. CAMPYAIR showed sensitivity and specificity values of 87.5% (95% CI 47.4%-99.7%) and 100% (95% CI 98%-100%), respectively. The positive predictive value of CAMPYAIR was 100% and its negative predictive value was 99.5% (95% CI 96.7%-99.9%); Kappa Cohen coefficient was 0.93 (95% CI 0.79-1.0). The high diagnostic performance and low technical requirements of the CAMPYAIR medium could permit Campylobacter culture in countries with limited resources.
Assuntos
Infecções por Campylobacter , Campylobacter , Meios de Cultura , Técnicas Microbiológicas , Meios de Cultura/normas , Aerobiose , Campylobacter/classificação , Campylobacter/crescimento & desenvolvimento , Campylobacter/isolamento & purificação , Infecções por Campylobacter/diagnóstico , Infecções por Campylobacter/microbiologia , Fezes/microbiologia , Valor Preditivo dos Testes , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/normasRESUMO
Diagnosis of bone and joint infections (BJI) relies on microbiological culture which has a long turnaround time and is challenging for certain bacterial species. Rapid molecular methods may alleviate these obstacles. Here, we investigate the diagnostic performance of IS-pro, a broad-scope molecular technique that can detect and identify most bacteria to the species level. IS-pro additionally informs on the amount of human DNA present in a sample, as a measure of leukocyte levels. This test can be performed in 4 h with standard laboratory equipment. Residual material of 591 synovial fluid samples derived from native and prosthetic joints from patients suspected of joint infections that were sent for routine diagnostics was collected and subjected to the IS-pro test. Bacterial species identification as well as bacterial load and human DNA load outcomes of IS-pro were compared to those of culture. At sample level, percent positive agreement (PPA) between IS-pro and culture was 90.6% (95% CI 85.7- to 94%) and negative percent agreement (NPA) was 87.7% (95% CI 84.1 to 90.6%). At species level PPA was 80% (95% CI 74.3 to 84.7%). IS-pro yielded 83 extra bacterial detections over culture for which we found supporting evidence for true positivity in 40% of the extra detections. Missed detections by IS-pro were mostly related to common skin species in low abundance. Bacterial and human DNA signals measured by IS-pro were comparable to bacterial loads and leukocyte counts reported by routine diagnostics. We conclude that IS-pro showed an excellent performance for fast diagnostics of bacterial BJI.
Assuntos
Artrite Infecciosa , Técnicas Microbiológicas , Infecções Relacionadas à Prótese , Humanos , Artrite Infecciosa/diagnóstico , Artrite Infecciosa/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Infecções Relacionadas à Prótese/diagnóstico , Infecções Relacionadas à Prótese/microbiologia , Testes de Diagnóstico Rápido/instrumentação , Testes de Diagnóstico Rápido/normas , Líquido Sinovial/citologia , Líquido Sinovial/microbiologia , Sensibilidade e Especificidade , DNA/genética , Técnicas Microbiológicas/instrumentação , Técnicas Microbiológicas/normasRESUMO
BD Phoenix CPO Detect panels can identify and classify carbapenemase-producing organisms (CPOs) simultaneously with antimicrobial susceptibility testing (AST) for Gram-negative bacteria. Detection and classification of carbapenemase producers were performed using the BD Phoenix CPO Detect panels NMIC/ID-441 for Enterobacterales, NMIC/ID-442 for nonfermenting bacteria, and NMIC-440 for both. The results were compared with those obtained using comparator methods. A total of 133 strains (32 Klebsiella pneumoniae, 37 Enterobacter cloacae complex, 33 Pseudomonas aeruginosa, and 31 Acinetobacter baumannii complex strains), including 60 carbapenemase producers (54 imipenemases [IMPs] and 6 OXA type), were analyzed. Using panels NMIC-440 and NMIC/ID-441 or NMIC/ID-442, all 54 IMP producers were accurately identified as CPOs (positive percent agreement [PPA], 100.0%; 54/54). Among the 54 IMP producers identified as CPOs using panels NMIC-440 and NMIC/ID-441, 12 and 14 Enterobacterales were not resistant to carbapenem, respectively. Among all 54 IMP producers, 48 (88.9%; 48/54) were correctly classified as Ambler class B using panel NMIC-440. Using panels NMIC-440 and NMIC/ID-442, all four OXA-23-like carbapenemase-producing A. baumannii complex strains (100.0%, 4/4) were correctly identified as CPOs, and three (75.0%, 3/4) were precisely classified as class D using panel NMIC-440. Both carbapenemase producers harboring the blaISAba1-OXA-51-like gene were incorrectly identified as non-CPOs using panels NMIC-440 and NMIC/ID-442. For detecting carbapenemase