Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

A Randomized Controlled Trial of an Electronic Clinical Decision Support Tool for Inpatient Antimicrobial Stewardship.

BACKGROUND: The weighted incidence syndromic combination antibiogram (WISCA) is an antimicrobial stewardship tool that utilizes electronic medical record data to provide real-time clinical decision support regarding empiric antibiotic prescription in the hospital setting. The aim of this study was to determine the impact of WISCA utilization for empiric antibiotic prescription on hospital length of stay (LOS). METHODS: We performed a crossover randomized controlled trial of the WISCA tool at 4 hospitals. Study participants included adult inpatients receiving empiric antibiotics for urinary tract infection (UTI), abdominal-biliary infection (ABI), pneumonia, or nonpurulent cellulitis. Antimicrobial stewardship (ASP) physicians utilized WISCA and clinical guidelines to provide empiric antibiotic recommendations. The primary outcome was LOS. Secondary outcomes included 30-day mortality, 30-day readmission, Clostridioides difficile infection, acquisition of multidrug-resistant gram-negative organism (MDRO), and antibiotics costs. RESULTS: In total, 6849 participants enrolled in the study. There were no overall differences in outcomes among the intervention versus control groups. Participants with cellulitis in the intervention group had significantly shorter mean LOS compared to participants with cellulitis in the control group (coefficient estimate = 0.53 [-0.97, -0.09], P = .0186). For patients with community acquired pneumonia (CAP), the intervention group had significantly lower odds of 30-day mortality compared to the control group (adjusted odds ratio [aOR] .58, 95% confidence interval [CI], .396, .854, P = .02). CONCLUSIONS: Use of WISCA was not associated with improved outcomes for UTI and ABI. Guidelines-based interventions were associated with decreased LOS for cellulitis and decreased mortality for CAP.

Validation of Active Surveillance Testing for Clostridium difficile Colonization Using the cobas Cdiff Test.

Clostridium difficile infection (CDI) is not declining in the United States. Nucleic acid amplification tests (NAAT) are used as part of active surveillance testing programs to prevent health care-associated infection. The objective of this study was to validate the cobas Cdiff Test on the cobas 4800 System (cobas) within a four-hospital system using prospectively collected perirectal swabs from asymptomatic patients at admission and during monthly intensive care unit (ICU) screening in an infection control CDI reduction program. Performance of the cobas was compared to that of toxigenic culture. Each positive cobas sample and the next following negative patient swab were cultured. The study design gave 273 samples processed by both cobas (137 positive and 136 negative) and culture (one negative swab was not cultured). Discrepant analysis was performed using a second NAAT, the Xpert C. difficile Epi test (Xpert). This strategy was compared to a medical record review for antibiotic receipt that would inhibit growth of C. difficile in colonic stool. None of the cobas-negative samples were culture positive. The cobas positive predictive value was 75.2% (95% confidence interval [CI], 66.9% to 82%) and positive percent agreement was 100% (95% CI, 96.0% to 100%). Overall agreement between cobas and direct toxigenic culture was 87.6% (95% CI, 83.1% to 91%). For the cobas-positive/culture-negative (discrepant) samples, 7 Xpert-positive samples were from patients receiving inhibitory antimicrobials; only 4 of 23 Xpert-negative samples received these agents (P = 0.00006). Our results support use of the cobas as a reliable assay for an active surveillance testing program to detect asymptomatic carriers of toxigenic C. difficile.

Multicenter Evaluation of the Xpert MRSA NxG Assay for Detection of Methicillin-Resistant Staphylococcus aureus in Nasal Swabs.

