Pharmacokinetics, Fecal Output, and Grimace Scores in Rabbits Given Long-acting Buprenorphine or Fentanyl for Postsurgical Analgesia.

The New Zealand white rabbit (Oryctolagus cuniculus) is a frequently used surgical model. Pain management after surgery is a critical aspect of animal welfare. Recently, a long-acting buprenorphine formulation (Ethiqa XR; EXR) was approved for use in rats and mice but has not yet been investigated in rabbits. The current study aimed to determine whether a single subcutaneous dose of 0.15 mg/kg of EXR could achieve and maintain therapeutic buprenorphine plasma concentrations (0.1 ng/mL) for 72 h in male and female rabbits. We also evaluated the safety profiles of EXR and the fentanyl patch (FP) by assessing fecal output after surgery, because opioids are known to decrease intestinal motility. Behavior and pain scores were compared for rabbits that received either EXR or the FP after undergoing an annulus puncture procedure to induce osteoarthritis. EXR at 0.15 mg/kg SC provided a shorter time to onset and sustained analgesia for 72 h in male and female rabbits, whereas the FP provided suboptimal analgesia after 48 h. Both EXR and FP reduced fecal output after surgery. Output returned to baseline levels within 72 h for the EXR group and remained slightly below baseline at 96 h after surgery for the fentanyl group. Grimace pain scores revealed no significant difference between treatment groups. These results suggest that EXR is a safe and effective option for postoperative pain management in rabbits.

Phosphate-binding pocket on cyclin B governs CDK substrate phosphorylation and mitotic timing.

Cell cycle progression is governed by complexes of the cyclin-dependent kinases (CDKs) and their regulatory subunits cyclin and Cks1. CDKs phosphorylate hundreds of substrates, often at multiple sites. Multisite phosphorylation depends on Cks1, which binds initial priming phosphorylation sites to promote secondary phosphorylation at other sites. Here, we describe a similar role for a recently discovered phosphate-binding pocket (PP) on B-type cyclins. Mutation of the PP in Clb2, the major mitotic cyclin of budding yeast, alters bud morphology and delays the onset of anaphase. Using phosphoproteomics in vivo and kinase reactions in vitro, we find that mutation of the PP reduces phosphorylation of several CDK substrates, including the Bud6 subunit of the polarisome and the Cdc16 and Cdc27 subunits of the anaphase-promoting complex/cyclosome. We conclude that the cyclin PP, like Cks1, controls the timing of multisite phosphorylation on CDK substrates, thereby helping to establish the robust timing of cell-cycle events.

Concentrations of remdesivir and GS-441524 in human milk from lactating individuals diagnosed with COVID-19.

IMPACT: Findings from this study provide further reassuring evidence that infant exposure through human milk received from lactating individuals who require treatment with remdesivir is negligible.

Oral Ingestion of an Iron-Containing Hand Warmer in a Pediatric Patient.

Hand warmer packets are common products used to provide a portable, nonflammable heat source via the exothermic oxidation of iron. We present the first reported case of pediatric hand warmer packet ingestion in a three-year-old male who developed an elevated serum iron concentration (peak 335 ug/dL) and gastrointestinal injury after ingesting the contents of a HOTHANDS hand warmer packet. He was treated with endoscopic gastric foreign body removal and lavage, as well as proton-pump inhibitors and whole bowel irrigation. Hand warmer packs contain reduced elemental iron powder, which has been shown to have a more favorable safety profile when compared to iron salts. The mechanism of toxicity for reduced iron is unknown, though it is thought to be due to conversion to more toxic iron ions in an acidic environment. While the current adult literature suggests that ingestion of a single hand warmer packet is without significant risk, our case demonstrates that even a partial ingestion carries a significant risk of both iron toxicity and direct gastrointestinal caustic injury in a young child. This case demonstrates the need for multidisciplinary care and consideration of urgent endoscopic foreign body removal and gastric lavage followed by whole bowel irrigation to mitigate the potential of severe iron toxicity.

