Live Organoid Cyclic Imaging.

Organoids are becoming increasingly relevant in biology and medicine for their physiological complexity and accuracy in modeling human disease. To fully assess their biological profile while preserving their spatial information, spatiotemporal imaging tools are warranted. While previously developed imaging techniques, such as four-dimensional (4D) live imaging and light-sheet imaging have yielded important clinical insights, these technologies lack the combination of cyclic and multiplexed analysis. To address these challenges, bioorthogonal click chemistry is applied to display the first demonstration of multiplexed cyclic imaging of live and fixed patient-derived glioblastoma tumor organoids. This technology exploits bioorthogonal click chemistry to quench fluorescent signals from the surface and intracellular of labeled cells across multiple cycles, allowing for more accurate and efficient molecular profiling of their complex phenotypes. Herein, the versatility of this technology is demonstrated for the screening of glioblastoma markers in patient-derived human glioblastoma organoids while conserving their viability. It is anticipated that the findings and applications of this work can be broadly translated into investigating physiological developments in other organoid systems.

Perfusion Window Chambers Enable Interventional Analyses of Tumor Microenvironments.

Intravital microscopy (IVM) allows spatial and temporal imaging of different cell types in intact live tissue microenvironments. IVM has played a critical role in understanding cancer biology, invasion, metastases, and drug development. One considerable impediment to the field is the inability to interrogate the tumor microenvironment and its communication cascades during disease progression and therapeutic interventions. Here, a new implantable perfusion window chamber (PWC) is described that allows high-fidelity in vivo microscopy, local administration of stains and drugs, and longitudinal sampling of tumor interstitial fluid. This study shows that the new PWC design allows cyclic multiplexed imaging in vivo, imaging of drug action, and sampling of tumor-shed materials. The PWC will be broadly useful as a novel perturbable in vivo system for deciphering biology in complex microenvironments.

ECAP-controlled closed-loop versus open-loop SCS for the treatment of chronic pain: 36-month results of the EVOKE blinded randomized clinical trial.

INTRODUCTION: The evidence for spinal cord stimulation (SCS) has been criticized for the absence of blinded, parallel randomized controlled trials (RCTs) and limited evaluations of the long-term effects of SCS in RCTs. The aim of this study was to determine whether evoked compound action potential (ECAP)-controlled, closed-loop SCS (CL-SCS) is associated with better outcomes when compared with fixed-output, open-loop SCS (OL-SCS) 36 months following implant. METHODS: The EVOKE study was a multicenter, participant-blinded, investigator-blinded, and outcome assessor-blinded, randomized, controlled, parallel-arm clinical trial that compared ECAP-controlled CL-SCS with fixed-output OL-SCS. Participants with chronic, intractable back and leg pain refractory to conservative therapy were enrolled between January 2017 and February 2018, with follow-up through 36 months. The primary outcome was a reduction of at least 50% in overall back and leg pain. Holistic treatment response, a composite outcome including pain intensity, physical and emotional functioning, sleep, and health-related quality of life, and objective neural activation was also assessed. RESULTS: At 36 months, more CL-SCS than OL-SCS participants reported ≥50% reduction (CL-SCS=77.6%, OL-SCS=49.3%; difference: 28.4%, 95% CI 12.8% to 43.9%, p<0.001) and ≥80% reduction (CL-SCS=49.3%, OL-SCS=31.3%; difference: 17.9, 95% CI 1.6% to 34.2%, p=0.032) in overall back and leg pain intensity. Clinically meaningful improvements from baseline were observed at 36 months in both CL-SCS and OL-SCS groups in all other patient-reported outcomes with greater levels of improvement with CL-SCS. A greater proportion of patients with CL-SCS were holistic treatment responders at 36-month follow-up (44.8% vs 28.4%), with a greater cumulative responder score for CL-SCS patients. Greater neural activation and accuracy were observed with CL-SCS. There were no differences between CL-SCS and OL-SCS groups in adverse events. No explants due to loss of efficacy were observed in the CL-SCS group. CONCLUSION: This long-term evaluation with objective measurement of SCS therapy demonstrated that ECAP-controlled CL-SCS resulted in sustained, durable pain relief and superior holistic treatment response through 36 months. Greater neural activation and increased accuracy of therapy delivery were observed with ECAP-controlled CL-SCS than OL-SCS. TRIAL REGISTRATION NUMBER: NCT02924129.

Multiplexed analysis of EV reveals specific biomarker composition with diagnostic impact.

