RESUMO
Despite the first-line recommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are pressing barriers for optimizing its use for the treatment of non-Escherichia coli Enterobacterales UTI. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for minimal inhibitory concentration (MIC) determination is largely impractical. Using 160 clinical Klebsiella pneumoniae isolates, we sought to understand rates of skipped wells and MIC imprecision in broth microdilution (BMD) and how that compares to rates of error using AD. Though the Clinical and Laboratory Standards Institute refers to the skipped well phenomena in their recommendation against the use of BMD, there is a paucity of data on its frequency. While AD and BMD produced similar MIC50/90 values (32/256 µg/mL for AD and 64/256 µg/mL for BMD), essential agreement was poor. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells at a given concentration, as the most frequent scientific error. Growth in concentrations above the measured MIC occurred in up to 3.3% of wells and was seen within three dilutions of the MIC for BMD. Observation of single colonies either at or beyond the measured MIC for AD was also common and occurred up to 8.3% and 2.5% of the time, respectively. The frequent scientific error in both testing methods should prompt re-evaluation of AD guidelines and expansion of MIC testing methods for fosfomycin susceptibility testing, as poor agreement with another method prone to scientific error should not be the main detractor from BMD use.IMPORTANCEDespite the recommendation of fosfomycin for uncomplicated urinary tract infections (UTIs), there are barriers for optimizing its use. There are no approved breakpoints for oral use against other Enterobacterales, and the recommended agar dilution (AD) reference method for MIC determination is largely impractical. The use of broth microdilution (BMD) for fosfomycin testing is not recommended by the Clinical and Laboratory Standards Institute due to unsatisfactory precision and skipped wells-occurrence of no-growth in a single well before the minimal inhibitory concentration (MIC)-and trailing endpoints. We sought to understand rates of skipped wells and growth at concentrations above measured MICs in BMD and how that compares to scientific error using AD. No-growth wells at concentrations below the MIC occurred in up to 10.9% of wells for BMD and single colonies at or beyond measured MICs for AD were also common. Frequent scientific error in both methods should prompt re-evaluation of both AD and BMD for fosfomycin susceptibility testing.
Assuntos
Antibacterianos , Fosfomicina , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Fosfomicina/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Antibacterianos/farmacologia , Humanos , Infecções Urinárias/microbiologia , Infecções Urinárias/tratamento farmacológico , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Ágar , Meios de Cultura/químicaRESUMO
Traditionally, cephalothin susceptibility results were used to predict the susceptibility of additional cephalosporins; however, in 2013-2014, the Clinical and Laboratory Standards Institute (CLSI) revisited this practice and determined that cefazolin is a more accurate proxy than cephalothin for uncomplicated urinary tract infections (uUTIs). Therefore, a cefazolin surrogacy breakpoint was established to predict the susceptibility of seven oral cephalosporins for Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis in the context of uUTIs. Clinical microbiology laboratories face several operational challenges when implementing the cefazolin surrogacy breakpoint, which may lead to confusion for the best path forward. Here, we review the historical context and data behind the surrogacy breakpoints, review PK/PD profiles for oral cephalosporins, discuss challenges in deploying the breakpoint, and highlight the limited clinical outcome data in this space.
Assuntos
Cefazolina , Infecções Urinárias , Humanos , Cefazolina/farmacologia , Cefazolina/uso terapêutico , Cefalosporinas/farmacologia , Cefalotina , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Testes de Sensibilidade Microbiana , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , Escherichia coli , MonobactamasRESUMO
A 65-year-old man with HIV sought treatment for fever, weight loss, and productive cough after returning to the United States from Liberia. Fungal cultures grew Emergomyces pasteurianus, and the patient's health improved after beginning voriconazole. We describe the clinical case and review the literature, treatment, and susceptibilities for E. pasteurianus.
