Xylazine awareness, desire, use and exposure: Preliminary findings from the Rhode Island community-based drug checking cohort study.

Background: Xylazine is an ⍺2 adrenergic receptor agonist and a veterinary sedative that can cause severe health complications yet interventions to detect and treat human exposure remain underdeveloped. Community-based drug checking services (DCS) involve the testing of small amounts of drugs to increase community knowledge of unregulated supplies and decrease harms. This study characterized xylazine awareness, desire, use and exposure among people who use drugs (PWUD) in Rhode Island, US. Methods: We analyzed data from an ongoing PWUD cohort study. In 2023, 125 PWUD were enrolled and surveyed. Using point-of-care Fourier Transform infrared spectroscopy (FTIR-S), we tested a drug sample from each participant onsite and confirmed the results offsite at a laboratory. Results were conveyed in real-time, along with harm reduction education, referrals to resources and care. Results: Virtually all participants (99.2 %) wanted to avoid xylazine exposure. Half (51.2 %) knew what xylazine was, and a quarter (26.1 %) suspected previous exposure. Xylazine exposure was primarily surmised through sedating (45.2 %) and ulcerative (29.0 %) effects. Only 8.8 % of participants submitted a sample that they expected to contain xylazine. Xylazine was detected in 14.5 % of samples using FTIR-S and in 21.4 % of samples using a dual laboratory approach of gas chromatography mass spectrometry (GC-MS) and liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Participants thought that these xylazine-positive samples were fentanyl (78.3 %), heroin (13.0 %), or Percocet® (8.7 %). Conclusion: Implementing point-of-care DCS at harm reduction organizations could be useful in rapidly increasing xylazine awareness and engaging at-risk individuals in prevention, harm reduction, treatment, and rapid care for xylazine-related wounds.

An urgent need for community lot testing of lateral flow fentanyl test strips marketed for harm reduction in Northern America.

BACKGROUND: Fentanyl test strips (FTS) are lateral flow immunoassay strips designed for detection of ng/mL levels of fentanyl in urine. In 2021, the US Centers for Disease Control and the Substance Abuse and Mental Health Administration stated that federal funds could be used for procurement of FTS for harm reduction strategies approved by the government such as drug checking. The market for FTS has expanded rapidly in the US and Canada. However, there is no regulatory oversight by either government to ensure proper function of FTS that are being marketed for drug checking. MAIN BODY: Many brands of FTS have rapidly entered the harm reduction market, creating concerns about the reproducibility and accuracy of their performance from brand to brand and lot to lot. Some examples are provided in this Comment. Similar problems with product quality were observed in the mid 2000's when lateral flow immunoassays for malaria were funded in many countries and again in 2020, when COVID-19 tests were in huge demand. The combination of high demand and low levels of regulation and enforcement led some manufacturers to join the goldrush without adequate field testing or quality assurance. We argue that the harm reduction community urgently needs to set a lot checking program in place. A set of simple protocols for conducting the tests and communicating the results have been developed, and are described in the following Perspectives paper in this issue. CONCLUSION: In the absence of governmental regulation and enforcement, the harm reduction community should implement a FTS lot checking program. Based on previous experience with the malaria diagnostic lot checking program, this inexpensive effort could identify products that are not suitable for harm reduction applications and provide valuable feedback to manufacturers. Dissemination of the results will help harm reduction organizations to ensure that FTS they use for drug checking are fit for the purpose.

Pilot findings on the real-world performance of xylazine test strips for drug residue testing and the importance of secondary testing methods.

Background: Xylazine is a sedative found increasingly in the illicit fentanyl supply that can cause hypotension, bradycardia, necrosis and death. This pilot examined the real-world performance of BTNX xylazine test strips (XTS) in drug residue samples. Methods: This study was nested within a drug checking service in Rhode Island. We tested unmeasured drug residue dissolved in 5 mL of distilled water using XTS and Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF-MS). Analyses compared XTS and LC-QTOF-MS results to calculate XTS detection of xylazine in residue. Results: Among 41 residue samples, xylazine was detected in 11% by the XTS and 44 % by the laboratory. The LC-QTOF-MS detected xylazine in 18 samples: 4 major, 9 minor, 5 trace by volume relative to the whole sample. The XTS disagreed with the LC-QTOF-MS by indicating a negative result in 77.8 % (N=14) of the samples but never indicated a positive when the LC-QTOF-MS reported xylazine's absence. The XTS correctly detected xylazine 22 % of the time, however, this increased to 100 % of the time if xylazine was a major active component. Conclusions: In this study, the BTNX XTS often disagreed with LC-QTOF-MS by indicating a negative result, likely due to the dilution levels used and sample composition. The XTS may not be accurate in detecting residual amounts of xylazine, especially if xylazine is not a dominant component of the tested sample. Given the novelty of BTNX's XTS products, we recommend XTS only be used in conjunction with other advanced drug checking modalities for residue testing.

