Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2024 Update by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM).

The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by experts in both adult and pediatric laboratory and clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including arboviral Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also addressed. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

A tyrosine phospho-switch within the Longin domain of VAMP721 modulates SNARE functionality.

The final step in secretion is membrane fusion facilitated by SNARE proteins that reside in opposite membranes. The formation of a trans-SNARE complex between one R and three Q coiled-coiled SNARE domains drives the final approach of the membranes providing the mechanical energy for fusion. Biological control of this mechanism is exerted by additional domains within some SNAREs. For example, the N-terminal Longin domain (LD) of R-SNAREs (also called Vesicle-associated membrane proteins, VAMPs) can fold back onto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine the subcellular localization via interaction with other trafficking-related proteins. Here, we provide cell-biological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionality of VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instability and subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects of vamp721vamp722 loss-of-function lines in spite of its wildtype-like localization within the secretory pathway and the ability to interact with cognate SNARE partners. Most importantly, it imposes a dominant negative phenotype interfering with root growth, normal secretion and cytokinesis in wildtype plants generating large aggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57F needs higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning that phosphorylation modulates SNARE function. We propose a model where short-lived phosphorylation of Y57 serves as a regulatory step to control VAMP721 activity, favoring its open state and interaction with cognate partners to ultimately drive membrane fusion.

NSF/αSNAP2-mediated cis-SNARE complex disassembly precedes vesicle fusion in Arabidopsis cytokinesis.

Eukaryotic membrane fusion requires trans-SNARE complexes bridging the gap between adjacent membranes1. Fusion between a transport vesicle and its target membrane transforms the trans- into a cis-SNARE complex. The latter interacts with the hexameric AAA+-ATPase N-ethylmaleimide-sensitive factor (NSF) and its co-factor alpha-soluble NSF attachment protein (αSNAP), forming a 20S complex2,3. ATPase activity disassembles the SNAP receptor (SNARE) complex into Qa-SNARE, which folds back onto itself, and its partners4,5. The fusion of identical membranes has a different sequence of events6. The fusion partners each have cis-SNARE complexes to be broken up by NSF and αSNAP. The Qa-SNARE monomers are then stabilized by interaction with Sec1/Munc18-type regulators (SM proteins) to form trans-SNARE complexes, as shown for the yeast vacuole7. Membrane fusion in Arabidopsis cytokinesis is formally akin to vacuolar fusion8. Membrane vesicles fuse with one another to form the partitioning membrane known as the cell plate. Cis-SNARE complexes of cytokinesis-specific Qa-SNARE KNOLLE and its SNARE partners are assembled at the endoplasmic reticulum and delivered by traffic via the Golgi/trans-Golgi network to the cell division plane9. The SM protein KEULE is required for the formation of trans-SNARE complexes between adjacent membrane vesicles10. Here we identify NSF and its adaptor αSNAP2 as necessary for the disassembly of KNOLLE cis-SNARE complexes, which is a prerequisite for KNOLLE-KEULE interaction in cytokinesis. In addition, we show that NSF is required for other trafficking pathways and interacts with the respective Q-SNAREs. The SNARE complex disassembly machinery is conserved in plants and plays a unique essential role in cytokinesis.

N-terminal domain of ARF-GEF GNOM prevents heterodimerization with functionally divergent GNL1 in Arabidopsis.

Evolutionary change following gene duplication can lead to functionally divergent paralogous proteins. If comprising identical subunits their random assortment would also form potentially detrimental heteromeric proteins. In Arabidopsis, the ARF GTPase guanine-nucleotide exchange factor GNOM is essential for polar recycling of auxin-efflux transporter PIN1 from endosomes to the basal plasma membrane whereas its paralog GNL1 mediates retrograde Golgi-endoplasmic reticulum traffic. Here we show that both GNOM and GNL1 form homodimers but no heterodimers. To assess the biological significance of this, we generated transgenic plants expressing engineered heterodimer-compatible GNOM variants. Those plants showed developmental defects such as the failure to produce lateral roots. To identify mechanisms underlying heterodimer prevention, we analyzed interactions of the N-terminal dimerization and cyclophilin-binding (DCB) domain. Each DCB domain interacted with the complementary fragment (ΔDCB) both of their own and of the paralogous protein. However, only DCBGNOM interacted with itself whereas DCBGNL1 failed to interact with itself and with DCBGNOM . GNOM variants in which the DCB domain was removed or replaced by DCBGNL1 revealed a role for DCB-DCB interaction in the prevention of GNOM-GNL1 heterodimers whereas DCB-ΔDCB interaction was essential for dimer formation and GNOM function. Our data suggest a model of early DCB-DCB interaction that facilitates GNOM homodimer formation, indirectly precluding formation of detrimental heterodimers.