producers, the overall PPA and negative percent agreement (NPA) between panel NMIC-440 and the comparator methods were 96.7% (58/60) and 71.2% (52/73), respectively, and the PPA and NPA between panels NMIC/ID-441 or NMIC/ID-442 and the comparator methods were 96.7% (58/60) and 74.0% (54/73), respectively. BD Phoenix CPO Detect panels can successfully screen carbapenemase producers, particularly IMP producers, regardless of the presence of carbapenem resistance and can be beneficial in routine AST workflows. IMPORTANCE Simple and efficient screening methods of detecting carbapenemase producers are required. BD Phoenix CPO Detect panels effectively screened carbapenemase producers, particularly IMP producers, with a high overall PPA. As the panels enable automatic screening for carbapenemase producers simultaneously with AST, the workflow from AST to confirmatory testing for carbapenemase production can be shortened. In addition, because carbapenem resistance varies among carbapenemase producers, the BD Phoenix CPO Detect panels, which can screen carbapenemase producers regardless of carbapenem susceptibility, can contribute to the accurate detection of carbapenemase producers. Our results report that these panels can help streamline the AST workflow before confirmatory testing for carbapenemase production in routine microbiological tests.
Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Técnicas Microbiológicas , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Testes de Sensibilidade Microbiana , Enterobacteriáceas Resistentes a Carbapenêmicos/classificação , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/normas , Reprodutibilidade dos Testes , Especificidade da EspécieRESUMO
Understanding the actual distribution of different Legionella species in water networks would help prevent outbreaks. Culture investigations followed by serological agglutination tests, with poly/monovalent antisera, still represent the gold standard for isolation and identification of Legionella strains. However, also MALDI-TOF and mip-gene sequencing are currently used. This study was conducted to genetically correlate strains of Legionella non pneumophila (L-np) isolated during environmental surveillance comparing different molecular techniques. Overall, 346 water samples were collected from the water system of four pavilions located in a hospital of the Apulia Region of Italy. Strains isolated from the samples were then identified by serological tests, MALDI-TOF, and mip-gene sequencing. Overall, 24.9% of water samples were positive for Legionella, among which the majority were Legionella pneumophila (Lpn) 1 (52.3%), followed by Lpn2-15 (20.9%), L-np (17.4%), Lpn1 + Lpn2-15 (7.1%), and L-np + Lpn1 (2.3%). Initially, L-np strains were identified as L. bozemanii by monovalent antiserum, while MALDI-TOF and mip-gene sequencing assigned them to L. anisa. More cold water than hot water samples were contaminated by L. anisa (p < 0.001). PFGE, RAPD, Rep-PCR, and SAU-PCR were performed to correlate L. anisa strains. Eleven out of 14 strains identified in all four pavilions showed 100% of similarity upon PFGE analysis. RAPD, Rep-PCR, and SAU-PCR showed greater discriminative power than PFGE.
Assuntos
Monitoramento Ambiental , Hospitais , Microbiologia da Água , Abastecimento de Água , Monitoramento Ambiental/métodos , Itália , Técnicas Microbiológicas/normas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Legionella/genética , Legionella/isolamento & purificação , Análise de Sequência de DNARESUMO
The rapidity, accuracy, and detection abilities of different laboratory methods (tube agglutination test (SAT), indirect ELISA, fluorescence polarization test (FPA), and blood culture methods) to detect Brucella in the laboratory. The study included 95 patients with documented and 42 patients with suspected brucellosis and 56 healthy control subjects. For the tests, the positive rates of Brucella infection detection in the confirmed group were significantly higher than in group with suspected infection (p<0.01) and in healthy controls (p<0.01). There was no significant difference between indirect ELISA and FPA in detecting antibodies to Brucella in acute (χ2=0.335), subacute (χ2=0.660), and chronic cases (χ2=5.332). Among the detection methods, indirect ELISA showed the highest sensitivity (98.9%), specificity (100%), and Youden index (0.989). The sensitivity and specificity of FPA were 96.8 and 96.4%, respectively. In order to easily and rapidly diagnose brucellosis in clinical practice, a combination of detection methods is recommended, in which Brucella antibodies are screened by FPA and then confirmed by indirect ELISA.