Comprehensive testing and rapid dissemination of local drug supply surveillance data in Rhode Island.

BACKGROUND: The North American overdose crisis has continued at unprecedented rates with more than 100,000 overdose deaths estimated to have occurred in the United States in 2022. Regional variations in overdose rates signify differences in local drug supplies. State-level drug supply surveillance systems have been limited in their ability to document and communicate the rapidly changing drug supplies which can hinder harm reduction efforts at the community level. We sought to address by piloting a two-year, community-engaged local drug supply surveillance program in Rhode Island (RI). METHODS: The first set of samples (n = 125) were collected from May 2022 to January 2023 across RI and included used paraphernalia (e.g., cookers), refuse (e.g., baggies), and product. Samples were tested using comprehensive toxicology testing approaches via liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Results were disseminated to participants and the broader public across platforms. RESULTS: Fentanyl was detected in 67.2% of all samples tested. 39.2% (n = 49) of samples were expected to be fentanyl. Xylazine was detected in 41.6% of all samples-always in combination with fentanyl-and no samples were expected to contain xylazine. In expected stimulant samples (n = 39), 10% contained fentanyl and/or analogues as major substances and 30.8% contained trace amounts of fentanyl and/or analogues. In expected stimulant samples, 15.4% contained xylazine with fentanyl. No opioids or benzodiazepines were detected in expected hallucinogen or dissociative samples (n = 7). In expected benzodiazepine samples (n = 8), no opioids were detected. CONCLUSIONS: Our results describe part of the local drug supply in Rhode Island, including a presence of NPS and adulterants (e.g., designer benzodiazepines, xylazine). Importantly, our findings underscore the feasibility of developing a community-driven drug supply surveillance database. Expanding drug supply surveillance initiatives is imperative for improving the health and safety of people who use drugs and informing public health approaches to addressing the overdose crisis.

Cannabis Use Patterns and Whole-Blood Cannabinoid Profiles of Emergency Department Patients With Suspected Cannabinoid Hyperemesis Syndrome.

STUDY OBJECTIVES: The objectives of this study were to characterize the detailed cannabis use patterns (eg, frequency, mode, and product) and determine the differences in the whole-blood cannabinoid profiles during symptomatic versus asymptomatic periods of participants with suspected cannabinoid hyperemesis syndrome recruited from the emergency department (ED) during a symptomatic episode. METHODS: This is a prospective observational cohort study of participants with symptomatic cyclic vomiting onset after chronic cannabis use. Standardized assessments were conducted to evaluate for lifetime and recent cannabis use, cannabis use disorder, and cannabis withdrawal symptoms. Quantitative whole-blood cannabinoid testing was performed at 2 times, first when symptomatic (ie, baseline) and at least 2 weeks after the ED visit when asymptomatic. The differences in cannabinoid concentrations were compared between symptomatic and asymptomatic testing. The study was conducted from September 2021 to August 2022. RESULTS: There was a difference observed between delta-9-tetrahydrocannabinol metabolites, but not the parent compound during symptomatic episodes and asymptomatic periods. Most participants (84%) reported using cannabis > once per day (median 3 times per day on weekdays, 4 times per day on weekends). Hazardous cannabis use was universal among participants; the mean cannabis withdrawal discomfort score was 13, indicating clinically significant rates of cannabis withdrawal symptoms with cessation of use. Most participants (79%) previously tried to stop cannabis use, but a few (13%) of them had sought treatment. CONCLUSION: Patients presenting to the ED with cannabinoid hyperemesis syndrome have high cannabis use disorder scores. Further studies are needed to better understand the influence of THC metabolism and concentrations on symptomatic cyclic vomiting.