Multicenter Evaluation of a Gradient Diffusion Method for Antimicrobial Susceptibility Testing of Helicobacter pylori.

Helicobacter pylori is an important human pathogen associated with peptic ulcer disease, dyspepsia, and gastric malignancy. Antimicrobial susceptibility testing (AST) is often requested for patients who fail eradication therapy. The Clinical and Laboratory Standards Institute (CLSI) reference method, agar dilution (AD), is not performed in most laboratories and maintaining organism viability during transit to a reference laboratory is difficult. We assessed the performance of the Etest (bioMérieux) as a method for H. pylori AST in comparison to AD. Etest MICs were determined for 83 H. pylori isolates at ARUP and Cleveland Clinic (CC). Categorical agreement (CA), very major, major, and minor errors (VME, ME, and mE) were determined for Etest using AD performed at Mayo Clinic Laboratories as the reference method. Testing on isolates with errors was repeated to determine final results summarized below. For clarithromycin, 66.3% of isolates were resistant (R) by AD; Etest results at each laboratory showed 1mE (1.2%) and 1 ME (3.8%). For tetracycline, only 2 isolates were R by AD; a single VME occurred at both sites (98.8% CA, 50% VME) with the same isolate. Applying EUCAST levofloxacin breakpoints to interpret ciprofloxacin results, 60.2% of isolates were R by AD; ARUP CA was 97.6% (1 ME (3%), 1 VME (2%)) and CC CA was 96.3% (1 ME (3%), 2 VMEs (4%)). Despite high error rates, the categorical agreement was acceptable (>90%) for all three antibiotics between AD and Etest. In-house susceptibility testing by gradient diffusion can allow for testing of fastidious organisms that may not survive transport to specialized laboratories; however, the method is not without technical challenges. Characterization of resistance mechanisms, increased AD dilutions, and testing from the same inoculum may determine if the observed errors reflect technical issues or breakpoints that need optimization. IMPORTANCE Routine antimicrobial susceptibility testing (AST) of Helicobacter pylori by agar dilution is difficult to perform and not practical in most clinical microbiology laboratories. The Etest gradient diffusion method can be a reliable alternative for H. pylori AST with the advantage of being a less laborious quantitative method. This work reveals that an optimized Etest method can provide acceptable performance for H. pylori AST and describes the challenges associated with this methodology.

Carbapenem-Resistant Acinetobacter baumannii in U.S. Hospitals: Diversification of Circulating Lineages and Antimicrobial Resistance.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is a major cause of health care-associated infections. CRAb is typically multidrug resistant, and infection is difficult to treat. Despite the urgent threat that CRAb poses, few systematic studies of CRAb clinical and molecular epidemiology have been conducted. The Study Network of Acinetobacter as a Carbapenem-Resistant Pathogen (SNAP) is designed to investigate the clinical characteristics and contemporary population structure of CRAb circulating in U.S. hospital systems using whole-genome sequencing (WGS). Analysis of the initial 120 SNAP patients from four U.S. centers revealed that CRAb remains a significant threat to hospitalized patients, affecting the most vulnerable patients and resulting in 24% all-cause 30-day mortality. The majority of currently circulating isolates belonged to ST2Pas, a part of clonal complex 2 (CC2), which is the dominant drug-resistant lineage in the United States and Europe. We identified three distinct sublineages within CC2, which differed in their antibiotic resistance phenotypes and geographic distribution. Most concerning, colistin resistance (38%) and cefiderocol resistance (10%) were common within CC2 sublineage C (CC2C), where the majority of isolates belonged to ST2Pas/ST281Ox. Additionally, we identified ST499Pas as the most common non-CC2 lineage in our study. Our findings suggest a shift within the CRAb population in the United States during the past 10 years and emphasize the importance of real-time surveillance and molecular epidemiology in studying CRAb dissemination and clinical impact. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii (CRAb) constitutes a major threat to public health. To elucidate the molecular and clinical epidemiology of CRAb in the United States, clinical CRAb isolates were collected along with data on patient characteristics and outcomes, and bacterial isolates underwent whole-genome sequencing and antibiotic susceptibility phenotyping. Key findings included emergence of new sublineages within the globally predominant clonal complex 2 (CC2), increased colistin and cefiderocol resistance within one of the CC2 sublineages, and emergence of ST499Pas as the dominant non-CC2 CRAb lineage in U.S. hospitals.