Health care-associated methicillin-resistant Staphylococcus aureus (MRSA) infections are a burden on the health care system. Clinical laboratories play a key role in reducing this burden, as the timely identification of MRSA colonization or infection facilitates infection control practices that are effective at limiting invasive MRSA infections. The Xpert MRSA NxG assay recently received FDA clearance for the direct detection of MRSA from nasal swabs. This multicenter study evaluated the clinical performance characteristics of the Xpert MRSA NxG assay with prospectively collected rayon nasal swabs (n = 1,103) and flocked swab (ESwab) nasal specimens (n = 846). Culture-based identification methods and antimicrobial susceptibility testing were used as the reference standards for comparison. According to the reference method, the positivity rates for MRSA in the population evaluated were 11.1% (122/1,103) for rayon swabs and 11.6% (98/846) for flocked swabs. The overall sensitivity and specificity of the rayon swabs were 91.0% (95% confidence interval [CI], 84.6 to 94.9%) and 96.9% (95% CI, 95.7 to 97.8%), respectively, across eight testing sites. The flocked swab specimens were 92.9% sensitive (95% CI, 86.0 to 96.5%) and 97.6% specific (95% CI, 96.2 to 98.5%) for MRSA detection across six testing sites. The sensitivity and specificity of the combined flocked and rayon swab data were 91.8% (95% CI, 87.4 to 94.8%) and 97.2% (95% CI, 96.3 to 97.9%), respectively. The positive predictive value (PPV) for rayon swabs was 78.7%, versus 83.5% for ESwabs. The negative predictive values (NPVs) for rayon swabs and ESwab specimens were 98.9% and 99.1%, respectively. In conclusion, the Xpert MRSA NxG assay is a sensitive and specific assay for the direct detection of MRSA from nasal swab specimens.

Activity of Uncleaved Caspase-8 Controls Anti-bacterial Immune Defense and TLR-Induced Cytokine Production Independent of Cell Death.

Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense.

Cell-Extrinsic TNF Collaborates with TRIF Signaling To Promote Yersinia-Induced Apoptosis.

Innate immune responses that are crucial for control of infection are often targeted by microbial pathogens. Blockade of NF-κB and MAPK signaling by the Yersinia virulence factor YopJ inhibits cytokine production by innate immune cells but also triggers cell death. This cell death requires RIPK1 kinase activity and caspase-8, which are engaged by TLR4 and the adaptor protein TRIF. Nevertheless, TLR4- and TRIF-deficient cells undergo significant apoptosis, implicating TLR4/TRIF-independent pathways in the death of Yersinia-infected cells. In this article, we report a key role for TNF/TNFR1 in Yersinia-induced cell death of murine macrophages, which occurs despite the blockade of NF-κB and MAPK signaling imposed by Yersinia on infected cells. Intriguingly, direct analysis of YopJ injection revealed a heterogeneous population of injection-high and injection-low cells, and demonstrated that TNF expression came from the injection-low population. Moreover, TNF production by this subpopulation was necessary for maximal apoptosis in the population of highly injected cells, and TNFR-deficient mice displayed enhanced susceptibility to Yersinia infection. These data demonstrate an important role for collaboration between TNF and pattern recognition receptor signals in promoting maximal apoptosis during bacterial infection, and demonstrate that heterogeneity in virulence factor injection and cellular responses play an important role in promoting anti-Yersinia immune defense.

Rapid Identification of Five Classes of Carbapenem Resistance Genes Directly from Rectal Swabs by Use of the Xpert Carba-R Assay.

Carbapenemase-producing organisms (CPO) have been identified by global health leaders as an urgent threat. Detection of patients with gastrointestinal carriage of CPO is necessary to interrupt their spread within health care facilities. In this multisite study, we assessed the performance of the Xpert Carba-R test, a rapid real-time quantitative PCR (qPCR) assay that detects five families of carbapenemase genes (blaIMP, blaKPC, blaNDM, blaOXA-48, and blaVIM) directly from rectal swab specimens. Using dual swabs, specimens from 755 patients were collected and tested prospectively. An additional 432 contrived specimens were prepared by seeding well-characterized carbapenem-susceptible and -nonsusceptible strains into a rectal swab matrix and inoculating them onto swabs prior to testing. Antimicrobial susceptibility testing, broth enriched culture, and DNA sequencing were performed by a central laboratory blind to the Xpert Carba-R results. The Xpert Carba-R assay demonstrated a positive percentage of agreement (PPA) between 60 and 100% for four targets (blaKPC, blaNDM, blaVIM, and blaOXA-48) and a negative percentage of agreement (NPA) ranging between 98.9 and 99.9% relative to the reference method (culture and sequencing of any carbapenem-nonsusceptible isolate). There were no prospective blaIMP-positive samples. Contrived specimens demonstrated a PPA between 95 and 100% and an NPA of 100% for all targets. Testing of rectal swabs directly using the Xpert Carba-R assay is effective for rapid detection and identification of CPO from hospitalized patients.

Evaluation of the cobas Cdiff Test for Detection of Toxigenic Clostridium difficile in Stool Samples.