Assuntos
Brucella/isolamento & purificação , Brucelose/diagnóstico , Técnicas Microbiológicas/métodos , Adolescente , Adulto , Idoso , Testes de Aglutinação , Anticorpos Antibacterianos/sangue , Criança , Pré-Escolar , China , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoensaio de Fluorescência por Polarização , Humanos , Lactente , Laboratórios , Ensaio de Proficiência Laboratorial , Masculino , Técnicas Microbiológicas/normas , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto JovemRESUMO
Brain abscesses are often polymicrobial and of unclear primary origin. Here, we compare the use of next-generation sequencing (NGS) technology with classical microbiological diagnostics for identification of clinically relevant microorganisms and describe the microbiome profiling with respect to the primary source of brain abscess. Thirty-six samples from 36 patients, with primary brain abscesses, were subjected to both culture- and 16S/18S rRNA Sanger sequencing-based diagnostics ("standard methods") and compared to a 16S/18S amplicon-based NGS, which were also subjected to a microbiome diversity analyses. Forty-seven species were identified with "standard methods" compared to 96 species with NGS, both confirming and adding to the number of species identified (p < 0.05). The variation of the brain abscess microbiome diversity was not continuous but could be stratified comparing the presumable origin of infection ("dental," "sinus," "disseminated," or "unknown"). Alpha diversity did not differ (p > 0.05) between groups while beta diversity differed significantly (p = 0.003) comparing disseminated vs the other presumable origin of infection. Interesting, clustering was also detected between "dental" and "sinusitis," although not significantly (p = 0.07). Microbiome-based diagnostics can increase sensitivity without losing specificity. The bacterial beta diversity differed between the presumably origin of the brain abscess and might help to clarify the primary source of infection.
Assuntos
Abscesso Encefálico/diagnóstico , Abscesso Encefálico/microbiologia , Técnicas Microbiológicas/métodos , Microbiota/genética , Adolescente , Adulto , Idoso , Criança , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Masculino , Técnicas Microbiológicas/normas , Pessoa de Meia-Idade , Estudos Prospectivos , RNA Ribossômico 16S/genética , RNA Ribossômico 18S/genética , Análise de Sequência de DNARESUMO
Esta Norma Técnica tem por objetivo estabelecer as orientações necessárias para a determinação da presença de bactérias coliformes totais no leite humano ordenhado pasteurizado, visando a garantia da qualidade em Bancos de Leite Humano e sua certificação.
Esta Norma Técnica tiene por objetivo establecer las orientaciones necesarias para determinar la presencia de bacterias coliformes totales en la leche humana pasteurizada, con el fin de garantizar la calidad de los Bancos de Leche Humana y su certificación.
Assuntos
Controle de Qualidade , Técnicas Microbiológicas/normas , Bancos de Leite Humano/normas , Coliformes , Extração de Leite/métodos , Pasteurização/normas , Leite Humano/microbiologiaRESUMO
ABSTRACT: Antibiotic stewardship (ABS) programs intend to improve outcomes of nosocomial infections and to counteract the emergence of further antimicrobial resistances. At the anesthesiologic-neurosurgical intensive care unit (ICU) of the University Medical Center Regensburg (Germany) we implemented a standard operating procedure (SOP) with clear instructions for the preanalytical handling and storage of microbiological samples. We intended to find out whether the instructions given in the SOP led to a higher rate of ideal material being sent to the laboratory and to overall better quality of the received results.We retraced retrospectively all samples taken in cases of suspected pneumonia, urinary tract infection, bloodstream infection, catheter infection associated with a central venous or arterial catheter and ventriculitis due to external ventricular drainage as well as all smears taken for the screening for multi-resistant bacteria within a time period of 1 year before to 1 year after the implementation of the SOP.In the case of suspected pneumonia and urinary tract infection, large amounts of ideal material were sent to the microbiological laboratory. A remarkable improvement after the implementation of the SOP, however, could only be observed regarding the number of urine samples taken from older urinary catheters, which was significantly lower in the "SOP group". Samples for microbiological diagnostics were taken much more often in the daytime, although storage of the probes did not lead to worse results.Concrete instructions enable adequate preanalytical handling of microbiological probes. However, we could not recognize substantial improvements probably due to a preexisting high process quality on the ICU. Microbiological diagnostics during the night shift has to be improved.
