Citations

ABSTRACT Metallo-beta-lactamase (MBL)-producing Enterobacterales are a global public health concern. Infections caused by these pathogens are often hard to treat, with few effective agents currently available. While aztreonam-avibactam only recently gained U.S. and European regulatory approval, laboratories have implemented laboratory-developed tests (LDTs) using existing aztreonam and ceftazidime-avibactam reagents. We sought to compare the performance of four phenotypic methods, up to three reagent manufacturers, and two media manufacturers. One hundred two MBL-producing Enterobacterales isolates were evaluated at two sites (Centers for Disease Control and Prevention [CDC] and Johns Hopkins University [JHU] School of Medicine). The performance of four methods was evaluated: broth disk elution (BDE), disk stacking (DS), gradient diffusion strip stacking (SS), and a strip-to-disk proximity (strip-disk) method, which were compared to reference broth microdilution (rBMD) tested in parallel. Respective category agreement for CDC and JHU was 92.2% and 96.1% for BDE, 51%–76.5% and 31%–49% for DS, 71.6%–86.3% and 83.3%–86.3% for SS, and 82.4%–87.3% and 80%–89.2% for the strip-disk method. The SS essential agreement ranged from 46.1% to 62.7% for JHU and 54.9% to 92.2% for CDC. Interlaboratory reproducibility was 93.1% for rBMD, 92.2% for BDE, 47.1%–53.1% for DS, 76.5%–80% for SS, and 87.3%–88.9% for strip-disk. BDE was the only method that exceeded acceptable performance of >90% accuracy and reproducibility. DS had the lowest and most variable performance between sites and among the different disk manufacturers used. LDTs for combination testing can be highly variable, and further work is needed to improve standardization and harmonization to achieve accurate and reliable results for clinical purposes. IMPORTANCE Carbapenem-resistant Enterobacterales are classified as an urgent public health threat in the Centers for Disease Control and Prevention’s 2019 Antibiotic Resistance Threats Report. Among the most concerning resistance mechanisms are carbapenemase enzymes encoded on mobile genetic elements, which can spread rapidly between bacteria. Of the various carbapenemases, metallo-beta-lactamases are particularly challenging, as they confer resistance to nearly all beta-lactams, and current treatment options remain limited. While aztreonam-avibactam has been U.S. Food and Drug Administration (FDA)-approved for treating certain infections with limited or no alternative treatment options, no FDA-cleared susceptibility test is yet available. In this context, laboratory-developed methods, such as combination testing of aztreonam and ceftazidime-avibactam, play a critical role in guiding therapy. Our study evaluates several of the more common laboratory-developed tests compared to reference broth microdilution and found that broth disk elution was the most accurate; in contrast, other methods had variable performance, some demonstrating limited reliability and being prone to overcalling resistance.

ABSTRACT Background Long‐term outcome data for stiff person spectrum disorder (SPSD) remain limited and sustained response to treatment varies. Methods In this retrospective cohort study, we included patients with probable or definite SPSD (Mayo Clinic criteria), evaluated at Mayo Clinic (12/1995‐07/2024) with ≥ 5 years of follow‐up after diagnosis. Primary outcome measures included the modified Rankin scale (mRS) and use of gait aids. Results Fifty‐four patients were included, 41 (76%) were female; median age at onset was 45 years (16–78). Most were antibody‐positive (51 [94%]), with high‐titer (serum, ≥ 20 nmol/L) GAD65‐IgG in 45 (83%). Nine patients had a stiff‐limb presentation (17%). The median interval from onset to diagnosis was 2 years (0–34), and median follow‐up was 9 years (5–26). Most patients (46 [85%]) received immune therapy; 37 (82%) reported improvement. All patients had significant impairment (mRS ≥ 2) at nadir, with 31 (57%) losing functional independence (mRS ≥ 3) and 40 (75%) requiring bilateral gait assistance. The median time from onset to nadir was 6 years (0–39). At last follow‐up, most (37 [69%]) were functionally independent (mRS ≤ 2), including 8 (15%) with minimal restrictions (mRS ≤ 1). Bilateral gait assistance was needed in 22 (41%). Among 40 patients with ≥ 10 years of follow‐up, 22 (56%) had an mRS ≤ 2 and 25 (63%) required a gait aid at last follow‐up. Poor outcome in this population was associated with bilateral gait assistance at nadir and older age ( p < 0.05). Conclusions Although over half of SPSD patients lose functional independence at disease nadir, many regain independence and achieve well‐controlled symptoms.

