Staphylococcus aureus antimicrobial susceptibility trends and cluster detection in Vermont: 2012-2018.

Objectives: This study presents demographic and temporal trends in the isolation of Staphylococcus aureus in Vermont clinical microbiology laboratories and explores the use of statistical algorithms and multi-resistance phenotypes to improve outbreak detection.Methods: Routine microbiology test results downloaded from Vermont clinical laboratory information systems were used to monitor S. aureus antimicrobial resistance trends. The integrated WHONET-SaTScan software used multi-resistance phenotypes to identify possible acute outbreaks with the space-time permutation model and slowly emerging geographic clusters using the spatial-only multinomial model.Results: Data were provided from seven hospital laboratories from 2012 to 2018 for 19,224 S. aureus isolates from 14,939 patients. Statistically significant differences (p ≤ 0.05) in methicillin-resistant S. aureus (MRSA) isolation were seen by age group, specimen type, and health-care setting. Among MRSA, multi-resistance profiles permitted the recognition and tracking of 6 common and 21 rare 'phenotypic clones.' We identified 43 acute MRSA clusters and 7 significant geographic clusters (p ≤ 0.05).Conclusions: There was significant heterogeneity in MRSA strains between facilities and the use of multi-resistance phenotypes facilitated the recognition of possible outbreaks. Comprehensive electronic surveillance of antimicrobial resistance utilizing routine clinical microbiology data with free software tools offers early recognition and tracking of emerging resistance threats.

Surveillance of antimicrobial resistance and evolving microbial populations in Vermont: 2011-2018.

OBJECTIVE: This study presents trends in organism isolation and antimicrobial resistance in routine microbiology test results from acute-care hospital microbiology laboratories in Vermont. METHODS: Organism identifications and antimicrobial susceptibility test results were captured from acute-care hospital laboratories to monitor geographic and temporal trends in resistance and emerging microbial threats with the free WHONET software. RESULTS: Data were provided from 12 acute care hospital laboratories from 2011 through 2018 for 318,833 isolates from 148,994 patients (70% female, 74% outpatient, and 63% urine). Significant differences (p < 0.05) in age, gender, and antimicrobial susceptibility results (e.g. Escherichia coli and levofloxacin) between outpatient and inpatient isolates were identified with temporal increases in certain species (e.g. Aerococcus urinae) and resistance (e.g. Streptococcus pneumoniae and erythromycin). The use of multi-resistance phenotypes demonstrated significant heterogeneity (p < 0.05) in MRSA strains between facilities, for example Staphylococcus aureus resistant to six priority antimicrobials were found in no critical access hospitals (fewer than 25 inpatient beds) but in all non-critical access hospitals. CONCLUSIONS: Comprehensive electronic surveillance of antimicrobial resistance utilizing routine clinical microbiology data with free software tools offers early recognition and tracking of emerging community and healthcare resistance threats at the local and state level.

Why surveillance of antimicrobial resistance needs to be automated and comprehensive.

OBJECTIVES: Surveillance of antimicrobial resistance (AMR) can now be automated to analyse the reports of microbiology laboratories continually without operator assistance. It can also be made comprehensive to monitor all the reports of all the world's microbiology laboratories. METHODS AND RESULTS: As illustrated through examples provided in this work, each clinical report can be scanned automatically by algorithms to suspect emerging problems and to prompt sampling to confirm such problems, now increasingly by nucleotide sequencing. An emerging problem may be an excess (clustering) of similar microbes owing to their spread among patients who are interrelated in some way, as by shared locations, caregivers or food products. Or it might be a microbe new to an area or to a laboratory but already seen nearby, such as Elizabethkingia anophelis or mcr-1-positive Escherichia coli. Automated early alerting of responders enables them to contain spread sooner and to avert infections downstream. 'Big Data' informatics now also enables surveillance of AMR to be made comprehensive, to monitor all reports of all the world's microbiology laboratories. Such orders of magnitude increase in analysed data would accordingly increase its granularity and thus detect many more global problems sooner. It would also reduce surveillance-blind areas where problems may now emerge and spread undetected. CONCLUSIONS: The world's microbiology laboratories need to integrate and analyse all of their reports for surveillance to make their own patients safer from existing and approaching problems otherwise hard to notice. Making automated surveillance an easy-to-adopt laboratory standard of care can make it comprehensive.

Multi-institute analysis of carbapenem resistance reveals remarkable diversity, unexplained mechanisms, and limited clonal outbreaks.

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

Statistical detection of geographic clusters of resistant Escherichia coli in a regional network with WHONET and SaTScan.