What do positive and negative Cutibacterium culture results in periprosthetic shoulder infection mean? A multi-institutional control study.

BACKGROUND: Deep tissue culture specimens obtained at the time of revision shoulder arthroplasty are commonly positive for Cutibacterium. Clinical interpretation of positive cultures can be difficult. This was a multi-institutional study evaluating the accuracy of cultures for Cutibacterium using positive control (PC) and negative control (NC) samples. The relationship between time to culture positivity and strength of culture positivity was also studied. METHODS: Eleven different institutions were each sent 12 blinded samples (10 PC and 2 NC samples). The 10 PC samples included 2 sets of 5 different dilutions of a Cutibacterium isolate from a failed total shoulder arthroplasty with a probable periprosthetic infection. At each institution, the samples were handled as if they were received from the operating room. Specimen growth, time to culture positivity, and strength of culture positivity (based on semiquantitative assessment) were reported. RESULTS: A total of 110 PC samples and 22 NC samples were tested. One hundred percent of specimens at the 4 highest dilutions were positive for Cutibacterium. At the lowest dilution, 91% of samples showed positive findings. Cutibacterium grew in 14% of NC samples. Cutibacterium grew in PC samples at an average of 4.0 ± 1.3 days, and all of these samples showed growth within 7 days. The time to positivity was significantly shorter (P < .001) and the strength of positivity was significantly higher (P < .001) in true-positive cultures compared with false-positive cultures. CONCLUSIONS: This multi-institutional study suggests that different institutions may report highly consistent rates of culture positivity for revision shoulder arthroplasty samples with higher bacterial loads. In contrast, with lower bacterial loads, the results are somewhat less consistent. Clinicians should consider using a shorter time to positivity and a higher strength of positivity as adjuncts in determining whether a tissue culture sample is a true positive.

A data management infrastructure for the integration of imaging and omics data in life sciences.

BACKGROUND: As technical developments in omics and biomedical imaging increase the throughput of data generation in life sciences, the need for information systems capable of managing heterogeneous digital assets is increasing. In particular, systems supporting the findability, accessibility, interoperability, and reusability (FAIR) principles of scientific data management. RESULTS: We propose a Service Oriented Architecture approach for integrated management and analysis of multi-omics and biomedical imaging data. Our architecture introduces an image management system into a FAIR-supporting, web-based platform for omics data management. Interoperable metadata models and middleware components implement the required data management operations. The resulting architecture allows for FAIR management of omics and imaging data, facilitating metadata queries from software applications. The applicability of the proposed architecture is demonstrated using two technical proofs of concept and a use case, aimed at molecular plant biology and clinical liver cancer research, which integrate various imaging and omics modalities. CONCLUSIONS: We describe a data management architecture for integrated, FAIR-supporting management of omics and biomedical imaging data, and exemplify its applicability for basic biology research and clinical studies. We anticipate that FAIR data management systems for multi-modal data repositories will play a pivotal role in data-driven research, including studies which leverage advanced machine learning methods, as the joint analysis of omics and imaging data, in conjunction with phenotypic metadata, becomes not only desirable but necessary to derive novel insights into biological processes.

The Implementation and Effect of Weekend Pharmacy-Driven Antimicrobial Stewardship Services at a Large Academic Medical Center.