Assuntos
Protocolos Clínicos/normas , Unidades de Terapia Intensiva/organização & administração , Técnicas Microbiológicas/normas , Gestão de Antimicrobianos , Infecção Hospitalar/microbiologia , Infecção Hospitalar/prevenção & controle , Alemanha , Humanos , Unidades de Terapia Intensiva/normas , Neurocirurgia , Estudos Retrospectivos , Fatores de TempoRESUMO
INTRODUCTION: Bacteremia is a major cause of morbidity and mortality in hospitalized patients. Predictors of mortality are critical for the management and survival of hospitalized patients. The objective of this study was to determine the factors related to blood culture positivity and the risk factors for mortality in patients whose blood cultures were collected. METHODS: A prospective 2-cohort study (derivation with 784 patients and validation with 380 patients) based on the Pitt bacteremia score for all patients undergoing blood culture collection. The score was obtained from multivariate analysis. The Kaplan-Meier survival curve of the cohort derivation and the cohort validation groups was calculated, and the difference was assessed using a log-rank test. Mortality-related factors were older age, extended hospitalization, > 10% of immature cells in the leukogram, lower mean blood pressure, elevated heart rate, elevated WBC count, and elevated respiratory rate. These continuous variables were dichotomized according to their significance level, and a cut-off limit was created. RESULTS: The area under the ROC curve (AUC) was 0.789. The score was validated in a group of 380 patients who were prospectively evaluated. CONCLUSION: Prolonged hospitalization, body temperature, and elevated heart rate were related to positive blood cultures. The Pitt score can be used to assess the risk of death; however it can be individualized according to the epidemiology of each hospital.
Assuntos
Bacteriemia , Hemocultura , Fungemia , Técnicas Microbiológicas , Adulto , Idoso , Bacteriemia/diagnóstico , Bacteriemia/epidemiologia , Bacteriemia/mortalidade , Brasil/epidemiologia , Estudos de Coortes , Fungemia/diagnóstico , Fungemia/epidemiologia , Fungemia/mortalidade , Humanos , Técnicas Microbiológicas/normas , Pessoa de Meia-Idade , Modelos Estatísticos , Valor Preditivo dos Testes , Estudos Prospectivos , Curva ROC , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de RiscoRESUMO
BACKGROUND: Neonatal sepsis accounts for a large proportion of neonatal deaths in sub-Saharan Africa. The lack of access to diagnostic testing and excessively long turnaround times to result contributes to delays in sepsis identification and initiation of appropriate treatment. This study aims to evaluate the novel InTrays COLOREX Screen and extended-spectrum beta-lactamase for rapid identification of bacterial pathogens causing sepsis and detection of resistance. METHODS: Neonates with suspected sepsis admitted to the Harare Central Hospital were prospectively enrolled. One blood culture was collected and incubated using the BacT/ALERT automated system. Positive blood cultures with potential pathogens identified by Gram stain were inoculated on the InTray COLOREX Screen and extended-spectrum beta-lactamase culture plates. RESULTS: A total of 216 neonates with suspected sepsis were recruited. Pathogens were isolated from blood cultures in 56 (25.9%) neonates of which 54 were Klebsiella pneumoniae. All K. pneumoniae were resistant to ceftriaxone and 53 (98%) were resistant to gentamicin. Sensitivity and specificity for ceftriaxone-resistant K. pneumoniae detection using InTrays were 100%. InTrays results were interpretable as early as 5-10 hours (median 7 hours, interquartile range 7-7) post blood culture positivity enabling rapid identification and notification of result and leading to a 60% reduction in time to result from blood culture collection. CONCLUSIONS: This study shows that the implementation of a novel culture method was feasible and reduced turnaround times for results by 60% compared with standard microbiologic techniques. An impact on patient outcomes and cost-effectiveness of this method needs to be demonstrated.