Exosomes and extracellular vesicles (EV) are increasingly being explored as circulating biomarkers, but their heterogenous composition will likely mandate the development of multiplexed EV technologies. Iteratively multiplexed analyses of near single EVs have been challenging to implement beyond a few colors during spectral sensing. Here we developed a multiplexed analysis of EV technique (MASEV) to interrogate thousands of individual EVs during 5 cycles of multi-channel fluorescence staining for 15 EV biomarkers. Contrary to the common belief, we show that: several markers proposed to be ubiquitous are less prevalent than believed; multiple biomarkers concur in single vesicles but only in small fractions; affinity purification can lead to loss of rare EV subtypes; and deep profiling allows detailed analysis of EV, potentially improving the diagnostic content. These findings establish the potential of MASEV for uncovering fundamental EV biology and heterogeneity and increasing diagnostic specificity.

Oxidative Desymmetrization Enables the Concise Synthesis of a trans-Cyclooctene Linker for Bioorthogonal Bond Cleavage.

Modified trans-cyclooctenes (TCO) are capable of highly efficient molecular manipulations in biological environments, driven by the bioorthogonal reaction with tetrazines (Tz). The development of click-cleavable TCO has fueled the field of in vivo chemistry and enabled the design of therapeutic strategies that have already started to enter the clinic. A key element for most of these approaches is the implementation of a cleavable TCO linker. So far, only one member of this class has been developed, a compound that requires a high synthetic effort, mainly to fulfill the multilayered demands on its chemical structure. To tackle this limitation, we developed a dioxolane-fused cleavable TCO linker (dcTCO) that can be prepared in only five steps by applying an oxidative desymmetrization to achieve diastereoselective introduction of the required functionalities. Based on investigation of the structure, reaction kinetics, stability, and hydrophilicity of dcTCO, we demonstrate its bioorthogonal application in the design of a caged prodrug that can be activated by in-situ Tz-triggered cleavage to achieve a remarkable >1000-fold increase in cytotoxicity.

HLA-II-Associated HIV-1 Adaptation Decreases CD4+ T-Cell Responses in HIV-1 Vaccine Recipients.

Epitopes with evidence of HLA-II-associated adaptation induce poorly immunogenic CD4+ T-cell responses in HIV-positive (HIV+) individuals. Many such escaped CD4+ T-cell epitopes are encoded by HIV-1 vaccines being evaluated in clinical trials. Here, we assessed whether this viral adaptation adversely impacts CD4+ T-cell responses following HIV-1 vaccination, thereby representing escaped epitopes. When evaluated in separate peptide pools, vaccine-encoded adapted epitopes (AE) induced CD4+ T-cell responses less frequently than nonadapted epitopes (NAE). We also demonstrated that in a polyvalent vaccine, where both forms of the same epitope were encoded, AE were less immunogenic. NAE-specific CD4+ T cells had increased, albeit low, levels of interferon gamma (IFN-γ) cytokine production. Single-cell transcriptomic analyses showed that NAE-specific CD4+ T cells expressed interferon-related genes, while AE-specific CD4+ T cells resembled a Th2 phenotype. Importantly, the magnitude of NAE-specific CD4+ T-cell responses, but not that of AE-specific responses, was found to positively correlate with Env-specific antibodies in a vaccine efficacy trial. Together, these findings show that HLA-II-associated viral adaptation reduces CD4+ T-cell responses in HIV-1 vaccine recipients and suggest that vaccines encoding a significant number of AE may not provide optimal B-cell help for HIV-specific antibody production. IMPORTANCE Despite decades of research, an effective HIV-1 vaccine remains elusive. Vaccine strategies leading to the generation of broadly neutralizing antibodies are likely needed to provide the best opportunity of generating a protective immune response against HIV-1. Numerous studies have demonstrated that T-cell help is necessary for effective antibody generation. However, immunogen sequences from recent HIV-1 vaccine efficacy trials include CD4+ T-cell epitopes that have evidence of immune escape. Our study shows that these epitopes, termed adapted epitopes, elicit lower frequencies of CD4+ T-cell responses in recipients from multiple HIV-1 vaccine trials. Additionally, the counterparts to these epitopes, termed nonadapted epitopes, elicited CD4+ T-cell responses that correlated with Env-specific antibodies in one efficacy trial. These results suggest that vaccine-encoded adapted epitopes dampen CD4+ T-cell responses, potentially impacting both HIV-specific antibody production and efficacious vaccine efforts.