An Opioid Hiding in Plain Sight: Loperamide-Induced False-Positive Fentanyl and Buprenorphine Immunoassay Results.

BACKGROUND: Loperamide (Imodium®), a commonly used anti-diarrheal, is a mu opioid receptor agonist that, like all opioids, reduces gastrointestinal tract peristalsis. Loperamide is considered to have low abuse potential as it does not produce an analgesic or euphoric effect due to low bioavailability and first-pass metabolism. However, reports of individuals misusing loperamide through the use of super-therapeutic doses, alone or in combination with P-glycoprotein and/or CYP450 enzyme inhibitors, is increasing. We hypothesized that loperamide could potentially cross-react with laboratory immunoassay drug screens. METHODS: Drug-free urine was spiked with loperamide or its principal metabolite, N-desmethyl loperamide (dLop), and assayed on multiple fentanyl and buprenorphine assays. Fentanyl immunoassay screen-positive results at one institution were examined by high-resolution mass spectrometry (MS) for the presence of loperamide and quantified by liquid chromatography- tandem MS when positive. RESULTS: Loperamide produced positive results on the Thermo DRI Fentanyl and Immunalysis Fentanyl assays at concentrations greater than 5.72 mg/L and 23.7 mg/L. dLop generated positive results for the Thermo DRI and Immunalysis fentanyl assays at concentrations exceeding 6.9 mg/L and 35.7 mg/L. dLop also produced positive buprenorphine results on the Thermo CEDIA buprenorphine assay at concentrations exceeding 12.2 mg/L. High-resolution MS analysis of 225 fentanyl immunoassay positives (Thermo DRI) yielded 5 specimens containing loperamide and/or dLop, 4 of which contained measurable quantities of fentanyl in addition to loperamide/dLop. CONCLUSIONS: Laboratories using these assays should be aware of the potential for false-positive screening results due to the presence of high concentrations of loperamide and its metabolite dLop.

Lacosamide induced Brugada I morphology in the setting of septicemia: A case report.

INTRODUCTION: Brugada syndrome may be unmasked by non-antiarrhythmic pharmaceuticals or drugs. Lacosamide is an antiepileptic agent with a novel mechanism of sodium channel inhibition and has the potential to cause cardiac sodium channel blockade. PATIENT CONCERNS: In this report, we describe the case of patient with a history of a seizure disorder who presented with Brugada I electrocardiogram morphology in the setting of septicemia. DIAGNOSIS: Brugada I electrocardiogram morphology was unmasked by lacosamide antiepileptic monotherapy. INTERVENTIONS: Lacosamide therapy was discontinued. OUTCOMES: Normalization of the electrocardiogram and resolution of Brugada morphology occurred on hospital day 1. CONCLUSION: Caution should be exercised in the use of lacosamide in those at risk for conduction delay, or in combination therapy with medications that impair renal clearance, metabolism of lacosamide, or that display inherent sodium channel blocking properties.

A case of unexplained duodenal ulcer and massive gastrointestinal bleed.

A 73-year-old man was displaying symptoms of massive gastrointestinal (GI) bleed. Surgical actions were performed to control the bleed caused by an erosive duodenal ulcer with duodenal perforation. When investigating the culprit of this case, the pain medications prescribed two weeks prior by a traditional Chinese medicine doctor raised attention. The patient's admission serum sample and the pain medications from unknown sources were analyzed using a clinically validated liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method. The NSAIDs diclofenac, piroxicam, and indomethacin were identified, as well as some other synthetic drugs and natural products. The patient's concurrent exposure to multiple NSAIDs significantly increased the risk of upper GI complications. It is reasonable to argue that the high-dose use of the NSAIDs was a major cause of the duodenal ulcer and GI bleed. In addition, the identified natural products such as atropine and ephedrine have well-documented toxicities. It is important to increase the visibility of unregulated medications, and the capability to perform untargeted mass spectrometry analysis provides a unique diagnostic advantage in cases where exposure to toxic substances is possible.

Real-time comprehensive toxicology testing in the clinical management of accidental pediatric capecitabine ingestion.