Nucleic acid amplification tests (NAATs) are reliable tools for the detection of toxigenic Clostridium difficile from unformed (liquid or soft) stool samples. The objective of this study was to evaluate performance of the cobas Cdiff test on the cobas 4800 system using prospectively collected stool specimens from patients suspected of having C. difficile infection (CDI). The performance of the cobas Cdiff test was compared to the results of combined direct and broth-enriched toxigenic culture methods in a large, multicenter clinical trial. Additional discrepancy analysis was performed by using the Xpert C. difficile Epi test. Sample storage was evaluated by using contrived and fresh samples before and after storage at -20°C. Testing was performed on samples from 683 subjects (306 males and 377 females); 113 (16.5%) of 683 subjects were positive for toxigenic C. difficile by direct toxigenic culture, and 141 of 682 subjects were positive by using the combined direct and enriched toxigenic culture method (reference method), for a prevalence rate of 20.7%. The sensitivity and specificity of the cobas Cdiff test compared to the combined direct and enriched culture method were 92.9% (131/141; 95% confidence interval [CI], 87.4% to 96.1%) and 98.7% (534/541; 95% CI, 97.4% to 99.4%), respectively. Discrepancy analysis using results for retested samples from a second NAAT (Xpert C. difficile/Epi test; Cepheid, Sunnyvale, CA) found no false-negative and 4 false-positive cobas Cdiff test results. There was no difference in positive and negative results in comparisons of fresh and stored samples. These results support the use of the cobas Cdiff test as a robust aid in the diagnosis of CDI.

Nonimpact of Decolonization as an Adjunctive Measure to Contact Precautions for the Control of Methicillin-Resistant Staphylococcus aureus Transmission in Acute Care.

This was an observational study comparing methicillin-resistant Staphylococcus aureus (MRSA) transmission with no decolonization of medical patients to required decolonization of all MRSA carriers during two consecutive periods: baseline with no decolonization of medical patients (16 months) and universal MRSA carrier decolonization (13 months). The setting was a one-hospital, 156-bed facility with 9,200 annual admissions. Regression models were used to compare rates of MRSA acquisition. The chi-square test was used to compare event frequencies. We used rates of MRSA clinical disease as an outcome monitor of the program. Analysis was done on 15,666 patients who had admission and discharge tests; 27.9% of inpatient days were occupied by a MRSA-positive patient (colonized patient-days) who received decolonization while hospitalized during the baseline period (this 27.9% represented those who had planned surgery) compared to 76.0% during the intervention period (P < 0.0001). The rate of MRSA transmission was 97 events (1.0%) for 9,415 admissions (2.0 transmission events/1,000 patient-days) during baseline and was 87 (1.4%) for 6,251 admissions (2.7 transmission events/1,000 patient-days) during intervention (P = 0.06; rate ratio, 0.74; 95% confidence interval [CI], 0.55 to 1.00). The MRSA nosocomial clinical disease rate was 5.9 infections/10,000 patient-days in the baseline period and was 7.2 infections/10,000 patient-days for the intervention period (rate ratio, 0.82; 95% CI, 0.46 to 1.45; P = 0.49). Decolonization of MRSA patients does not add benefit when contact precautions are used for patients colonized with MRSA in acute (hospital) care.

Methicillin-Resistant Staphylococcus aureus Control in the 21st Century: Laboratory Involvement Affecting Disease Impact and Economic Benefit from Large Population Studies.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a global health care problem. Large studies (e.g., >25,000 patients) show that active surveillance testing (AST) followed by contact precautions for positive patients is an effective approach for MRSA disease control. With this approach, the clinical laboratory will be asked to select what AST method(s) to use and to provide data monitoring outcomes of the infection prevention interventions. This minireview summarizes evidence for MRSA disease control, reviews the involvement of the laboratory, and provides examples of how to undertake a program cost analysis. Health care organizations with total MRSA clinical infections of >0.3/1,000 patient days or bloodstream infections of >0.03/1,000 patient days should implement a MRSA control plan.

Economic evaluation of laboratory testing strategies for hospital-associated Clostridium difficile infection.