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Micoses , Onygenales , Humanos , Estados Unidos , Idoso , Micoses/microbiologia , Libéria , VoriconazolRESUMO
BACKGROUND: Metagenomic next generation sequencing (mNGS) is becoming increasingly available for pathogen detection directly from clinical specimens. These tests use target-independent, shotgun sequencing to detect potentially unlimited organisms. The promise of this methodology to aid infection diagnosis is demonstrated through early case reports and clinical studies. However, the optimal role of mNGS in clinical microbiology remains uncertain. CONTENT: We reviewed studies reporting clinical use of mNGS for pathogen detection from various specimen types, including cerebrospinal fluid, plasma, lower respiratory specimens, and others. Published clinical study data were critically evaluated and summarized to identify promising clinical indications for mNGS-based testing, to assess the clinical impact of mNGS for each indication, and to recognize test limitations. Based on these clinical studies, early testing recommendations are made to guide clinical utilization of mNGS for pathogen detection. Finally, current barriers to routine clinical laboratory implementation of mNGS tests are highlighted. SUMMARY: The promise of direct-from-specimen mNGS to enable challenging infection diagnoses has been demonstrated through early clinical studies of patients with meningitis or encephalitis, invasive fungal infections, community acquired pneumonia, and other clinical indications. However, the proportion of patient cases with positive clinical impact due to mNGS testing is low in published studies and the cost of testing is high, emphasizing the importance of improving our understanding of 'when to test' and for which patients mNGS testing is appropriate.
Assuntos
Líquidos Corporais/microbiologia , Líquidos Corporais/parasitologia , Sequenciamento de Nucleotídeos em Larga Escala/normas , Metagenômica/normas , Alveolados/genética , Bactérias/genética , Infecções Bacterianas/diagnóstico , Fungos/genética , Humanos , Micoses/diagnóstico , Infecções por Protozoários/diagnósticoRESUMO
OBJECTIVES: Rapid and accurate mold identification is critical for guiding therapy for mold infections. MALDI-TOF MS has been widely adopted for bacterial and yeast identification; however, few clinical laboratories have applied this technology for routine mold identification due to limited database availability and lack of standardized processes. Here, we evaluated the versatility of the NIH Mold Database in a multicenter evaluation. METHODS: The NIH Mold Database was evaluated by eight US academic centers using a solid media extraction method and a challenge set of 80 clinical mold isolates. Multiple instrument parameters important for spectra optimization were evaluated, leading to the development of two specialized acquisition programs (NIH method and the Alternate-B method). RESULTS: A wide range in performance (33-77%) was initially observed across the eight centers when routine spectral acquisition parameters were applied. Use of the NIH or the Alternate-B specialized acquisition programs, which are different than those used routinely for bacterial and yeast spectral acquisition (MBT_AutoX), in combination with optimized instrument maintenance, improved performance, illustrating that acquisition parameters may be one of the key limiting variable in achieving successful performance. CONCLUSION: Successful mold identification using the NIH Database for MALDI-TOF MS on Biotyper systems was demonstrated across multiple institutions for the first time following identification of critical program parameters combined with instrument optimization. This significantly advances our potential to implement MALDI-TOF MS for mold identification across many institutions. Because instrument variability is inevitable, development of an instrument performance standard specific for mold spectral acquisition is suggested to improve reproducibility across instruments.
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OBJECTIVE: To evaluate the diagnostic yield of fungal smears and cultures from bronchial lavage and wash specimens obtained from immunocompetent patients in the intensive care unit (ICU) because respiratory tract samples from patients in the ICU often undergo extensive microbiological testing. PATIENTS AND METHODS: In total, we enrolled 112 immunocompetent adult patients treated in the medical and surgical ICU between July 1, 2016, and June 30, 2017. We evaluated whether the results of fungal smears and cultures of specimens obtained from bronchoscopy and bronchoalveolar lavage changed patient care. RESULTS: In total, 131 bronchoscopic specimens and 31 bronchoalveolar lavage specimens were tested for fungi. Cultures were held for an estimated 4680 culture-days. Two results changed patient therapy. In both cases, other routine tests provided the same information as fungal culture before these results were returned. CONCLUSION: In immunocompetent, critically ill patients, fungal culture of respiratory tract specimens does not add diagnostic value. Routine fungal culture of respiratory tract specimens should be discouraged in this population.
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Antifúngicos/uso terapêutico , Líquido da Lavagem Broncoalveolar/microbiologia , Fungos/isolamento & purificação , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/microbiologia , Adulto , Lavagem Broncoalveolar/métodos , Broncoscopia/métodos , Estudos de Coortes , Estado Terminal , Técnicas de Cultura , Feminino , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Prognóstico , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/imunologia , Estudos Retrospectivos , Medição de Risco , Resultado do Tratamento , Procedimentos Desnecessários/métodosRESUMO
This article summarizes recent advances in the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to new areas of infectious diseases diagnostics. We discuss progress toward routine identification of mycobacteria and filamentous fungi and direct identification of pathogens from clinical specimens. Of greatest interest is the use of MALDI-TOF MS for identifying organisms from positive blood cultures and from clinical specimens such as urine. Last, We highlight some exciting new possibilities for MALDI-TOF MS phenotypic susceptibility testing for bacteria and yeast.