INTRODUCTION: Capecitabine is an orally bioavailable prodrug of the chemotherapeutic agent, fluorouracil. Fluorouracil is converted to several active metabolites that induce a cytotoxic effect. Capecitabine toxicity can be life-threatening with a delayed presentation from ingestion. An oral antidote, uridine triacetate, exists but requires the administration of 20 total doses over a course of five days. CASE REPORT: In this report, we describe a case where timely coordination with a clinical toxicology laboratory was utilized to drive clinical decision making and management. Two children were brought to the emergency department shortly after suspected capecitabine ingestion. MANAGEMENT AND OUTCOME: Patients were admitted to the hospital and started on uridine triacetate. Real-time comprehensive toxicology testing of the children's blood was used to rule out capecitabine toxicity and prevent several unnecessary days of hospitalization and doses of antidote. Patients were discharged safely. DISCUSSION: Real-time comprehensive toxicology testing on a patient's blood may be a valuable resource in ruling out or confirming toxic exposure in accidental pediatric ingestion of chemotherapeutic agents like capecitabine when performed in a timely manner.

3D-Printed Hydrogel Composites for Predictive Temporal (4D) Cellular Organizations and Patterned Biogenic Mineralization.

Materials chemistries for hydrogel scaffolds that are capable of programming temporal (4D) attributes of cellular decision-making in supported 3D microcultures are described. The scaffolds are fabricated using direct-ink writing (DIW)-a 3D-printing technique using extrusion to pattern scaffolds at biologically relevant diameters (≤ 100 µm). Herein, DIW is exploited to variously incorporate a rheological nanoclay, Laponite XLG (LAP), into 2-hydroxyethyl methacrylate (HEMA)-based hydrogels-printing the LAP-HEMA (LH) composites as functional modifiers within otherwise unmodified 2D and 3D HEMA microstructures. The nanoclay-modified domains, when tested as thin films, require no activating (e.g., protein) treatments to promote robust growth compliances that direct the spatial attachment of fibroblast (3T3) and preosteoblast (E1) cells, fostering for the latter a capacity to direct long-term osteodifferentiation. Cell-to-gel interfacial morphologies and cellular motility are analyzed with spatial light interference microscopy (SLIM). Through combination of HEMA and LH gels, high-resolution DIW of a nanocomposite ink (UniH) that translates organizationally dynamic attributes seen with 2D gels into dentition-mimetic 3D scaffolds is demonstrated. These analyses confirm that the underlying materials chemistry and geometry of hydrogel nanocomposites are capable of directing cellular attachment and temporal development within 3D microcultures-a useful material system for the 4D patterning of hydrogel scaffolds.

Three-dimensional mesostructures as high-temperature growth templates, electronic cellular scaffolds, and self-propelled microrobots.

Recent work demonstrates that processes of stress release in prestrained elastomeric substrates can guide the assembly of sophisticated 3D micro/nanostructures in advanced materials. Reported application examples include soft electronic components, tunable electromagnetic and optical devices, vibrational metrology platforms, and other unusual technologies, each enabled by uniquely engineered 3D architectures. A significant disadvantage of these systems is that the elastomeric substrates, while essential to the assembly process, can impose significant engineering constraints in terms of operating temperatures and levels of dimensional stability; they also prevent the realization of 3D structures in freestanding forms. Here, we introduce concepts in interfacial photopolymerization, nonlinear mechanics, and physical transfer that bypass these limitations. The results enable 3D mesostructures in fully or partially freestanding forms, with additional capabilities in integration onto nearly any class of substrate, from planar, hard inorganic materials to textured, soft biological tissues, all via mechanisms quantitatively described by theoretical modeling. Illustrations of these ideas include their use in 3D structures as frameworks for templated growth of organized lamellae from AgCl-KCl eutectics and of atomic layers of WSe2 from vapor-phase precursors, as open-architecture electronic scaffolds for formation of dorsal root ganglion (DRG) neural networks, and as catalyst supports for propulsive systems in 3D microswimmers with geometrically controlled dynamics. Taken together, these methodologies establish a set of enabling options in 3D micro/nanomanufacturing that lie outside of the scope of existing alternatives.

3D-Printed pHEMA Materials for Topographical and Biochemical Modulation of Dorsal Root Ganglion Cell Response.