Clostridium difficile infection (CDI) is the most common cause of infectious diarrhea in health care settings, and for patients presumed to have CDI, their isolation while awaiting laboratory results is costly. Newer rapid tests for CDI may reduce this burden, but the economic consequences of different testing algorithms remain unexplored. We used decision analysis from the hospital perspective to compare multiple CDI testing algorithms for adult inpatients with suspected CDI, assuming patient management according to laboratory results. CDI testing strategies included combinations of on-demand PCR (odPCR), batch PCR, lateral-flow diagnostics, plate-reader enzyme immunoassay, and direct tissue culture cytotoxicity. In the reference scenario, algorithms incorporating rapid testing were cost-effective relative to nonrapid algorithms. For every 10,000 symptomatic adults, relative to a strategy of treating nobody, lateral-flow glutamate dehydrogenase (GDH)/odPCR generated 831 true-positive results and cost $1,600 per additional true-positive case treated. Stand-alone odPCR was more effective and more expensive, identifying 174 additional true-positive cases at $6,900 per additional case treated. All other testing strategies were dominated by (i.e., more costly and less effective than) stand-alone odPCR or odPCR preceded by lateral-flow screening. A cost-benefit analysis (including estimated costs of missed cases) favored stand-alone odPCR in most settings but favored odPCR preceded by lateral-flow testing if a missed CDI case resulted in less than $5,000 of extended hospital stay costs and <2 transmissions, if lateral-flow GDH diagnostic sensitivity was >93%, or if the symptomatic carrier proportion among the toxigenic culture-positive cases was >80%. These results can aid guideline developers and laboratory directors who are considering rapid testing algorithms for diagnosing CDI.

Sensitivity of surveillance testing for multidrug-resistant Gram-negative bacteria in the intensive care unit.

We tested intensive care unit patients for colonization with multidrug-resistant Gram-negative bacilli (MDR GNB) and compared the results with those of concurrent clinical cultures. The sensitivity of the surveillance test for detecting MDR GNB was 58.8% (95% confidence interval, 48.6 to 68.5%). Among 133 patients with positive surveillance tests, 61% had no prior clinical culture with MDR GNB.

Crystal structure of the cowpox virus-encoded NKG2D ligand OMCP.

The NKG2D receptor is expressed on the surface of NK, T, and macrophage lineage cells and plays an important role in antiviral and antitumor immunity. To evade NKG2D recognition, herpesviruses block the expression of NKG2D ligands on the surface of infected cells using a diverse repertoire of sabotage methods. Cowpox and monkeypox viruses have taken an alternate approach by encoding a soluble NKG2D ligand, the orthopoxvirus major histocompatibility complex (MHC) class I-like protein (OMCP), which can block NKG2D-mediated cytotoxicity. This approach has the advantage of targeting a single conserved receptor instead of numerous host ligands that exhibit significant sequence diversity. Here, we show that OMCP binds the NKG2D homodimer as a monomer and competitively blocks host ligand engagement. We have also determined the 2.25-Å-resolution crystal structure of OMCP from the cowpox virus Brighton Red strain, revealing a truncated MHC class I-like platform domain consisting of a beta sheet flanked with two antiparallel alpha helices. OMCP is generally similar in structure to known host NKG2D ligands but has notable variations in regions typically used to engage NKG2D. Additionally, the determinants responsible for the 14-fold-higher affinity of OMCP for human than for murine NKG2D were mapped to a single loop in the NKG2D ligand-binding pocket.

Perceived Admiration and Transition to Parenthood for Black and White Married Couples.

Perceived admiration was examined in this study as a mediator of marital quality and transition to parenthood among Black American and White American couples. Both positive and negative dimensions of marital quality were assessed for husbands (N = 148) and wives (N = 155) during their first and third years of marriage in a large-scale survey. Findings revealed that transitioning Black American husbands reported lower marital tension than transitioning White American husbands. Perceived admiration mediated the link between transition to parenthood and marital wellbeing for wives, and between transition to parenthood and marital tension for husbands. Results suggest that perceived admiration plays a critical role in understanding the transition to parenthood, regardless of race. Insights are offered for practitioners who provide relationship or parental counseling and education to couples during the transition to parenthood.

Nasal Carriage of Epidemic Methicillin-Resistant Staphylococcus aureus 15 (EMRSA-15) Clone Observed in Three Chicago-Area Long-Term Care Facilities.

The spread of pandemic methicillin-resistant Staphylococcus aureus (MRSA) clones such as USA300 and EMRSA-15 is a global health concern. As a part of a surveillance study of three long-term care facilities in the Greater Chicago area, phenotypic and molecular characterization of nasal MRSA isolates was performed. We report a cluster of pandemic EMRSA-15, an MRSA clone rarely reported from the United States, detected during this study.

Multiple broad-spectrum Beta-lactamase targets for comprehensive surveillance.