BACKGROUND: In September 2018, pharmacy antimicrobial stewardship (AMS) services were expanded to include weekends at this academic medical center. Activities performed by AMS pharmacists on the weekends include blood culture rapid diagnostic (RDT) review, antiretroviral therapy (ART) review, prospective audit and feedback (PAF) utilizing clinical decision support, vancomycin dosing, and operational support. The purpose of this study was to assess the operational and clinical impact of these expanded AMS services. METHODS: This single-center, quasi-experimental study included data from weekends before (9/2017-11/2017) and after (9/2018-11/2018) implementation. The descriptive primary outcome was the number of activities completed for each AMS activity type in the post-implementation group only. Secondary outcomes were time to AMS opportunity resolution, time to escalation or de-escalation following PAF or RDT alert, time to resolution of miscellaneous AMS related opportunities, length of stay (LOS), and antimicrobial use outcomes. RESULTS: During the post-implementation period 1258 activities were completed, averaging 97/weekend. Inclusion criteria for time to resolution outcomes were met by 72 patients pre-implementation and 59 patients post. The median (IQR) time to AMS opportunity resolution decreased from 18.5 hours pre-intervention (7.7-35.7) to 8.5 hours post-intervention (IQR 1.8-14.0), p < 0.01. Time to escalation was 11.6 hours compared to 1.7 hours (p = 0.1), de-escalation 16.7 hours compared to 10.8 hours (p = 0.03), and miscellaneous opportunity 40.8 hours compared to 13.2 hours (p = 0.01). No differences were observed in LOS or antimicrobial use outcomes. CONCLUSION: Presence of pharmacist-driven weekend AMS services significantly reduced time to resolution of AMS opportunities. These data support the value of weekend AMS services.

Multicenter evaluation of the VITEK MS matrix-assisted laser desorption/ionization-time of flight mass spectrometry system for identification of bacteria, including Brucella, and yeasts.

The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has proven to be rapid and accurate for the majority of clinical isolates. Some gaps remain concerning rare, emerging, or highly pathogenic species, showing the need to continuously expand the databases. In this multicenter study, we evaluated the accuracy of the VITEK MS v3.2 database in identifying 1172 unique isolates compared to identification by DNA sequence analysis. A total of 93.6% of the isolates were identified to species or group/complex level. A remaining 5.2% of the isolates were identified to the genus level. Forty tests gave a result of no identification (0.9%) and 12 tests (0.3%) gave a discordant identification compared to the reference identification. VITEK MS is also the first MALDI-TOF MS system that is able to delineate the four members of the Acinetobacter baumannii complex at species level without any specific protocol or special analysis method. These findings demonstrate that the VITEK MS v3.2 database is highly accurate for the identification of bacteria and fungi encountered in the clinical laboratory as well as emerging species like Candida auris and the highly pathogenic Brucella species.

Real-time polymerase chain reaction (PCR) cycle threshold and Clostridioides difficile infection outcomes.

OBJECTIVE: Clostridioides difficile infection (CDI) causes significant morbidity and mortality; however, the diagnosis of CDI remains controversial. The primary aim of our study was to evaluate the association of polymerase chain reaction (PCR) cycle threshold (Ct) values with CDI disease severity, recurrence, and mortality among adult patients with CDI. DESIGN: Retrospective cohort study. SETTING: Single tertiary-care hospital. PATIENTS: Adult patients diagnosed with hospital-onset, healthcare facility-associated CDI from June 2014 to September 2015. METHODS: We performed a retrospective chart review of included patients. Univariate and multivariable logistic regression methods were used to evaluate the association between Ct values and CDI severity, 8-week recurrence, and 30-day mortality. RESULTS: Among 318 included patients, 51% were male and the mean age was 62 years; ~32% of the patients developed severe CDI and 11% developed severe-complicated CDI. The 30-day all-cause mortality rate was 11% and the 8-week recurrence rate was 9.5%. The overall mean Ct value was 32.9 (range, 23-40). Multivariable analyses showed that lower values of PCR Ct were associated with increased odds of 30-day morality (odds ratio [OR] 0.83; 95% confidence interval [CI], 0.72-0.96) but were not independently associated with CDI severity (OR, 0.99; 95% CI, 0.90-1.09) or recurrence (OR, 0.88; 95% CI, 0.77-1.00). CONCLUSIONS: Our findings suggest that PCR Ct values at the time of diagnosis may have a limited predictive value and utility in clinical decision making for inpatients with CDI. Larger, prospective studies across different patient populations are needed to confirm our findings.

