Publications by Year: 2026

Laboratory-developed tests (LDTs) play a critical role in meeting unmet diagnostic needs, particularly for rare infections and high-acuity or immunocompromised patient populations. However, current US regulatory and reimbursement frameworks have constrained innovation and delayed implementation of many essential laboratory diagnostic tests. Here, we propose a pragmatic, collaborative model focusing on infectious disease molecular diagnostics that maintains analytical rigor while allowing clinical validity to be supported by evidence from the medical literature and clinical judgment. To address the resource constraints faced by hospital laboratories, we envision a voluntary, national repository where laboratories and manufacturers contribute standardized analytical and, where appropriate, clinical validation data for both newly developed and modified Food and Drug Administration (FDA)-cleared assays. Data elements such as accuracy, limit of detection, analytical measurement range, analytical specificity, and inclusivity compared with reference determinations would be aggregated within groups representing technically identical assays, enabling cumulative validation and shared use of high-quality evidence. By leveraging these shared data sets, hospital laboratories could implement LDTs in-house with minimal redundant validation, enabling broader access to testing near the point of patient care and faster turnaround times. Manufacturers could use the same data to support applications for expanded intended use of existing FDA-cleared and -approved tests. This framework would strengthen hospital laboratory diagnostic capacity, accelerate test implementation, and improve patient outcomes and healthcare system efficiency.

UNLABELLED: Agar dilution is a reference susceptibility testing method uniquely or preferentially recommended for certain antimicrobials. However, the effort required to pour individual agar plates spanning a doubling dilution range precludes its practical implementation in hospital clinical laboratories. Here, we describe an on-demand replacement for agar dilution, specifically substituting Poloxamer 407 (also known as Pluronic F-127) for Bacto agar as the solidifying agent. Notably, 20% Poloxamer 407 solutions (e.g., with Mueller-Hinton broth) remain liquid at refrigerated temperatures but solidify upon warming, enabling facile setup of poloxamer dilution testing in Petri dish or microwell format. For fosfomycin susceptibility testing, poloxamer dilution and reference agar dilution showed excellent categorical agreement (CA) and essential agreement (EA) for Escherichia coli (100% and 87%, respectively, n = 31). For other Enterobacterales, excluding Klebsiella spp., CA and EA were both 82% (n = 17, respectively). For Pseudomonas aeruginosa, CA and EA were 60% and 100% (n = 10), respectively, with the lower CA reflecting the large number of strains tested with minimal inhibitory concentrations near categorical breakpoints. There were no very major errors, while major errors were only observed for Klebsiella spp. Additionally, poloxamer dilution substantially reduced the number of skipped dilutions sixfold for E. coli (P < 0.0001) and inhibited swarming of Proteus spp. We conclude that poloxamer dilution and agar dilution, an imperfect gold standard, have essentially equivalent practical performance and that poloxamer dilution can therefore serve as an on-demand alternative testing methodology in clinical laboratories for fosfomycin testing of gram-negative pathogens. A broader exploration of poloxamer dilution's utility is thus warranted.

IMPORTANCE: Accurate antibiotic susceptibility testing is essential for guiding treatment of bacterial infections. For the antibiotic fosfomycin, used to treat Escherichia coli urinary tract infections, the most reliable testing method requires solid media prepared by hand for each antibiotic concentration, which is too time-consuming for most clinical laboratories to perform. Our study shows that replacing agar with an alternative temperature-sensitive gelling agent called poloxamer enables laboratories to prepare solid test plates rapidly without special equipment. This approach, which is essentially identical to traditional agar dilution, provides a practical means for performing reference-quality minimal inhibitory concentration (MIC) testing near the point of patient care, as demonstrated for fosfomycin, for which current FDA-cleared methods do not provide MIC data. This strategy may also be applicable to other drugs for which agar dilution is the preferred testing method, supporting expedited testing to inform treatment decisions for bacterial infections.

In this study, we investigated the impact of G protein-coupled receptor (GPCR) signaling on the intracellular replication of the model pathogen Brucella neotomae. Building on a prior chemical genetics screen, we identified agonists of the Gαi-coupled adenosine A1 and dopamine D4 receptors as potent inhibitors of intracellular Brucella replication. In contrast, agonists of Gαs-coupled adenosine A2A or dopamine D1 receptors, as well as antagonists of A1 or D4 receptors, either failed to inhibit or enhanced intracellular replication. Wild-type B. neotomae induced a rapid, type IV secretion system-dependent increase in host-cell cAMP during early infection. ENBA and cilostamide prevented this infection-associated cAMP increase and completely inhibited intracellular growth; this effect was partially reversed by cell-permeable cAMP analogs Using a real-time NanoBRET biosensor, we detected rapid Gαs activation within minutes of infection that was sustained during wild-type but not ΔvirB4 infection and was abrogated by ENBA or cilostamide. Disruption of early Gαs-cAMP signaling redirected BCVs to replication-incompatible phagolysosomal and autophagy-associated compartments. Collectively, these data support a model in which early GPCR signaling dynamics, balancing Gαs and Gαi pathways, are critical for establishment of productive intracellular Brucella infection.