Assuntos
Carga Bacteriana/métodos , Carga Bacteriana/normas , Cefalosporinas/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/diagnóstico , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/normas , Sepse Neonatal/diagnóstico , Adulto , Hemocultura/métodos , Hemocultura/normas , Resistência às Cefalosporinas , Feminino , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/sangue , Humanos , Recém-Nascido , Masculino , Técnicas Microbiológicas/instrumentação , Mães , Sepse Neonatal/microbiologia , ZimbábueRESUMO
Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Biofilm formation has been implicated in both pharyngeal and dermal GAS infections. In vitro, plate-based assays have shown that several GAS M-types form biofilms, and multiple GAS virulence factors have been linked to biofilm formation. Although the contributions of these plate-based studies have been valuable, most have failed to mimic the host environment, with many studies utilising abiotic surfaces. GAS is a human specific pathogen, and colonisation and subsequent biofilm formation is likely facilitated by distinct interactions with host tissue surfaces. As such, a host cell-GAS model has been optimised to support and grow GAS biofilms of a variety of GAS M-types. Improvements and adjustments to the crystal violet biofilm biomass assay have also been tailored to reproducibly detect delicate GAS biofilms. We propose 72 h as an optimal growth period for yielding detectable biofilm biomass. GAS biofilms formed are robust and durable, and can be reproducibly assessed via staining/washing intensive assays such as crystal violet with the aid of methanol fixation prior to staining. Lastly, SEM imaging of GAS biofilms formed by this model revealed GAS cocci chains arranged into three-dimensional aggregated structures with EPS matrix material. Taken together, we outline an efficacious GAS biofilm pharyngeal cell model that can support long-term GAS biofilm formation, with biofilms formed closely resembling those seen in vivo.
Assuntos
Biofilmes/crescimento & desenvolvimento , Faringe/microbiologia , Streptococcus pyogenes/fisiologia , Calibragem , Técnicas de Cultura de Células/normas , Células Cultivadas , Humanos , Técnicas Microbiológicas/normas , Modelos Biológicos , Faringe/citologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/crescimento & desenvolvimento , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismoRESUMO
Numerous metagenome-wide association studies (MWAS) for urolithiasis have been published, leading to the discovery of potential interactions between the microbiome and urolithiasis. However, questions remain about the reproducibility, applicability and physiological relevance of these data owing to discrepancies in experimental technique and a lack of standardization in the field. One barrier to interpreting MWAS is that experimental biases can be introduced at every step of the experimental pipeline, including sample collection, preservation, storage, processing, sequencing, data analysis and validation. Thus, the introduction of standardized protocols that maintain the flexibility to achieve study-specific objectives is urgently required. To address this need, the first international consortium for microbiome in urinary stone disease - MICROCOSM - was created and consensus panel members were asked to participate in a consensus meeting to develop standardized protocols for microbiome studies if they had published an MWAS on urolithiasis. Study-specific protocols were revised until a consensus was reached. This consensus group generated standardized protocols, which are publicly available via a secure online server, for each step in the typical clinical microbiome-urolithiasis study pipeline. This standardization creates the benchmark for future studies to facilitate consistent interpretation of results and, collectively, to lead to effective interventions to prevent the onset of urolithiasis, and will also be useful for investigators interested in microbiome research in other urological diseases.
Assuntos
Técnicas Microbiológicas/normas , Microbiota , Urolitíase/microbiologia , HumanosRESUMO
BACKGROUND: This study illustrates for the first time the performance (sensitivity and selectivity) of the selective medium BCYEα +AB suggested by the new edition of ISO 11731 for legionella isolation and enumeration. We compared the efficacy of the selective BCYEα +AB medium with that of the highly selective MWY medium. RESULTS: Legionella spp. was detected in 48.2 and 47.1% of the samples by BCYEα +AB and MWY agar, respectively. For optimal detection of Legionella spp., most protocols recommend using selective media to reduce the number of non-Legionella bacteria. Agreement between the two media was 86.7%. CONCLUSIONS: According to the results, both media have a very similar performance and they both have advantages and disadvantages over each other. In AB medium there is the risk of being less selective so more interfering microbiota may grow but in MWY medium there is the risk of being too selective. The low selectivity of the AB medium could be resolved if other treatments are applied after filtration, e.g. acid and/or heat treatment, but it must be taken into account that these treatments still reduce the number of viable Legionella. In conclusion, we recommend using MWY as a selective medium for the detection of Legionella spp. as it is easier discern suspected colonies and facilitate the final Legionella spp.