De-escalation of Empiric Antibiotics Following Negative Cultures in Hospitalized Patients With Pneumonia: Rates and Outcomes.

BACKGROUND: For patients at risk for multidrug-resistant organisms, IDSA/ATS guidelines recommend empiric therapy against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas. Following negative cultures, the guidelines recommend antimicrobial de-escalation. We assessed antibiotic de-escalation practices across hospitals and their associations with outcomes in hospitalized patients with pneumonia with negative cultures. METHODS: We included adults admitted with pneumonia in 2010-2015 to 164 US hospitals if they had negative blood and/or respiratory cultures and received both anti-MRSA and antipseudomonal agents other than quinolones. De-escalation was defined as stopping both empiric drugs on day 4 while continuing another antibiotic. Patients were propensity adjusted for de-escalation and compared on in-hospital 14-day mortality, late deterioration (ICU transfer), length-of-stay (LOS), and costs. We also compared adjusted outcomes across hospital de-escalation rate quartiles. RESULTS: Of 14 170 patients, 1924 (13%) had both initial empiric drugs stopped by hospital day 4. Hospital de-escalation rates ranged from 2-35% and hospital de-escalation rate quartile was not significantly associated with outcomes. At hospitals in the top quartile of de-escalation, even among patients at lowest risk for mortality, the de-escalation rates were <50%. In propensity-adjusted analysis, patients with de-escalation had lower odds of subsequent transfer to ICU (adjusted odds ratio, .38; 95% CI, .18-.79), LOS (adjusted ratio of means, .76; .75-.78), and costs (.74; .72-.76). CONCLUSIONS: A minority of eligible patients with pneumonia had antibiotics de-escalated by hospital day 4 following negative cultures and de-escalation rates varied widely between hospitals. To adhere to recent guidelines will require substantial changes in practice.

Multicenter Performance Evaluation of the Simplexa Bordetella Direct Kit in Nasopharyngeal Swab Specimens.

Detection of Bordetella pertussis and Bordetella parapertussis using molecular methods is sensitive and specific with a short turnaround time compared to other diagnostic methods. In this multicenter study, we compared the performance of the Simplexa Bordetella Direct kit to those of other molecular assays in detecting and differentiating B. pertussis and B. parapertussis in nasopharyngeal swab specimens. The limits of detection (LODs) were 150 CFU/ml or 3 fg/µl of DNA for B. pertussis and 1,500 CFU/ml or 10 fg/µl of DNA for B. parapertussis A total of 1,103 fresh and residual frozen specimens from eight clinical sites were tested. Combining the data from individual clinical sites using different comparative assays, the overall positive percent agreement (PPA) and negative percent agreement (NPA) for B. pertussis were 98.7% and 97.3%, respectively. The overall PPA and NPA for B. parapertussis were 96.7% and 100%, respectively. For prospective fresh specimens, the overall PPA and NPA for both targets were 97.7% and 99.3%, respectively. For retrospective frozen specimens, the overall PPA and NPA for both targets were 92.6% and 93.2%, respectively. The percentage of invalid results was 1.0%. A cross-reactivity study using 74 non-Bordetella bacterial species and five yeast species revealed that the Simplexa Bordetella Direct kit was 100% specific. The hands-on time and assay run time of the Simplexa Bordetella Direct kit are favorable compared to those of other commercial and laboratory-developed tests. In summary, the Simplexa Bordetella Direct kit has a performance comparable to those of other molecular assays for the detection of B. pertussis and B. parapertussis.

Ecto-Nucleotide Triphosphate Diphosphohydrolase-2 (NTPDase2) Deletion Increases Acetaminophen-Induced Hepatotoxicity.