BACKGROUND: While antimicrobial resistance threatens the prevention, treatment, and control of infectious diseases, systematic analysis of routine microbiology laboratory test results worldwide can alert new threats and promote timely response. This study explores statistical algorithms for recognizing geographic clustering of multi-resistant microbes within a healthcare network and monitoring the dissemination of new strains over time. METHODS: Escherichia coli antimicrobial susceptibility data from a three-year period stored in WHONET were analyzed across ten facilities in a healthcare network utilizing SaTScan's spatial multinomial model with two models for defining geographic proximity. We explored geographic clustering of multi-resistance phenotypes within the network and changes in clustering over time. RESULTS: Geographic clustering identified from both latitude/longitude and non-parametric facility groupings geographic models were similar, while the latter was offers greater flexibility and generalizability. Iterative application of the clustering algorithms suggested the possible recognition of the initial appearance of invasive E. coli ST131 in the clinical database of a single hospital and subsequent dissemination to others. CONCLUSION: Systematic analysis of routine antimicrobial resistance susceptibility test results supports the recognition of geographic clustering of microbial phenotypic subpopulations with WHONET and SaTScan, and iterative application of these algorithms can detect the initial appearance in and dissemination across a region prompting early investigation, response, and containment measures.

Esophageal Actinomycoses Mimicking Malignancy.

Actinomycosis is caused by anaerobic bacteria and rarely affects the esophagus. We present a case of esophageal actinomycosis in a 55-year old woman that mimicked malignancy. The patient presented with dysphagia and weight loss. Preoperative esophagogastroscopic biopsy revealed purulent material, but was inconclusive. Endoscopic ultrasonography suggested esophageal cancer, and chest computed tomography showed a mass in the lower esophagus surrounded by inflammation. The patient underwent esophagogastrectomy, and histopathology examination of the specimen revealed distal esophageal actinomycosis. Preoperative diagnosis of esophageal actinomycosis is difficult, but clinicians should be aware of its unusual presentations and its ability to mimic malignancy.

Origin and proliferation of multiple-drug resistance in bacterial pathogens.

SUMMARY: Many studies report the high prevalence of multiply drug-resistant (MDR) strains. Because MDR infections are often significantly harder and more expensive to treat, they represent a growing public health threat. However, for different pathogens, different underlying mechanisms are traditionally used to explain these observations, and it is unclear whether each bacterial taxon has its own mechanism(s) for multidrug resistance or whether there are common mechanisms between distantly related pathogens. In this review, we provide a systematic overview of the causes of the excess of MDR infections and define testable predictions made by each hypothetical mechanism, including experimental, epidemiological, population genomic, and other tests of these hypotheses. Better understanding the cause(s) of the excess of MDR is the first step to rational design of more effective interventions to prevent the origin and/or proliferation of MDR.

The world's microbiology laboratories can be a global microbial sensor network.

The microbes that infect us spread in global and local epidemics, and the resistance genes that block their treatment spread within and between them. All we can know about where they are to track and contain them comes from the only places that can see them, the world's microbiology laboratories, but most report each patient's microbe only to that patient's caregiver. Sensors, ranging from instruments to birdwatchers, are now being linked in electronic networks to monitor and interpret algorithmically in real-time ocean currents, atmospheric carbon, supply-chain inventory, bird migration, etc. To so link the world's microbiology laboratories as exquisite sensors in a truly lifesaving real-time network their data must be accessed and fully subtyped. Microbiology laboratories put individual reports into inaccessible paper or mutually incompatible electronic reporting systems, but those from more than 2,200 laboratories in more than 108 countries worldwide are now accessed and translated into compatible WHONET files. These increasingly web-based files could initiate a global microbial sensor network. Unused microbiology laboratory byproduct data, now from drug susceptibility and biochemical testing but increasingly from new technologies (genotyping, MALDI-TOF, etc.), can be reused to subtype microbes of each genus/species into sub-groupings that are discriminated and traced with greater sensitivity. Ongoing statistical delineation of subtypes from global sensor network data will improve detection of movement into any patient of a microbe or resistance gene from another patient, medical center or country. Growing data on clinical manifestations and global distributions of subtypes can automate comments for patient's reports, select microbes to genotype and alert responders.