Assuntos
Bactérias/isolamento & purificação , Técnicas Microbiológicas/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , HumanosRESUMO
The recent development of commercial panel-based molecular diagnostics for the rapid detection of pathogens in positive blood culture bottles, respiratory specimens, stool, and cerebrospinal fluid has resulted in a paradigm shift in clinical microbiology and clinical practice. This review focuses on U.S. Food and Drug Administration (FDA)-approved/cleared multiplex molecular panels with more than five targets designed to assist in the diagnosis of bloodstream, respiratory tract, gastrointestinal, or central nervous system infections. While these panel-based assays have the clear advantages of a rapid turnaround time and the detection of a large number of microorganisms and promise to improve health care, they present certain challenges, including cost and the definition of ideal test utilization strategies (i.e., optimal ordering) and test interpretation.
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Infecções/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Humanos , Técnicas de Diagnóstico Molecular/normas , Técnicas de Diagnóstico Molecular/tendências , Estados Unidos , United States Food and Drug AdministrationRESUMO
UNLABELLED: The genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and enzymatic probing. We then designed CRISPR spacer sequences targeting T4 and found that wild-type T4 containing glc-HMC was insensitive to attack by CRISPR-Cas9 but mutants with unmodified cytosine were sensitive. Phage with HMC showed only intermediate sensitivity. While this work was in progress, another group reported examples of heavily engineered CRISRP-Cas9 complexes that could, in fact, overcome the effects of T4 DNA modification, indicating that modifications can inhibit but do not always fully block attack. IMPORTANCE: Bacteria were recently found to have a form of adaptive immunity, the CRISPR-Cas systems, which use nucleic acid pairing to recognize and cleave genomic DNA of invaders such as bacteriophage. Historic work with tailed phages has shown that phage DNA is often modified by covalent attachment of large chemical groups. Here we demonstrate that DNA modification in phage T4 inhibits attack by the CRISPR-Cas9 system. This finding provides insight into mechanisms of host-virus competition and also a new set of tools that may be useful in modulating the activity of CRISPR-Cas9 in genome engineering applications.
Assuntos
Bacteriófago T4/crescimento & desenvolvimento , Bacteriófago T4/genética , Sistemas CRISPR-Cas , DNA Viral/química , DNA Viral/metabolismo , Escherichia coli/virologia , Citosina/química , Citosina/metabolismo , DNA Viral/genética , Viabilidade Microbiana , Dados de Sequência Molecular , Análise de Sequência de DNA , Ensaio de Placa ViralRESUMO
BACKGROUND: Kallikrein 6 (KLK6) is a newly identified member of the kallikrein family of secreted serine proteases that prior studies indicate is elevated at sites of central nervous system (CNS) inflammation and which shows regulated expression with T cell activation. Notably, KLK6 is also elevated in the serum of multiple sclerosis (MS) patients however its potential roles in immune function are unknown. Herein we specifically examine whether KLK6 alters immune cell survival and the possible mechanism by which this may occur. METHODOLOGY/PRINCIPAL FINDINGS: Using murine whole splenocyte preparations and the human Jurkat T cell line we demonstrate that KLK6 robustly supports cell survival across a range of cell death paradigms. Recombinant KLK6 was shown to significantly reduce cell death under resting conditions and in response to camptothecin, dexamethasone, staurosporine and Fas-ligand. Moreover, KLK6-over expression in Jurkat T cells was shown to generate parallel pro-survival effects. In mixed splenocyte populations the vigorous immune cell survival promoting effects of KLK6 were shown to include both T and B lymphocytes, to occur with as little as 5 minutes of treatment, and to involve up regulation of the pro-survival protein B-cell lymphoma-extra large (Bcl-XL), and inhibition of the pro-apoptotic protein Bcl-2-interacting mediator of cell death (Bim). The ability of KLK6 to promote survival of splenic T cells was also shown to be absent in cell preparations derived from PAR1 deficient mice. CONCLUSION/SIGNIFICANCE: KLK6 promotes lymphocyte survival by a mechanism that depends in part on activation of PAR1. These findings point to a novel molecular mechanism regulating lymphocyte survival that is likely to have relevance to a range of immunological responses that depend on apoptosis for immune clearance and maintenance of homeostasis.