Ecto-nucleotidase triphosphate diphosphohydrolase-2 (NTPDase2) is an ecto-enzyme that is expressed on portal fibroblasts in the liver that modulates P2 receptor signaling by regulating local concentrations of extracellular ATP and ADP. NTPDase2 has protective properties in liver fibrosis and may impact bile duct epithelial turnover. Here, we study the role of NTPDase2 in acute liver injury using an experimental model of acetaminophen (APAP) intoxication in mice with global deletion of NTPDase2. Acute liver toxicity was caused by administration of acetaminophen in wild type (WT) and NTPDase2-deficient (Entpd2 null) mice. The extent of liver injury was compared by histology and serum alanine transaminase (ALT). Markers of inflammation, regeneration and fibrosis were determined by qPCR). We found that Entpd2 expression is significantly upregulated after acetaminophen-induced hepatotoxicity. Entpd2 null mice showed significantly more necrosis and higher serum ALT compared to WT. Hepatic expression of IL-6 and PDGF-B are higher in Entpd2 null mice. Our data suggest inducible and protective roles of portal fibroblast-expressed NTPDase2 in acute necrotizing liver injury. Further studies should investigate the relevance of these purinergic pathways in hepatic periportal and sinusoidal biology as such advances in understanding might provide possible therapeutic targets.

Assessment of the Accuracy of Using ICD-9 Diagnosis Codes to Identify Pneumonia Etiology in Patients Hospitalized With Pneumonia.

Importance: Administrative databases may offer efficient clinical data collection for studying epidemiology, outcomes, and temporal trends in health care delivery. However, such data have seldom been validated against microbiological laboratory results. Objective: To assess the validity of International Classification of Diseases, Ninth Revision (ICD-9) organism-specific administrative codes for pneumonia using microbiological data (test results for blood or respiratory culture, urinary antigen, or polymerase chain reaction) as the criterion standard. Design, Setting, and Participants: Cross-sectional diagnostic accuracy study conducted between February 2017 and June 2019 using data from 178 US hospitals in the Premier Healthcare Database. Patients were aged 18 years or older admitted with pneumonia and discharged between July 1, 2010, and June 30, 2015. Data were analyzed from February 14, 2017, to June 27, 2019. Exposures: Organism-specific pneumonia identified from ICD-9 codes. Main Outcomes and Measures: Sensitivity, specificity, positive predictive value, and negative predictive value of ICD-9 codes using microbiological data as the criterion standard. Results: Of 161 529 patients meeting inclusion criteria (mean [SD] age, 69.5 [16.2] years; 51.2% women), 35 759 (22.1%) had an identified pathogen. ICD-9-coded organisms and laboratory findings differed notably: for example, ICD-9 codes identified only 14.2% and 17.3% of patients with laboratory-detected methicillin-sensitive Staphylococcus aureus and Escherichia coli, respectively. Although specificities and negative predictive values exceeded 95% for all codes, sensitivities ranged downward from 95.9% (95% CI, 95.3%-96.5%) for influenza virus to 14.0% (95% CI, 8.8%-20.8%) for parainfluenza virus, and positive predictive values ranged downward from 91.1% (95% CI, 89.5%-92.6%) for Staphylococcus aureus to 57.1% (95% CI, 39.4%-73.7%) for parainfluenza virus. Conclusions and Relevance: In this study, ICD-9 codes did not reliably capture pneumonia etiology identified by laboratory testing; because of the high specificities of ICD-9 codes, however, administrative data may be useful in identifying risk factors for resistant organisms. The low sensitivities of the diagnosis codes may limit the validity of organism-specific pneumonia prevalence estimates derived from administrative data.

Multicenter evaluation of the RAPIDEC® CARBA NP assay for the detection of carbapenemase production in clinical isolates of Enterobacterales and Pseudomonas aeruginosa.