The world´s microbiology laboratories can be a global microbial sensor network / Los laboratorios de microbiología del mundo pueden convertirse en una red de detección microbiana

The microbes that infect us spread in global and local epidemics, and the resistance genes that block their treatment spread within and between them. All we can know about where they are to track and contain them comes from the only places that can see them, the world´s microbiology laboratories, but most report each patient´s microbe only to that patient´s caregiver. Sensors, ranging from instruments to birdwatchers, are now being linked in electronic networks to monitor and interpret algorithmically in real-time ocean currents, atmospheric carbon, supply-chain inventory, bird migration, etc. To so link the world´s microbiology laboratories as exquisite sensors in a truly lifesaving real-time network their data must be accessed and fully subtyped. Microbiology laboratories put individual reports into inaccessible paper or mutually incompatible electronic reporting systems, but those from more than 2,200 laboratories in more than 108 countries worldwide are now accessed and translated into compatible WHONET files. These increasingly web-based files could initiate a global microbial sensor network. Unused microbiology laboratory byproduct data, now from drug susceptibility and biochemical testing but increasingly from new technologies (genotyping, MALDI-TOF, etc.), can be reused to subtype microbes of each genus/species into sub-groupings that are discriminated and traced with greater sensitivity. Ongoing statistical delineation of subtypes from global sensor network data will improve detection of movement into any patient of a microbe or resistance gene from another patient, medical center or country. Growing data on clinical manifestations and global distributions of subtypes can automate comments for patient´s reports, select microbes to genotype and alert responders.

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Cefepime vs other antibacterial agents for the treatment of Enterobacter species bacteremia.

BACKGROUND: Carbapenems are recommended for treatment of Enterobacter infections with AmpC phenotypes. Although isolates are typically susceptible to cefepime in vitro, there are few data supporting its clinical efficacy. METHODS: We reviewed all cases of Enterobacter species bacteremia at 2 academic hospitals from 2005 to 2011. Outcomes of interest were (1) persistent bacteremia ≥1 calendar day and (2) in-hospital mortality. We fit logistic regression models, adjusting for clinical risk factors and Pitt bacteremia score and performed propensity score analyses to compare the efficacy of cefepime and carbapenems. RESULTS: Three hundred sixty-eight patients experienced Enterobacter species bacteremia and received at least 1 antimicrobial agent, of whom 52 (14%) died during hospitalization. Median age was 59 years; 19% were neutropenic, and 22% were in an intensive care unit on the day of bacteremia. Twenty-nine (11%) patients had persistent bacteremia for ≥1 day after antibacterial initiation. None of the 36 patients who received single-agent cefepime (0%) had persistent bacteremia, as opposed to 4 of 16 (25%) of those who received single-agent carbapenem (P < .01). In multivariable models, there was no association between carbapenem use and persistent bacteremia (adjusted odds ratio [aOR], 1.52; 95% CI, .58-3.98; P = .39), and a nonsignificant lower odds ratio with cefepime use (aOR, 0.52; 95% CI, .19-1.40; P = .19). In-hospital mortality was similar for use of cefepime and carbapenems in adjusted regression models and propensity-score matched analyses. CONCLUSIONS: Cefepime has a similar efficacy as carbapenems for the treatment of Enterobacter species bacteremia. Its use should be further explored as a carbapenem-sparing agent in this clinical scenario.

Critical role of the tumor suppressor tuberous sclerosis complex 1 in dendritic cell activation of CD4 T cells by promoting MHC class II expression via IRF4 and CIITA.

Dendritic cell (DC) maturation is characterized by upregulation of cell-surface MHC class II (MHC-II) and costimulatory molecules, and production of a variety of cytokines that can shape both innate and adaptive immunity. Paradoxically, transcription of the MHC-II genes, as well as its activator, CIITA, is rapidly silenced during DC maturation. The mechanisms that control CIITA/MHC-II expression and silencing have not been fully understood. We report in this article that the tumor suppressor tuberous sclerosis complex 1 (TSC1) is a critical regulator of DC function for both innate and adaptive immunity. Its deficiency in DCs results in increased mammalian target of rapamycin (mTOR) complex 1 but decreased mTORC2 signaling, altered cytokine production, impaired CIITA/MHC-II expression, and defective Ag presentation to CD4 T cells after TLR4 stimulation. We demonstrate further that IFN regulatory factor 4 can directly bind to CIITA promoters, and decreased IFN regulatory factor 4 expression is partially responsible for decreased CIITA/MHC-II expression in TSC1-deficient DCs. Moreover, we identify that CIITA/MHC-II silencing during DC maturation requires mTOR complex 1 activity. Together, our data reveal unexpected roles of TSC1/mTOR that control multifaceted functions of DCs.