Bacterial antiviral defense pathways encode eukaryotic-like ubiquitination systems.

Ubiquitination and related pathways play crucial roles in protein homeostasis, signaling, and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2, and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6 but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here, we demonstrate that a bacterial antiviral immune system encodes a complete ubiquitination pathway. Two structures of a bacterial E1:E2:Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 encodes an N-terminal inactive adenylation domain (IAD) and a C-terminal active adenylation domain (AAD) with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C-terminus positioned for adenylation, and the E1 CYS domain poised nearby for thioester formation. A second structure mimics an E1-to-E2 transthioesterification state, with the E1 CYS domain rotated outward and its catalytic cysteine adjacent to the bound E2. We show that a deubiquitinase (DUB) in the same pathway pre-processes the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.

Directed evolution unlocks oxygen reactivity for a nicotine-degrading flavoenzyme.

The flavoenzyme nicotine oxidoreductase (NicA2) is a promising injectable treatment to aid in the cessation of smoking, a behavior responsible for one in ten deaths worldwide. NicA2 acts by degrading nicotine in the bloodstream before it reaches the brain. Clinical use of NicA2 is limited by its poor catalytic activity in the absence of its natural electron acceptor CycN. Without CycN, NicA2 is instead oxidized slowly by dioxygen (O2), necessitating unfeasibly large doses in a therapeutic setting. Here, we report a genetic selection strategy that directly links CycN-independent activity of NicA2 to growth of Pseudomonas putida S16. This selection enabled us to evolve NicA2 variants with substantial improvement in their rate of oxidation by O2. The encoded mutations cluster around a putative O2 tunnel, increasing flexibility and accessibility to O2 in this region. These mutations further confer desirable clinical properties. A variant form of NicA2 is tenfold more effective than the wild type at degrading nicotine in the bloodstream of rats.

Evaluation of Field Sobriety Tests for Identifying Drivers Under the Influence of Cannabis: A Randomized Clinical Trial.

Importance: With increasing medicinal and recreational cannabis legalization, there is a public health need for effective and unbiased evaluations for determining whether a driver is impaired due to Δ9-tetrahydrocannabinol (THC) exposure. Field sobriety tests (FSTs) are a key component of the gold standard law enforcement officer-based evaluations, yet controlled studies are inconclusive regarding their efficacy in detecting whether a person is under the influence of THC. Objective: To examine the classification accuracy of FSTs with respect to cannabis exposure and driving impairment (as determined via a driving simulation). Design, Setting, and Participants: This double-blind, placebo-controlled parallel randomized clinical trial was conducted from February 2017 to June 2019 at the Center for Medicinal Cannabis Research, University of California, San Diego. Participants were aged 21 to 55 years and had used cannabis in the past month. Data were analyzed from August 2021 to April 2023. Intervention: Participants were randomized 1:1:1 to placebo (0.02% THC), 5.9% THC cannabis, or 13.4% THC cannabis smoked ad libitum. Main Outcome and Measures: The primary end point was law enforcement officer determination of FST impairment at 4 time points after smoking. Additional measures included officer estimation as to whether participants were in the THC or placebo group as well as driving simulator data. Officers did not observe driving performance. Results: The study included 184 participants (117 [63.6%] male; mean [SD] age, 30 [8.3] years) who had used cannabis a mean (SD) of 16.7 (9.8) days in the past 30 days; 121 received THC and 63, placebo. Officers classified 98 participants (81.0%) in the THC group and 31 (49.2%) in the placebo group as FST impaired (difference, 31.8 percentage points; 95% CI, 16.4-47.2 percentage points; P < .001) at 70 minutes after smoking. The THC group performed significantly worse than the placebo group on 8 of 27 individual FST components (29.6%) and all FST summary scores. However, the placebo group did not complete a median of 8 (IQR, 5-11) FST components as instructed. Of 128 participants classified as FST impaired, officers suspected 127 (99.2%) as having received THC. Driving simulator performance was significantly associated with results of select FSTs (eg, ≥2 clues on One Leg Stand was associated with impairment on the simulator: odds ratio, 3.09; 95% CI, 1.63-5.88; P < .001). Conclusions and Relevance: This randomized clinical trial found that when administered by highly trained officers, FSTs differentiated between individuals receiving THC vs placebo and driving abilities were associated with results of some FSTs. However, the high rate at which the participants receiving placebo failed to adequately perform FSTs and the high frequency that poor FST performance was suspected to be due to THC-related impairment suggest that FSTs, absent other indicators, may be insufficient to denote THC-specific impairment in drivers. Trial Registration: ClinicalTrials.gov Identifier: NCT02849587.