Carbapenem-resistant Gram-negative bacilli are a major public health problem. Accurate and rapid detection of carbapenemase-producing organisms can facilitate appropriate infection prevention measures. The objective was to evaluate the performance of the RAPIDEC® CARBA NP assay (RAPIDEC), a screening assay that utilizes a pH indicator to detect carbapenem hydrolysis within 2 h. A multicenter study evaluated 306 clinical bacterial strains of Enterobacterales (n = 257) and Pseudomonas aeruginosa (n = 49). The RAPIDEC was compared to a composite reference standard-the Clinical Laboratory Standards Institute (CLSI) Carba NP assay, PCR for specific carbapenemase genes (blaKPC, blaNDM, blaOXA-48-like, blaVIM and blaIMP), and phenotypic carbapenem susceptibility testing. The assay was evaluated using two culture incubation times for the bacterial isolates: "routine"(cultures incubated 18-24 h) and "short" (cultures incubated 4-5 h). For the routine incubation, the overall percent agreement was 98.7% with a positive percent agreement (PPA) of 99.6% and a negative percent agreement (NPA) of 97.4%; there were five false positives and one false negative. For the short incubation, the overall percent agreement was 98.0% with a PPA of 98.5% and a NPA of 97.3%; there were five false positives and four false negatives. RAPIDEC results for the P. aeruginosa isolates were 100% concordant with the reference standard for both incubation times. The RAPIDEC assay is an accurate and rapid (≤ 2 h) assay for the detection of the most common carbapenemases in clinical isolates. Growth from a short incubation culture may be used to reliably detect carbapenemase production in clinical strains.

Coordinated Activation of ARF1 GTPases by ARF-GEF GNOM Dimers Is Essential for Vesicle Trafficking in Arabidopsis.

Membrane trafficking maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations, such as sites of action or degradation. The formation of membrane vesicles requires the activation of the ADP-ribosylation factor ARF GTPase by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs form dimers in vivo. This feature is conserved across eukaryotes, although its biological significance is unknown. Here, we demonstrate the proximity of ARF1•GTPs in vivo by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy, mediated through coordinated activation by dimers of Arabidopsis (Arabidopsis thaliana) ARF-GEF GNOM, which is involved in polar recycling of the auxin transporter PIN-FORMED1. Mutational disruption of ARF1 spacing interfered with ARF1-dependent trafficking but not with coat protein recruitment. A mutation impairing the interaction of one of the two SEC7 domains of the GNOM ARF-GEF dimer with its ARF1 substrate reduced the efficiency of coordinated ARF1 activation. Our results suggest a model of coordinated activation-dependent membrane insertion of ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

Mass-spectrometry-based draft of the Arabidopsis proteome.

Plants are essential for life and are extremely diverse organisms with unique molecular capabilities1. Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions.

Pneumococcal Urinary Antigen Testing in United States Hospitals: A Missed Opportunity for Antimicrobial Stewardship.

BACKGROUND: The Infectious Diseases Society of America recommends pneumococcal urinary antigen testing (UAT) when identifying pneumococcal infection would allow for antibiotic de-escalation. However, the frequencies of UAT and subsequent antibiotic de-escalation are unknown. METHODS: We conducted a retrospective cohort study of adult patients admitted with community-acquired or healthcare-associated pneumonia to 170 US hospitals in the Premier database from 2010 to 2015, to describe variation in UAT use, associations of UAT results with antibiotic de-escalation, and associations of de-escalation with outcomes. RESULTS: Among 159 894 eligible admissions, 24 757 (15.5%) included UAT performed (18.4% of intensive care unit [ICU] and 15.3% of non-ICU patients). Among hospitals with ≥100 eligible patients, UAT proportions ranged from 0% to 69%. Compared to patients with negative UAT, 7.2% with positive UAT more often had a positive Streptococcus pneumoniae culture (25.4% vs 1.9%, P < .001) and less often had resistant bacteria (5.2% vs 6.8%, P < .05). Of patients initially treated with broad-spectrum antibiotics, most were still receiving broad-spectrum therapy 3 days later, but UAT-positive patients more often had coverage narrowed (38.4% vs 17.0% UAT-negative and 14.6% untested patients, P < .001). Hospital rate of UAT was strongly correlated with de-escalation following a positive test. Only 3 patients de-escalated after a positive UAT result were subsequently admitted to ICU. CONCLUSIONS: UAT is not ordered routinely in pneumonia, even in ICU. A positive UAT result was associated with less frequent resistant organisms, but usually did not lead to antibiotic de-escalation. Increasing UAT and narrowing therapy after a positive UAT result are opportunities for improved antimicrobial stewardship.