Biochemical phenotypes to discriminate microbial subpopulations and improve outbreak detection.

BACKGROUND: Clinical microbiology laboratories worldwide constitute an invaluable resource for monitoring emerging threats and the spread of antimicrobial resistance. We studied the growing number of biochemical tests routinely performed on clinical isolates to explore their value as epidemiological markers. METHODOLOGY/PRINCIPAL FINDINGS: Microbiology laboratory results from January 2009 through December 2011 from a 793-bed hospital stored in WHONET were examined. Variables included patient location, collection date, organism, and 47 biochemical and 17 antimicrobial susceptibility test results reported by Vitek 2. To identify biochemical tests that were particularly valuable (stable with repeat testing, but good variability across the species) or problematic (inconsistent results with repeat testing), three types of variance analyses were performed on isolates of K. pneumonia: descriptive analysis of discordant biochemical results in same-day isolates, an average within-patient variance index, and generalized linear mixed model variance component analysis. RESULTS: 4,200 isolates of K. pneumoniae were identified from 2,485 patients, 32% of whom had multiple isolates. The first two variance analyses highlighted SUCT, TyrA, GlyA, and GGT as "nuisance" biochemicals for which discordant within-patient test results impacted a high proportion of patient results, while dTAG had relatively good within-patient stability with good heterogeneity across the species. Variance component analyses confirmed the relative stability of dTAG, and identified additional biochemicals such as PHOS with a large between patient to within patient variance ratio. A reduced subset of biochemicals improved the robustness of strain definition for carbapenem-resistant K. pneumoniae. Surveillance analyses suggest that the reduced biochemical profile could improve the timeliness and specificity of outbreak detection algorithms. CONCLUSIONS: The statistical approaches explored can improve the robust recognition of microbial subpopulations with routinely available biochemical test results, of value in the timely detection of outbreak clones and evolutionarily important genetic events.

Incomplete intrapulmonary lymph node retrieval after routine pathologic examination of resected lung cancer.

PURPOSE: Pathologic nodal stage affects prognosis in patients with surgically resected non-small-cell lung cancer (NSCLC). Unlike examination of mediastinal lymph nodes (LNs), which depends on surgical practice, accurate examination of intrapulmonary (N1) nodes depends primarily on pathology practice. We investigated the completeness of N1 LN examination in NSCLC resection specimens and its potential impact on stage. PATIENTS AND METHODS: We performed a case-control study of a special pathologic examination (SPE) protocol using thin gross dissection with retrieval and microscopic examination of all LN-like material on remnant NSCLC resection specimens after routine pathologic examination (RPE). We compared LNs retrieved by the SPE protocol with nodes examined after RPE of the same lung specimens and with those of an external control cohort. RESULTS: We retrieved additional LNs in 66 (90%) of 73 patient cases and discovered metastasis in 56 (11%) of 514 retrieved LNs from 27% of all patients. We found unexpected LN metastasis in six (12%) of 50 node-negative patients. Three other patients had undetected satellite metastatic nodules. Pathologic stage was upgraded in eight (11%) of 73 patients. The time required for the SPE protocol decreased significantly with experience, with no change in the number of LNs found. CONCLUSION: Standard pathology practice frequently leaves large numbers of N1 LNs unexamined, a clinically significant proportion of which harbor metastasis. By improving N1 LN examination, SPE can have an impact on prognosis and adjuvant management. We suggest adoption of the SPE to improve pathologic staging of resected NSCLC.

Chondromyxoid fibroma of the orbit.

A 51-year-old woman with a history of migraine headaches was found to have an incidental right orbital mass on MRI during neurologic evaluation for headaches. The orbital mass was a well-defined, lobulated, intraosseous soft tissue lesion with circumscribed margins. Clinically, there was noted proptosis, tenderness to palpation, and slight limitation to right abduction. An orbitotomy with incisional biopsy revealed a lesion arising within the lateral orbital rim extending to the subperiosteal space. Intraoperative frozen sections indicated a low grade sarcoma, possibly metastatic. The extraosseous component was excised, and the bone was curetted until all visible tumor was removed. A diagnosis of chondromyxoid fibroma was made. The patient did well until 5 months postoperatively, when right-sided proptosis returned due to recurrent tumor. Repeat surgical resection with removal of the lateral orbital rim was performed. Histopathology was consistent with recurrent chondromyxoid fibroma.

Use of a surgical specimen-collection kit to improve mediastinal lymph-node examination of resectable lung cancer.