Assuntos
Ágar/química , Ágar/normas , Meios de Cultura/normas , Água Potável/microbiologia , Hospitais , Legionella/isolamento & purificação , Meios de Cultura/química , Legionella/crescimento & desenvolvimento , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/normas , Microbiologia da ÁguaRESUMO
Globally, it is estimated that there are 2 billion cases of diarrhoeal disease each year, with 525,000 children under the age of 5 years, dying from diarrhoea. This also affects 1 in 5 people in the UK each year. Rapid diagnosis, appropriate treatment and infection control measures are, therefore, particularly important. Currently, Public Health Wales and England Microbiology Division test for five key bacterial gastrointestinal pathogens, i.e. Escherichia coli O157 (VTEC), Shigella dysenteriae, Salmonella spp., Campylobacter spp. and Clostridioides difficile. There is, however, a poor success rate with identification of these pathogens, leaving the patient at risk from untreated infections. This study has developed effective and reliable tools with a high positive outcome for diagnosis of diarrhoeal infection. The study blindly analysed 592 samples, with the most abundant species being Shigella sonnei at 15%, and the top genus Bacteroides at 26%. Campylobacter spp. had an abundance of 4%, Clostridium difficile 3%, and Salmonella spp. 0.2%. There were also significant differences in abundance at genus level, between the Flemish Gut project and diarrhoea samples, with respect to Shigella (0.2%) and Campylobacter (0.1%). The project introduced a novel Shigella spp. (Escherichia) threshold of 5.32% to determine (Escherichia) a healthy or unhealthy community. A DMBiome model was developed to integrate the 5.32% threshold of Shigella spp., the Public Health laboratory tested pathogens, and two emerging enteropathogens. The overall positive outcome was that 89% of all samples were diagnosed with diarrhoea infections, leaving 11% unknown.
Assuntos
Bactérias , Diarreia , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas Microbiológicas , Shigella , Bactérias/genética , Bactérias/isolamento & purificação , Campylobacter/genética , Diarreia/diagnóstico , Diarreia/microbiologia , Fezes/microbiologia , Humanos , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/normas , Shigella/genética , Shigella/isolamento & purificaçãoRESUMO
Barbour-Stoenner-Kelly II (BSK-II) and BSK-H media were used for cultivation and isolation of fastidious Borrelia species - the causative agents of Lyme borreliosis. Culture media have a limited shelf life and require adequate storage. Our goal was to assess how the growth of Borrelia would be affected by prolonged storage of media and inadequate storage conditions (BSK-H stored at +4 °C for 2.5 years and BSK-II stored at -20 °C for 11 years). Growth of different Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae and Borrelia valaisiana strains was assessed during 2 weeks of incubation at 33 °C. Monitored parameters included cell count per mL, morphology and motility. The results of this study have shown weaker growth of borrelia strains in BSK-H at +4 °C (median final cell number of 1.5 × 106 /mL) than in BSK-II at -20 °C (median final cell number of 7.75 × 106 /mL) and in fresh BSK-H media (median final cell number of 8.95 × 106 /mL). Duration of storage of media had no impact on Borrelia morphology and motility. Our results indicate that temperature of -20 °C is optimal for long-term storage of medium, BSK-II stored for 11 years provided effective support to growth of Borrelia and may be employed for cultivation.
Assuntos
Borrelia burgdorferi/crescimento & desenvolvimento , Meios de Cultura , Técnicas Microbiológicas/métodos , Técnicas Microbiológicas/normasRESUMO
Chlamydophila pneumoniae is an intracellular pathogen responsible for respiratory tract infections. The isolation of the microorganism from clinical specimens is essential for a diagnosis. However, the identification of C. pneumoniae by cell cultures is very difficult besides strongly depending on the sample conditions. The study aimed to investigate, in adult patients with pharyngotonsillitis, the frequency of Chlamydophila pneumoniae detection by cell cultures and three conventional PCRs (a conventional PCR targeting the 16S rRNA gene and two nested PCRs, targeting the 16S rRNA gene and the ompA gene, respectively). The presence of chlamydial inclusion in cell cultures was observed in 11/94 samples (11.70%) by IFA. C. pneumoniae DNA was detected in 12/94 (12.76%) specimens by the 16S rRNA gene nested PCR, 4/94 (4.26%) by ompA gene nested PCR, and in 2/94 (2.13%) by 16S rRNA single-step PCR. Our data show poor agreement between the three applied DNA-amplification methods; in fact, only 16S rRNA gene nested PCR showed a statistically significant difference. Moreover, this result allowed us to achieve a definitive confirmation of the previous finding and to avoid the risk of an overestimation of the C. pneumoniae as a pathogen in pharyngotonsillitis.