Spectrophotometric analysis of purple urine secondary to methylene blue and hydroxocobalamin co-administration.

BACKGROUND: The development of purple urine after methylene blue (methylthioninium chloride) and hydroxocobalamin co-administration is a rare clinical entity that has not been fully elucidated. A 47-year-old male presented to the emergency department with hypotension, cyanosis, and depressed mental status. The patient was noted to have profound peripheral and central cyanosis, as well as chocolate-colored arterial blood. He was treated with both methylene blue and hydroxocobalamin and developed purple urine for approximately 1 week. METHODS: Color chromatography was performed by placing the patient's urine directly onto absorbent filter paper. Urine spectrophotometry was performed utilizing the NanoDrop One/One C UV-Vis Spectrophotometer. RESULTS: Color chromatography of the urine was demonstrated clear separation of distinct red and blue phases. Urine spectrophotometry demonstrated near perfect overlap between the methylene blue + hydroxocobalamin absorbance spectrum and the patient's purple urine absorbance spectrum. CONCLUSION: Purple urine secondary to methylene blue and hydroxocobalamin co-administration is due to combined urinary excretion of methylene blue (blue) and hydroxocobalamin (red), and not a novel purple metabolite. We anticipate that this is going to be an increasingly common clinical entity as the roles of both hydroxocobalamin and methylene blue expand from toxicologic antidotes to adjunct therapies for vasoplegia, poor cardiac output, and sepsis.

Severe arsenic poisoning due to Ayurvedic supplements.

Patients that are taking Ayurvedic supplements have an increased risk of heavy metal toxicity. Lead, arsenic, and mercury are frequently identified in these supplements and can cause clinically significant toxicity. Clinicians should screen patients routinely for use of non-pharmaceutical medications and supplements.

Variable Delta-9-Tetrahydrocannabinol Pharmacokinetics and Pharmacodynamics After Cannabis Smoking in Regular Users.

BACKGROUND: Despite its federally restricted status, cannabis is widely used medicinally and recreationally. The pharmacokinetics (PK) and central nervous system (CNS) effects of tetrahydrocannabinol (THC), the major psychoactive cannabinoid, are not well understood. The objective of this study was to develop a population PK model of inhaled THC, including sources of variability, and to conduct an exploratory analysis of potential exposure-response relationships. METHODS: Regular adult cannabis users smoked a single cannabis cigarette containing 5.9% THC (Chemovar A) or 13.4% THC (Chemovar B) ad libitum. THC concentrations in whole blood were measured and used to develop a population PK model to identify potential factors contributing to interindividual variability in THC PK and to describe THC disposition. Relationships between model-predicted exposure and heart rate, change in composite driving score on a driving simulator, and perceived highness were evaluated. RESULTS: From the 102 participants, a total of 770 blood THC concentrations were obtained. A two-compartment structural model adequately fit the data. Chemovar and baseline THC (THC BL ) were found to be significant covariates for bioavailability, with Chemovar A having better THC absorption. The model predicted that heavy users-those with the highest THC BL -would have significantly higher absorption than those with lighter previous use. There was a statistically significant relationship between exposure and heart rate, and exposure and perceived highness. CONCLUSIONS: THC PK is highly variable and related to baseline THC concentrations and different chemovars. The developed population PK model showed that heavier users had higher THC bioavailability. To better understand the factors affecting THC PK and dose-response relationships, future studies should incorporate a wide range of doses, multiple routes of administration, and different formulations relevant to typical community use.