INTRODUCTION: Pathologic examination of mediastinal lymph nodes (MLNs) after resection of non-small-cell lung cancer is critical in the determination of prognosis and postoperative management. Although systematic nodal dissection is recommended, the quality of pathologic lymph-node staging often falls short of recommendations in practice. We tested the feasibility of improving pathologic lymph-node staging of resectable non-small-cell lung cancer by using a prelabeled specimen-collection kit. METHODS: Case-control study with comparison of 51 resections, using a special lymph-node collection kit, with 51 controls matched for surgeon, extent of resection, pathologist, and T category. Appropriate statistical methods were used for all comparisons. RESULTS: The median number of MLNs examined increased from one in the control group, to six in the case group (p < 0.001). The percentage of resections attaining the National Comprehensive Cancer Network-recommended quality of MLN examination, and the proportion that would have been eligible for recent landmark postresection adjuvant therapy trials increased significantly (p < 0.001). The duration of surgery and postoperative complication rates were similar between cases and controls. Eighteen percent of kit cases had positive MLN, compared with 8% of controls. CONCLUSIONS: The use of a specialized specimen-collection kit for MLN examination was feasible, markedly improved MLN staging, and showed a trend toward increased detection of patients with MLN metastasis, with only a modest increase in duration of surgery, and no increase in perioperative morbidity, mortality, or hospital length of stay.

The role and regulation of mTOR in T-lymphocyte function.

The conversion of naïve T cells into effector T cells is initiated by stimulation through the T-cell receptor (TCR). Upon activation, T cells undergo significant morphological and functional changes, putting new metabolic demands on the cell. Past research has identified the mammalian target of rapamycin (mTOR) as a critical regulator of cell metabolism, and the development of new genetic models has begun to reveal an important role for this pathway in the homeostasis and function of T lymphocytes. In this review, we focus on the most recent findings that demonstrate the ability of mTOR to regulate T-cell activation, CD8(+) memory cell formation and function, and helper T lineage differentiation. Furthermore, we highlight the importance of tight control of mTOR signaling by tuberous sclerosis complex 1 for T-cell homeostasis, and the regulation of mTOR signaling by diacylglycerol kinases and the RasGRP1-Ras-Erk1/2 pathway in the context of TCR signaling.

The role of tuberous sclerosis complex 1 in regulating innate immunity.

The mechanisms that control TLR-induced responses, including endotoxin tolerance, have been not well understood. The tuberous sclerosis complex 1 (TSC1) is a tumor suppressor that inhibits the mammalian target of rapamycin (mTOR). We show in this study that deficiency of TSC1 results in enhanced activation of not only mTOR complex 1 (mTORC1), but also JNK1/2, following LPS stimulation in macrophages. TSC1-deficient macrophages produce elevated proinflammatory cytokines and NO in response to multiple TLR ligands. Such enhanced TLR-induced responses can be inhibited by reducing mTORC1 and JNK1/2 activities with chemical inhibitors or small hairpin RNA, suggesting that TSC1 negatively controls TLR responses through both mTORC1 and JNK1/2. The impact of TSC1 deficiency appeared not limited to TLRs, as NOD- and RIG-I/MDA-5-induced innate responses were also altered in TSC1-deficient macrophages. Furthermore, TSC1 deficiency appears to cause impaired induction of endotoxin tolerance in vitro and in vivo, which is correlated with increased JNK1/2 activation and can be reversed by JNK1/2 inhibition. Our results reveal a critical role of TSC1 in regulating innate immunity by negative control of mTORC1 and JNK1/2 activation.

Differential regulation of primary and memory CD8 T cell immune responses by diacylglycerol kinases.

The manipulation of signals downstream of the TCR can have profound consequences for T cell development, function, and homeostasis. Diacylglycerol (DAG) produced after TCR stimulation functions as a secondary messenger and mediates the signaling to Ras-MEK-Erk and NF-κB pathways in T cells. DAG kinases (DGKs) convert DAG into phosphatidic acid, resulting in termination of DAG signaling. In this study, we demonstrate that DAG metabolism by DGKs can serve a crucial function in viral clearance upon lymphocytic choriomeningitis virus infection. Ag-specific CD8(+) T cells from DGKα(-/-) and DGKζ(-/-) mice show enhanced expansion and increased cytokine production after lymphocytic choriomeningitis virus infection, yet DGK-deficient memory CD8(+) T cells exhibit impaired expansion after rechallenge. Thus, DGK activity plays opposing roles in the expansion of CD8(+) T cells during the primary and memory phases of the immune response, whereas consistently inhibiting antiviral cytokine production.