Real-time PCR testing for blaKPC, blaNDM, blaVIM, blaIMP, and blaCTX-M was performed on rectal swabs obtained from residents of two long-term acute-care facilities. While blaKPC was detected in 69/102 swabs (67.6%), testing for four other targets increased the positivity rate for a broad-spectrum ß-lactamase to 73.5% (McNemar's P = 0.03).

Rectal screening for Klebsiella pneumoniae carbapenemases: comparison of real-time PCR and culture using two selective screening agar plates.

Klebsiella pneumoniae carbapenemases (KPCs) have recently been described in Chicago, IL, especially among residents of long-term acute care hospitals (LTACHs). These patients are frequently transferred to local Chicago hospitals for higher acuity of medical care, and rapid detection and isolation of KPC-colonized LTACH residents may interrupt the introduction of KPCs into acute care hospitals. We evaluated the performance of a real-time PCR for bla(KPC) from enrichment broth versus direct plating of rectal surveillance swabs on two selective culture media, CHROMagar extended-spectrum-ß-lactamase (ESBL) and vancomycin, amphotericin B, ceftazidime, and clindamycin (VACC) plates. Rectal surveillance swabs were collected as part of a point prevalence study of KPC carriage rates among 95 residents of two Chicago area LTACHs. Discrepant results between PCR and culture were resolved by subculturing the enrichment broth. Overall, 66 of 95 patients (69.5%) were colonized with KPCs, using the cumulative results of culture as a reference standard. Real-time PCR from enrichment broth was positive in 64 of 66 (97%) colonized patients, including nine surveillance swabs that were missed by both selective culture media. PCR demonstrated higher sensitivity, 97.0%, than culture using either CHROMagar or VACC plates (both with sensitivity of 77.3%). In addition, turnaround time was significantly shorter for the PCR-based method than for culture, with a mean of 24 h versus 64 to 72 h for CHROMagar and VACC plates (P < 0.0001). Overall, PCR for bla(KPC) represents the best screening test for KPCs with significantly higher sensitivity and with less hands-on time, resulting in a shorter time to results.

Multicenter clinical evaluation of the portrait toxigenic C. difficile assay for detection of toxigenic Clostridium difficile strains in clinical stool specimens.

We compared the Portrait Toxigenic C. difficile Assay, a new semiautomated sample-to-result molecular test, to a toxigenic bacterial culture/cell cytotoxin neutralization assay (TBC/CCNA) for the detection of toxigenic Clostridium difficile in 549 stool specimens. Stool specimens were also tested by one of three alternative FDA-cleared molecular tests for toxigenic C. difficile (Xpert C. difficile, Illumigene C. difficile, or GeneOhm Cdiff). The sensitivities and specificities of the molecular tests compared to TBC/CCNA were as follows: 98.2% and 92.8% for the Portrait assay, 100% and 91.7% for the Xpert assay, 93.3% and 95.1% for the Illumigene assay, and 97.4% and 98.5% for the GeneOhm assay, respectively. The majority of Portrait false-positive results (20/31; 64.5%) were also positive for C. difficile by an alternative molecular test, suggesting an increased sensitivity compared to the culture-based "gold standard" method. The Portrait test detected an assay input of 30 CFU in 100% of spiked samples and detected an input of 10 CFU in 96.7% of samples tested.

Methicillin-resistant staphylococcus aureus nosocomial infection has a distinct epidemiological position and acts as a marker for overall hospital-acquired infection trends.

An ongoing healthcare debate is whether controlling hospital-acquired infection (HAI) from methicillin-resistant Staphylococcus aureus (MRSA) will result in lowering the global HAI rate, or if MRSA will simply be replaced by another pathogen and there will be no change in overall disease burden. With surges in drug-resistant hospital-acquired pathogens during the COVID-19 pandemic, this remains an important issue. Using a dataset of more than 1 million patients in 51 acute care facilities across the USA, and with the aid of a threshold model that models the nonlinearity in outbreaks of diseases, we show that MRSA is additive to the total burden of HAI, with a distinct 'epidemiological position', and does not simply replace other microbes causing HAI. Critically, as MRSA is reduced it is not replaced by another pathogen(s) but rather lowers the overall HAI burden. The analysis also shows that control of MRSA is a benchmark for how well all non-S. aureus nosocomial infections in the same hospital are prevented. Our results are highly relevant to healthcare epidemiologists and policy makers when assessing the impact of MRSA on hospitalized patients. These findings further stress the major importance of MRSA as a unique cause of nosocomial infections, as well as its pivotal role as a biomarker in demonstrating the measured efficacy (or lack thereof) of an organization's Infection Control program.