Driving Under the Influence of Cannabis: Impact of Combining Toxicology Testing with Field Sobriety Tests.

BACKGROUND: Cannabis is increasingly used both medically and recreationally. With widespread use, there is growing concern about how to identify cannabis-impaired drivers. METHODS: A placebo-controlled randomized double-blinded protocol was conducted to study the effects of cannabis on driving performance. One hundred ninety-one participants were randomized to smoke ad libitum a cannabis cigarette containing placebo or delta-9-tetrahydrocannabinol (THC) (5.9% or 13.4%). Blood, oral fluid (OF), and breath samples were collected along with longitudinal driving performance on a simulator (standard deviation of lateral position [SDLP] and car following [coherence]) over a 5-hour period. Law enforcement officers performed field sobriety tests (FSTs) to determine if participants were impaired. RESULTS: There was no relationship between THC concentrations measured in blood, OF, or breath and SDLP or coherence at any of the timepoints studied (P > 0.05). FSTs were significant (P < 0.05) for classifying participants into the THC group vs the placebo group up to 188 minutes after smoking. Seventy-one minutes after smoking, FSTs classified 81% of the participants who received active drug as being impaired. However, 49% of participants who smoked placebo (controls) were also deemed impaired at this same timepoint. Combining a 2 ng/mL THC cutoff in OF with positive findings on FSTs reduced the number of controls classified as impaired to zero, 86 minutes after smoking the placebo. CONCLUSIONS: Requiring a positive toxicology result in addition to the FST observations substantially improved the classification accuracy regarding possible driving under the influence of THC by decreasing the percentage of controls classified as impaired.

Evaluating the performance of the Roche FEN2 fentanyl immunoassay and its clinical implementation: The role of LDT-based mass spectrometry testing.

Introduction: While laboratory-developed tests (LDTs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) are widely employed to support the development of FDA-cleared drug immunoassays, their significance in the clinical implementation and evaluation of such assays is often overlooked. This paper reports on the important role of LC-MS/MS LDTs in demonstrating improved performance of the Roche FEN2 fentanyl immunoassay compared with the Thermo DRI fentanyl immunoassay. Methods: The FEN2 assay was implemented according to the manufacturer's instructions and its performance was compared to the existing DRI assay using LC-MS/MS as a reference. Clinical sensitivity and specificity were determined using 250 consecutive random patient specimens. Spiking experiments were conducted to determine cross-reactivity with 31 fentanyl analogs. Select DRI false-positive samples were analyzed by the FEN2 assay via time-of-flight mass spectrometry method (LC-QTOF). Results: The FEN2 assay showed improved clinical sensitivity compared to the DRI (98% vs 61%) in 250 consecutive patient samples due to its ability to detect norfentanyl. It also showed better clinical specificity by correctly classifying select DRI false-positive results. Upon implementation in clinical practice, the FEN2 resulted in a higher screening positivity rate than the DRI (17.3% vs 13.3%) and a greater LC-MS/MS confirmation rate of immunoassay-positive samples (96.8% vs 88.8%, respectively). Conclusion: The use of LC-MS/MS LDTs demonstrated that the FEN2 assay has greater clinical sensitivity and is less prone to false-positives than the DRI assay. These findings support the use of FEN2 in routine clinical practice and emphasize the role of mass spectrometry-based LDTs in clinical toxicology testing.

Comparison of two highly sensitive benzodiazepine immunoassay lab developed tests for urine drug testing in clinical specimens.