Human acute inflammatory recovery is defined by co-regulatory dynamics of white blood cell and platelet populations.

Inflammation is the physiologic reaction to cellular and tissue damage caused by trauma, ischemia, infection, and other pathologic conditions. Elevation of white blood cell count (WBC) and altered levels of other acute phase reactants are cardinal signs of inflammation, but the dynamics of these changes and their resolution are not well established. Here we studied inflammatory recovery from trauma, ischemia, and infection by tracking longitudinal dynamics of clinical laboratory measurements in hospitalized patients. We identified a universal recovery trajectory defined by exponential WBC decay and delayed linear growth of platelet count (PLT). Co-regulation of WBC-PLT dynamics is a fundamental mechanism of acute inflammatory recovery and provides a generic approach for identifying high-risk patients: 32x relative risk (RR) of adverse outcomes for cardiac surgery, 9x RR of death from COVID-19, 9x RR of death from sepsis, and 5x RR of death from myocardial infarction.

Tetrazine-Triggered Bioorthogonal Cleavage of trans-Cyclooctene-Caged Phenols Using a Minimal Self-Immolative Linker Strategy.

Bond-cleavage reactions triggered by bioorthogonal tetrazine ligation have emerged as strategies to chemically control the function of (bio)molecules and achieve activation of prodrugs in living systems. While most of these approaches make use of caged amines, current methods for the release of phenols are limited by unfavorable reaction kinetics or insufficient stability of the Tz-responsive reactants. To address this issue, we have implemented a self-immolative linker that enables the connection of cleavable trans-cyclooctenes (TCO) and phenols via carbamate linkages. Based on detailed investigation of the reaction mechanism with several Tz, revealing up to 96 % elimination after 2 hours, we have developed a TCO-caged prodrug with 750-fold reduced cytotoxicity compared to the parent drug and achieved in situ activation upon Tz/TCO click-to-release.

Multiplexed single-cell analysis of FNA allows accurate diagnosis of salivary gland tumors.

Diagnosing salivary gland tumors (SGTs) through fine-needle aspiration (FNA) biopsies is challenging due to the overlapping cytomorphologic features between benign and malignant tumors. The authors developed an innovative, multiplexed cycling technology for the rapid analyses of single cells obtained from FNA that can facilitate the molecular analyses and diagnosis of SGTs. Antibodies against 29 protein markers associated with 7 SGT subtypes were validated and chemically modified via custom linker-bio-orthogonal probes (FAST). Single-cell homogenates and FNA samples were profiled by FAST cyclic imaging and computational analysis. A prediction model was generated using a training set of 151,926 cells from primary SGTs (N = 26) and validated on a separate cohort (N = 30). Companion biomarker testing, such as neurotrophic tyrosine receptor kinase (NTRK), was also assessed with the FAST technology. The FAST molecular diagnostic assay was able to distinguish between benign and malignant SGTs with an accuracy of 0.86 for single-cell homogenate samples and 0.88 for FNA samples. Profiling of multiple markers as compared to a single marker increased the diagnostic accuracy (0.82 as compared to 0.65-0.74, respectively), independent of the cell number sampled. NTRK expression was also assessed by the FAST assay, highlighting the potential therapeutic application of this technology. Application of the novel multiplexed single-cell technology facilitates rapid biomarker testing from FNA samples at low cost. The customizable and modular FAST-FNA approach has relevance to multiple pathologies and organ systems where cytologic samples are often scarce and/or indeterminate resulting in improved diagnostic workflows and timely therapeutic clinical decision-making.

In Vivo Click Chemistry Enables Multiplexed Intravital Microscopy.

The ability to observe cells in live organisms is essential for understanding their function in complex in vivo milieus. A major challenge today has been the limited ability to perform higher multiplexing beyond four to six colors to define cell subtypes in vivo. Here, a click chemistry-based strategy is presented for higher multiplexed in vivo imaging in mouse models. The method uses a scission-accelerated fluorophore exchange (SAFE), which exploits a highly efficient bioorthogonal mechanism to completely remove fluorescent signal from antibody-labeled cells in vivo. It is shown that the SAFE-intravital microscopy imaging method allows 1) in vivo staining of specific cell types in dorsal and cranial window chambers of mice, 2) complete un-staining in minutes, 3) in vivo click chemistries at lower (µm) and thus non-toxic concentrations, and 4) the ability to perform in vivo cyclic imaging. The potential utility of the method is demonstrated by 12 color imaging of immune cells in live mice.