Background/Objectives: Hearing loss is frequently misunderstood by communication partners and healthcare providers of patients with hearing loss alike, leading to gaps in counseling, medical adherence, and empathy. Hearing loss simulation is a technique that can clarify patient experience by illustrating how hearing loss affects daily listening and speech discrimination. The current research explores the influences of a web-based hearing loss simulator on how hearing loss is understood by communication partners, audiologists, and medical providers/students. Methods: Using a convergent parallel mixed-methods design, 72 participants (communication partners, audiologists, and medical providers/residents) completed a guided trial with the hearing simulator, either one-on-one or in group settings, and subsequently completed a questionnaire regarding their experiences. The simulation trial included demonstrations of hearing loss and amplification across four listening environments: speech in quiet, in noise, at a distance, as well as listening to instrumental music. Results: Communication partners reported that they gained a better understanding of their partner’s hearing loss, with all expressing increased feelings of empathy towards their partner and individuals with hearing loss. When compared to audiologists, communication partners were significantly more surprised by the impact of hearing loss on speech understanding. Medical providers and students emphasized the value of the simulator as a counseling tool and highly recommended that other providers use the simulator with their patients. Conclusions: Simulating hearing loss can be beneficial for counseling purposes and medical training. These findings suggest that hearing loss simulation can enhance empathy and communication across clinical and educational contexts.

Complete revascularization improves outcomes in patients with ST-segment elevation myocardial infarction (STEMI) and multivessel coronary artery disease (MVD); however, the optimal timing of noninfarct-related artery intervention during the index procedure, immediate revascularization (IR) versus a staged approach (SR), remains uncertain. We conducted a systematic review and meta-analysis of randomized controlled trials comparing SR and IR in patients with STEMI and MVD. The primary outcome was major adverse cardiovascular events. Secondary outcomes included all-cause and cardiovascular mortality, recurrent myocardial infarction, unplanned ischemia-driven revascularization, stent thrombosis, stroke, major bleeding, acute kidney injury, and heart failure hospitalization. Random-effects models with Hartung–Knapp adjustment were used. Trial sequential analysis assessed evidence conclusiveness, and the certainty of evidence was evaluated using the Grading of Recommendations Assessment, Development, and Evaluation. Eight randomized trials, including 5077 patients (2556 SR; 2521 IR), were analyzed. No significant differences were observed between SR and IR for major adverse cardiovascular events [odds ratios (ORs), 1.07; 95% confidence intervals (CI), 0.76–1.49], recurrent myocardial infarction (OR, 1.30; 95% CI, 0.80–2.14), cardiovascular mortality (OR, 0.76; 95% CI, 0.51–1.13), or all-cause mortality (OR, 0.75; 95% CI, 0.54–1.06). Rates of stent thrombosis, stroke, unplanned ischemia-driven revascularization, major bleeding, acute kidney injury, and heart failure hospitalization were also comparable. Trial sequential analysis indicated insufficient information size to draw definitive conclusions, and the certainty of evidence ranged from very low to low. In patients with STEMI and MVD, staged and immediate complete revascularization provide comparable ischemic, safety, and mortality outcomes. Current evidence supports clinical equipoise, and revascularization timing should be individualized pending results from adequately powered trials.