Background: The VALID Act is a legislative effort that, if enacted, would alter the regulatory requirements of laboratory developed tests (LDTs) used for clinical testing in the United States. Benzodiazepines, which are primarily excreted into urine as glucuronidated metabolites such as lorazepam, cross-react poorly with FDA-cleared immunoassays, leading to false-negatives. This shortfall can be addressed with LDTs created by adding glucuronidase to the immunoassay reagents producing "high sensitivity" assays that detect glucuronidated metabolites. Methods: Precision and stability of two high-sensitivity (HS) benzodiazepine immunoassays from Roche and Thermo Scientific were evaluated using manufacturer-supplied quality control (QC) material and glucuronidated QC material. The immunoassays were directly compared to an LC-MS/MS LDT benzodiazepine assay to determine clinical sensitivity/specificity using urine specimens (n = 82 for Thermo Scientific; n = 265 for Roche). The clinical impact of the HS LDT immunoassay was determined by analyzing clinical testing results 60 days before and after its implementation. Results: The precision and clinical sensitivity/specificity of the HS-Thermo Scientific and HS-Roche benzodiazepine assays were acceptable. The reagent stability of the HS-Thermo Scientific immunoassay was poor, whereas the HS-Roche immunoassay was stable. After implementation of the HS-Roche benzodiazepine immunoassay as an LDT, there was a 30-fold increase (p-value: < 0.00001) in the percentage of lorazepam confirmations. Conclusions: We demonstrate the development and validation of an immunoassay LDT with improved sensitivity for glucuronidated benzodiazepines. This LDT can detect glucuronidated benzodiazepines in clinical urine specimens and is stable for 60 days. Importantly, we were able to validate the immunoassay as an LDT by utilizing an LC-MS/MS LDT.

Development of Potent and Highly Selective Epoxyketone-Based Plasmodium Proteasome Inhibitors.

Here, we present remarkable epoxyketone-based proteasome inhibitors with low nanomolar in vitro potency for blood-stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,000-fold greater selectivity for erythrocytic-stage P. falciparum over HepG2 and H460 cells, which is largely driven by the accommodation of the parasite proteasome for a D-amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. We isolated the proteasome from P. falciparum cell extracts and determined that the best compound is 171-fold more potent at inhibiting the ß5 subunit of P. falciparum proteasome when compared to the same subunit of the human constitutive proteasome. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti-malarial drugs.

Follow-Up: Effects of a Pandemic and Isolation on Alcohol and Psychoactive Medication Use in a Population of Rehabilitation and Pain Patients.

OBJECTIVE: The conjunction of the coronavirus disease lockdown and the use of illicit drugs suggests the potential increase in drug usage and opioid deaths. Because of other studies, we felt the need to examine if the lockdown has caused a change in the drug intake of our population of substance abuse and pain management patients. Our initial study indicated no increase in the use of illicit and antianxiety drugs. This study is a continuation of that work. MATERIALS: Urine drug testing is a strategy to reduce harm to patients in pain management and substance abuse treatment programs. We analyzed trends in the clinical drug testing patterns of urine specimens sent by substance abuse and pain clinics to monitor their patients. These specimens were tested by a national clinical laboratory using LC-MS/MS definitive methods. The time frame of these comparative observations was the past six years, including the two years of the pandemic. RESULTS: We observed a 30% reduction in test requests during the second quarter of 2020, the number of test requests and specimens submitted was similar during other times of the six-year period. The observed drug use pattern was similar to the earlier study. Among the patients tested, positivity decreased greatly for the illicit drugs heroin and cocaine but increased for methamphetamine and fentanyl. Use of the antidepressant and anxiolytic drugs remained consistent or declined for some drugs, relative to pre-pandemic patterns. The percent of patients prescribed the opiates morphine and oxycodone decreased, while the use of hydrocodone increased. Positivity for the drug gabapentin increased greatly. The use of alcohol did not increase significantly during the lockdown period. CONCLUSION: In summary, these findings demonstrate relatively consistent drug use, with decreased positivity for high-risk drugs and dangerous drug combinations. We speculate that monitoring of these patients mitigates the possibility of drug misuse and potential overdose and is in concordance with the goals of these monitoring programs.

Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB.

The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle-structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phospholipase D1 (PLD1) preferentially interacts with two closely related but poorly studied Arf family GTPases, ARL11 and ARL14, showing that PLD1 is activated by ARL11/14 and may recruit these GTPases to membrane vesicles, and that PLD1 and ARL11 collaborate to promote macrophage phagocytosis. Moreover, ARL5A and ARL5B were found to interact with and recruit phosphatidylinositol 4-kinase beta (PI4KB) at trans-Golgi, thus promoting PI4KB's function in PI4P synthesis and protein secretion.