Spatiotemporal multiplexed immunofluorescence imaging of living cells and tissues with bioorthogonal cycling of fluorescent probes.

Cells in complex organisms undergo frequent functional changes, but few methods allow comprehensive longitudinal profiling of living cells. Here we introduce scission-accelerated fluorophore exchange (SAFE), a method for multiplexed temporospatial imaging of living cells with immunofluorescence. SAFE uses a rapid bioorthogonal click chemistry to remove immunofluorescent signals from the surface of labeled cells, cycling the nanomolar-concentration reagents in seconds and enabling multiple rounds of staining of the same samples. It is non-toxic and functional in both dispersed cells and intact living tissues. We demonstrate multiparameter (n ≥ 14), non-disruptive imaging of murine peripheral blood mononuclear and bone marrow cells to profile cellular differentiation. We also show longitudinal multiplexed imaging of bone marrow progenitor cells as they develop into neutrophils over 6 days and real-time multiplexed cycling of living mouse hepatic tissues. We anticipate that SAFE will find broad utility for investigating physiologic dynamics in living systems.

TNIK Inhibition Has Dual Synergistic Effects on Tumor and Associated Immune Cells.

Treatment with checkpoint inhibitors can be extraordinarily effective in a fraction of patients, particularly those whose tumors are pre-infiltrated by T cells. In others, efficacy is considerably lower, which has led to interest in developing strategies for sensitization to immunotherapy. Using various colorectal cancer mouse models, it is shown that the use of Traf2 and Nck-interacting protein kinase inhibitors (TNIKi) unexpectedly increases tumor infiltration by PD-1+ CD8+ T cells, thus contributing to tumor control. This appears to happen by two independent mechanisms, by inducing immunogenic cell death and separately by directly activating CD8. The use of TNIKi achieves complete tumor control in 50% of mice when combined with checkpoint inhibitor targeting PD-1. These findings reveal immunogenic properties of TNIKi and indicate that the proportion of colorectal cancers responding to checkpoint therapy can be increased by combining it with immunogenic kinase inhibitors.

Integrated Analytical System for Clinical Single-Cell Analysis.

High-dimensional analyses of cancers can potentially be used to better define cancer subtypes, analyze the complex tumor microenvironment, and perform cancer cell pathway analyses for drug trials. Unfortunately, integrated systems that allow such analyses in serial fine needle aspirates within a day or at point-of-care currently do not exist. To achieve this, an integrated immunofluorescence single-cell analyzer (i2SCAN) for deep profiling of directly harvested cells is developed. By combining a novel cellular imaging system, highly cyclable bioorthogonal FAST antibody panels, and integrated computational analysis, it is shown that same-day analysis is possible in thousands of harvested cells. It is demonstrated that the i2SCAN approach allows comprehensive analysis of breast cancer samples obtained by fine needle aspiration or core tissues. The method is a rapid, robust, and low-cost solution to high-dimensional analysis of scant clinical specimens.

T cell receptor sequencing identifies prior SARS-CoV-2 infection and correlates with neutralizing antibodies and disease severity.

BACKGROUNDMeasuring the immune response to SARS-CoV-2 enables assessment of past infection and protective immunity. SARS-CoV-2 infection induces humoral and T cell responses, but these responses vary with disease severity and individual characteristics.METHODSA T cell receptor (TCR) immunosequencing assay was conducted using small-volume blood samples from 302 individuals recovered from COVID-19. Correlations between the magnitude of the T cell response and neutralizing antibody (nAb) titers or indicators of disease severity were evaluated. Sensitivity of T cell testing was assessed and compared with serologic testing.RESULTSSARS-CoV-2-specific T cell responses were significantly correlated with nAb titers and clinical indicators of disease severity, including hospitalization, fever, and difficulty breathing. Despite modest declines in depth and breadth of T cell responses during convalescence, high sensitivity was observed until at least 6 months after infection, with overall sensitivity ~5% greater than serology tests for identifying prior SARS-CoV-2 infection. Improved performance of T cell testing was most apparent in recovered, nonhospitalized individuals sampled > 150 days after initial illness, suggesting greater sensitivity than serology at later time points and in individuals with less severe disease. T cell testing identified SARS-CoV-2 infection in 68% (55 of 81) of samples with undetectable nAb titers (<1:40) and in 37% (13 of 35) of samples classified as negative by 3 antibody assays.CONCLUSIONThese results support TCR-based testing as a scalable, reliable measure of past SARS-CoV-2 infection with clinical value beyond serology.TRIAL REGISTRATIONSpecimens were accrued under trial NCT04338360 accessible at clinicaltrials.gov.FUNDINGThis work was funded by Adaptive Biotechnologies, Frederick National Laboratory for Cancer Research, NIAID, Fred Hutchinson Joel Meyers Endowment, Fast Grants, and American Society for Transplantation and Cell Therapy.