ABSTRACT Background High-titer glutamic acid decarboxylase 65 (GAD65) antibodies are found in patients with GAD65 antibody-associated neurological disorders, including stiff-person syndrome (SPS), GAD65 cerebellar ataxia (CA), and GAD65 epilepsy. Given the intracellular localization of the antigenic target, a direct pathogenic role for GAD65 autoantibodies is unlikely. Instead, the autoantibody may be a biomarker for the existence of pathogenic anti-GAD65 autoreactive CD8 + T cells. Methods PBMC-derived dendritic cells (DCs) from 20 GAD65 antibody-positive patients with neurological manifestations (SPS, n=10; CA, n=7; epilepsy, n=3) and 15 healthy controls were pulsed with full-length GAD65 protein, full-length GAD67 protein, overlapping 15-mer peptide pools spanning GAD65 and GAD67, individual GAD65 15-mer peptides, or GAD65 9-mer peptides selected from predicted class I binding hotspots. T cell activation was quantified by flow cytometry-based activation-induced marker (AIM) assays using CD69 upregulation. Class I and class II HLA haplotypes were determined by high resolution typing. NetMHCpan v4.2c was used to generate residue-level peptide:HLA binding density maps across GAD65, and candidate 9-mers were validated for HLA binding by peptide:MHC monomer affinity testing. GAD65-peptide-HLA-restricted CD8 + T cells were identified by co-staining with two separately assembled tetramers carrying the same peptide:HLA complex on different fluorophores (APC and BV421), with double-positive events scored as antigen-specific. HLA-restricted cytotoxicity was measured by coculture of patient CD8 + T cells with GAD65-expressing HEK-293T cells reconstituted with defined HLA class I alleles using AAV-delivered Cre-dependent HLA-2A-eGFP cassettes. Results CD8 + T cells from GAD65 antibody-positive patients showed increased activation in response to DCs pulsed with full-length GAD65 relative to healthy controls (P=0.0157, Welch’s t-test), whereas responses to GAD67 did not differ significantly between groups. CD4 + T cells responded to both GAD65 (P=0.0004) and GAD67 (P=0.0051). Peptide pool screening of GAD65 identified discrete CD8 + and CD4 + immunogenic regions, with refinement using individual 15-mers localizing CD8 + activity to multiple subdomains within GAD65 (205-300) , GAD65 (316-435) , and GAD65 (447-520) . HLA class I haplotyping in 16 non-Hispanic White GAD65 + patients revealed enrichment of HLA-B*08:01 (3.0-fold) and HLA-B*40:01 (4.1-fold) relative to USA NMDP European Caucasian reference frequencies (both BH q < 0.05), with suggestive enrichment of HLA-C*03:04 (2.9-fold; q = 0.09). Additionally, 44% of patients carried the HLA-A*01:01, HLA-B*08:01, and HLA-C*07:01 8.1 ancestral haplotype, approximately four-fold higher than the expected population frequency. Dual-fluorophore tetramer staining identified CD8 + T cells in GAD65 + subjects that bound a subset of HLA-A*11:01- and HLA-B*08:01-restricted GAD65 9-mers, with the clearest disease-skewed signals localized to GAD65 (213-221) , GAD65 (257-265) , and GAD65 (529-537) . In an HLA-reconstituted target-cell killing assay, CD8 + T cells from an HLA-B*08:01-positive GAD65 + patient mediated antigen- and HLA-restricted depletion of GAD65-expressing HEK-293T cells, with HLA-restricted target loss also observed in single-donor experiments across additional HLA-A, -B, and -C contexts. Conclusions Patients with GAD65 antibody-associated neurological disorders harbor circulating CD8 + T cells that recognize discrete HLA class I-restricted GAD65 peptides and that are capable of cytotoxicity against GAD65-expressing HLA-matched target cells. We characterize the immunogenetic and cellular features of class I-restricted CD8 + T cell responses in GAD65 autoimmunity, including overrepresentation of the 8.1 ancestral haplotype, complementing the historical focus on antibodies and CD4 + T cell help, and we provide a panel of validated GAD65 peptide:HLA tetramers for prospective isolation, clonotypic analysis, and longitudinal monitoring of candidate pathogenic CD8 + T cell populations across the GAD65 antibody-associated neurological disease spectrum.

Abstract We describe early experience with beta-lactam therapeutic drug monitoring (TDM) in a high-volume outpatient parenteral antimicrobial therapy program. In selected patients, TDM informed dose optimization and toxicity mitigation. Illustrative cases highlight feasibility and emerging lessons supporting beta-lactam TDM in outpatient care.

Abstract Endothelial-to-mesenchymal transition (EndMT) is a biological process through which lung vascular endothelial cells (ECs) transdifferentiate into mesenchymal-like cells. EndMT has recently been implicated in the development and progression of pulmonary vascular remodeling in pulmonary hypertension (PH); however, its underlying regulatory mechanisms remain incompletely understood. MicroRNAs (miRNAs) are key post-transcriptional regulators of EC gene expression and cellular responses to various stimuli. Notably, microRNA-153 (miR-153) has been shown to directly target SNAI1 to modulate epithelial-to-mesenchymal transition (EMT), a process closely related to EndMT and extensively studied in cancer. Whether miR-153 also participates in EndMT regulation, however, remains unknown. In this study, we demonstrate that 72-hour hypoxic exposure induces SNAI1-mediated EndMT in human lung vascular ECs. Hypoxia also increased cell proliferation and disrupted intercellular junctions, leading to enhanced endothelial permeability. Reduced miR-153 expression was observed in both hypoxia- and TGF-β1-induced EndMT, as well as in ECs isolated from PH patients exhibiting an EndMT phenotype. Similar to hypoxia, TGF-β1 promoted EC permeability. Loss of miR-153 enhanced SNAI1-mediated EndMT, endothelial survival, and permeability under normoxic conditions, whereas miR-153 overexpression attenuated EndMT induced by hypoxia or TGF-β1. However, miR-153 restoration did not completely restore endothelial barrier integrity disrupted by these stimuli. In vitro findings were validated in experimental PH model. In conclusion, miR-153 serves as a critical regulator of EndMT, maintaining endothelial identity and barrier function. Therapeutic delivery of miR-153 may therefore represent a novel strategy to inhibit EndMT and attenuate pulmonary vascular remodeling in PH.