Mass Spectrometry-Based Detection of Beta Lactam Hydrolysis Enables Rapid Detection of Beta Lactamase Mediated Antibiotic Resistance.

OBJECTIVE: Antibiotic resistance by beta lactamase expression is a serious and growing threat. We aimed to determine whether beta-lactamase activity is detectable in urine specimens to enable faster identification of resistance. METHODS: Urine specimens from patients with extended spectrum beta lactamase (ESBL)-expressing urinary infections were incubated with beta lactam antibiotics. Beta lactam hydrolysis was determined by mass spectrometry methods. RESULTS: Ceftriaxone hydrolysis was observed in 45 of 45 ESBL-containing specimens from patients not treated with a beta lactamase inhibitor before specimen collection. Ceftriaxone hydrolysis was not observed in 108 of 108 non-ESBL-containing specimens. Spiking studies show that beta lactam hydrolysis can be observed within 30 minutes. Beta lactam hydrolysis is evidenced by mass spectrometry preceded by either liquid chromatography or matrix-assisted laser desorption ionization specimen processing methods. CONCLUSION: Clinically significant beta lactamase activity is detectable directly from urine specimens. The described methods would enable the detection of beta lactam resistance 24 to 48 hours sooner than culture based methods.

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways.

Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between diverse mechanisms of DPC repair. Here, we show that several SUMO biogenesis factors (Ulp1, Siz2, Slx5, and Slx8) control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1Δ wss1Δ mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation inhibits alternative DPC repair mechanisms, including Ddi1. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair.

Qua-Alluding to the Past: A Case of Methaqualone Analog Ingestion.

Methaqualone, known previously under the brand name Quaalude, is a Schedule I sedative hypnotic drug that may cause neurotoxicity in overdose, characterized by somnolence, hyperreflexia and muscular hyperactivity. We present a case of a 21-year-old male who reportedly ingested methaqualone in addition to insufflation of street cocaine. He subsequently developed hypoxia, hyperreflexia, myoclonus, and altered mental status. His laboratory results were notable for the presence of methemoglobinemia, which was most likely due to a cocaine contaminant. Laboratory analysis of the alleged methaqualone pills identified the substance as SL-164, a dichlorinated methaqualone analog. Urine toxicology results were positive for SL-164 (and presumed metabolites) as well as for cocaine and tetrahydrocannabinol metabolites. The patient was treated with supplemental oxygen and a benzodiazepine (lorazepam) and observed in the Emergency Department (ED) until his symptoms resolved. This case highlights current community access to methaqualone analogs. The case also focuses on laboratory techniques used to identify the methaqualone analog.

A Rapid Assay for SARS-CoV-2 Neutralizing Antibodies That Is Insensitive to Antiretroviral Drugs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike pseudotyped virus (PSV) assays are widely used to measure neutralization titers of sera and of isolated neutralizing Abs (nAbs). PSV neutralization assays are safer than live virus neutralization assays and do not require access to biosafety level 3 laboratories. However, many PSV assays are nevertheless somewhat challenging and require at least 2 d to carry out. In this study, we report a rapid (<30 min), sensitive, cell-free, off-the-shelf, and accurate assay for receptor binding domain nAb detection. Our proximity-based luciferase assay takes advantage of the fact that the most potent SARS-CoV-2 nAbs function by blocking the binding between SARS-CoV-2 and angiotensin-converting enzyme 2. The method was validated using isolated nAbs and sera from spike-immunized animals and patients with coronavirus disease 2019. The method was particularly useful in patients with HIV taking antiretroviral therapies that interfere with the conventional PSV assay. The method provides a cost-effective and point-of-care alternative to evaluate the potency and breadth of the predominant SARS-CoV-2 nAbs elicited by infection or vaccines.