Longitudinal analysis of T cell receptor repertoires reveals shared patterns of antigen-specific response to SARS-CoV-2 infection.

T cells play a prominent role in orchestrating the immune response to viral diseases, but their role in the clinical presentation and subsequent immunity to SARS-CoV-2 infection remains poorly understood. As part of a population-based survey of the municipality of Vo', Italy, conducted after the initial SARS-CoV-2 outbreak, we sampled the T cell receptor (TCR) repertoires of the population 2 months after the initial PCR survey and followed up positive cases 9 and 15 months later. At 2 months, we found that 97.0% (98 of 101) of cases had elevated levels of TCRs associated with SARS-CoV-2. T cell frequency (depth) was increased in individuals with more severe disease. Both depth and diversity (breadth) of the TCR repertoire were positively associated with neutralizing antibody titers, driven mostly by CD4+ T cells directed against spike protein. At the later time points, detection of these TCRs remained high, with 90.7% (78 of 96) and 86.2% (25 of 29) of individuals having detectable signal at 9 and 15 months, respectively. Forty-three individuals were vaccinated by month 15 and showed a significant increase in TCRs directed against spike protein. Taken together, these results demonstrate the central role of T cells in mounting an immune defense against SARS-CoV-2 that persists out to 15 months.

Single-EV analysis (sEVA) of mutated proteins allows detection of stage 1 pancreatic cancer.

Tumor cell-derived extracellular vesicles (EVs) are being explored as circulating biomarkers, but it is unclear whether bulk measurements will allow early cancer detection. We hypothesized that a single-EV analysis (sEVA) technique could potentially improve diagnostic accuracy. Using pancreatic cancer (PDAC), we analyzed the composition of putative cancer markers in 11 model lines. In parental PDAC cells positive for KRASmut and/or P53mut proteins, only ~40% of EVs were also positive. In a blinded study involving 16 patients with surgically proven stage 1 PDAC, KRASmut and P53mut protein was detectable at much lower levels, generally in <0.1% of vesicles. These vesicles were detectable by the new sEVA approach in 15 of the 16 patients. Using a modeling approach, we estimate that the current PDAC detection limit is at ~0.1-cm3 tumor volume, below clinical imaging capabilities. These findings establish the potential for sEVA for early cancer detection.

Durability of Clinical and Quality-of-Life Outcomes of Closed-Loop Spinal Cord Stimulation for Chronic Back and Leg Pain: A Secondary Analysis of the Evoke Randomized Clinical Trial.

Importance: Chronic pain is debilitating and profoundly affects health-related quality of life. Spinal cord stimulation (SCS) is a well-established therapy for chronic pain; however, SCS has been limited by the inability to directly measure the elicited neural response, precluding confirmation of neural activation and continuous therapy. A novel SCS system measures the evoked compound action potentials (ECAPs) to produce a real-time physiological closed-loop control system. Objective: To determine whether ECAP-controlled, closed-loop SCS is associated with better outcomes compared with fixed-output, open-loop SCS at 24 months following implant. Design, Setting, and Participants: The Evoke study was a double-blind, randomized, controlled, parallel arm clinical trial with 36 months of follow-up. Participants were enrolled from February 2017 to 2018, and the study was conducted at 13 US investigation sites. SCS candidates with chronic, intractable back and leg pain refractory to conservative therapy, who consented, were screened. Key eligibility criteria included overall, back, and leg pain visual analog scale score of 60 mm or more; Oswestry Disability Index score of 41 to 80; stable pain medications; and no previous SCS. Analysis took place from October 2020 to April 2021. Interventions: ECAP-controlled, closed-loop SCS was compared with fixed-output, open-loop SCS. Main Outcomes and Measures: Reported here are the 24-month outcomes of the trial, which include all randomized patients in the primary and safety analyses. The primary outcome was a reduction of 50% or more in overall back and leg pain assessed at 3 and 12 months (previously published). Results: Of 134 randomized patients, 65 (48.5%) were female and the mean (SD) age was 55.2 (10.6) years. At 24 months, significantly more closed-loop than open-loop patients were responders (≥50% reduction) in overall pain (53 of 67 [79.1%] in the closed-loop group; 36 of 67 [53.7%] in the open-loop group; difference, 25.4% [95% CI, 10.0%-40.8%]; P = .001). There was no difference in safety profiles between groups (difference in rate of study-related adverse events: 6.0 [95% CI, -7.8 to 19.7]). Improvements were also observed in health-related quality of life, physical and emotional functioning, and sleep, in parallel with opioid reduction or elimination. Objective neurophysiological measurements substantiated the clinical outcomes and provided evidence of activation of inhibitory pain mechanisms. Conclusions and Relevance: ECAP-controlled, closed-loop SCS, which elicited a more consistent neural response, was associated with sustained superior pain relief at 24 months, consistent with the 3- and 12-month outcomes.

Twelve-Month results from multicenter, open-label, randomized controlled clinical trial comparing differential target multiplexed spinal cord stimulation and traditional spinal cord stimulation in subjects with chronic intractable back pain and leg pain.

BACKGROUND: Spinal cord stimulation (SCS) is a well-established treatment for chronic intractable pain of the trunk and/or limbs; however, low back pain (LBP) is difficult to treat using traditional SCS. Differential Target Multiplexed spinal cord stimulation (DTM SCS) is an advanced approach inspired from animal studies demonstrating improved pain-related behavior and pain-relevant biological processes. OBJECTIVE: The purpose of this study was to compare the effectiveness of DTM SCS and traditional SCS in treating chronic LBP and leg pain (LP). METHODS: This prospective, postmarket randomized controlled trial compared DTM SCS to traditional SCS in patients with chronic LBP and LP. Primary end point was LBP responder rate (percentage of subjects with ≥ 50% relief) at 3 months. Noninferiority and superiority were assessed. Other outcomes included mean change in back and leg pain, responder rates, disability, global health, satisfaction, and safety profile throughout the 12-month follow-up. RESULTS: One hundred twenty-eight subjects were randomized across 12 centers (67 DTM SCS and 61 traditional SCS). Of the 94 patients implanted, 46 subjects in each group completed the 3-month assessment. LBP responder rate of 80.1% with DTM SCS was superior to 51.2% with traditional SCS (p = 0.0010). Mean LBP reduction (5.36 cm) with DTM SCS was greater than reduction (3.37 cm) with traditional SCS (p < 0.0001). These results were sustained at 6 months and 12 months. Safety profiles were similar between treatment groups. CONCLUSION: Superiority of DTM SCS compared with traditional SCS for chronic LBP was demonstrated. Clinical improvements provided by DTM SCS were sustained over 12 months and are expected to significantly impact the management of chronic LBP.

Implementation and Evaluation of Gradient Strip Antimicrobial Susceptibility Testing in US Public Health Laboratories to Respond to Resistant Gonorrhea.

BACKGROUND: Gradient strip antimicrobial susceptibility testing using Etest is conducted by local public health jurisdictions participating in the Strengthening the US Response to Resistant Gonorrhea (SURRG) program to inform public health responses to resistant gonorrhea. Proficiency testing results across the participating laboratories were analyzed and a comparison of Etest with the agar dilution method was conducted. METHODS: Laboratories participating in SURRG performed Etest for azithromycin (AZM), cefixime (CFX), and ceftriaxone (CRO). Concurrence between minimum inhibitory concentrations (MICs) obtained with Etest versus the agar dilution method using corresponding isolates was defined as ±1 double dilution. Specific levels of reduced susceptibility were termed "alerts" and included isolates with the following MICs: ≥2.0 µg/mL (AZM), ≥0.25 µg/mL (CFX), and ≥0.125 µg/mL (CRO). Categorical (alert/nonalert) agreement was calculated for MICs determined using Etest and agar dilution methods. RESULTS: Strengthening the US Response to Resistant Gonorrhea laboratories had high proficiency testing scores (≥98%) and low levels of interlaboratory variations in MICs. The overall concurrence of MICs (essential agreement) determined using agar dilution, and Etest was 96% (CRO), 96% (CFX), and 95% (AZM). Depending on the antibiotic tested, between 27% and 66% of isolates with alert MICs determined by Etest also had alert MICs using the reference agar dilution methodology; however, most of these alert MICs were detected at threshold levels. CONCLUSIONS: This study demonstrates that MICs produced by SURRG laboratories using Etest have a high level of concurrence with agar dilution. Although confirmation of specific alert MICs varied, Etest facilities rapid detection and response to emerging resistant gonorrhea.