Understanding and controlling the interactions occurring between cells and engineered materials are central challenges toward progress in the development of biomedical devices. In this work, we describe materials for direct ink writing (DIW), an extrusion-based type of 3D printing, that embed a custom synthetic protein (RGD-PDL) within the microfilaments of 3D-hydrogel scaffolds to modify these interactions and differentially direct tissue-level organization of complex cell populations in vitro. The RGD-PDL is synthesized by modifying poly-d-lysine (PDL) to varying extents with peptides containing the integrin-binding motif Arg-Gly-Asp (RGD). Compositional gradients of the RGD-PDL presented by both patterned and thin-film poly(2-hydroxyethyl) methacrylate (pHEMA) substrates allow the patterning of cell-growth compliance in a grayscale form. The surface chemistry-dependent guidance of cell growth on the RGD-PDL-modified pHEMA materials is demonstrated using a model NIH-3T3 fibroblast cell line. The formation of a more complex cellular system-organotypic primary murine dorsal root ganglion (DRG)-in culture is also achieved on these scaffolds, where distinctive forms of cell growth and migration guidance are seen depending on their RGD-PDL content and topography. This experimental platform for the study of physicochemical factors on the formation and the reorganization of organotypic cultures offers useful capabilities for studies in tissue engineering, regenerative medicine, and diagnostics.

Quantitative Reflection Imaging for the Morphology and Dynamics of Live Aplysia californica Pedal Ganglion Neurons Cultured on Nanostructured Plasmonic Crystals.

We describe a reflection imaging system that consists of a plasmonic crystal, a common laboratory microscope, and band-pass filters for use in the quantitative imaging and in situ monitoring of live cells and their substrate interactions. Surface plasmon resonance (SPR) provides a highly sensitive method to monitor changes in physicochemical properties occurring at metal-dielectric interfaces. Polyelectrolyte thin films deposited using the layer-by-layer (LBL) self-assembly method provide a reference system for calibrating the reflection contrast changes that occur when the polyelectrolyte film thickness changes and provide insight into the optical responses that originate from the multiple plasmonic features supported by this imaging system. Finite-difference time-domain (FDTD) simulations of the optical responses measured experimentally from the polyelectrolyte reference system are used to provide a calibration of the optical system for subsequent use in quantitative studies investigating live cell dynamics in cultures supported on a plasmonic crystal substrate. Live Aplysia californica pedal ganglion neurons cultured in artificial seawater were used as a model system through which to explore the utility of this plasmonic imaging technique. Here, the morphology of cellular peripheral structures ≲80 nm in thickness were quantitatively analyzed, and the dynamics of their trypsin-induced surface detachment were visualized. These results illustrate the capacities of this system for use in investigations of the dynamics of ultrathin cellular structures within complex bioanalytical environments.

Deterministic Integration of Biological and Soft Materials onto 3D Microscale Cellular Frameworks.

Complex 3D organizations of materials represent ubiquitous structural motifs found in the most sophisticated forms of matter, the most notable of which are in life-sustaining hierarchical structures found in biology, but where simpler examples also exist as dense multilayered constructs in high-performance electronics. Each class of system evinces specific enabling forms of assembly to establish their functional organization at length scales not dissimilar to tissue-level constructs. This study describes materials and means of assembly that extend and join these disparate systems-schemes for the functional integration of soft and biological materials with synthetic 3D microscale, open frameworks that can leverage the most advanced forms of multilayer electronic technologies, including device-grade semiconductors such as monocrystalline silicon. Cellular migration behaviors, temporal dependencies of their growth, and contact guidance cues provided by the nonplanarity of these frameworks illustrate design criteria useful for their functional integration with living matter (e.g., NIH 3T3 fibroblast and primary rat dorsal root ganglion cell cultures).

Programming Mechanical and Physicochemical Properties of 3D Hydrogel Cellular Microcultures via Direct Ink Writing.

3D hydrogel scaffolds are widely used in cellular microcultures and tissue engineering. Using direct ink writing, microperiodic poly(2-hydroxyethyl-methacrylate) (pHEMA) scaffolds are created that are then printed, cured, and modified by absorbing 30 kDa protein poly-l-lysine (PLL) to render them biocompliant in model NIH/3T3 fibroblast and MC3T3-E1 preosteoblast cell cultures. Spatial light interference microscopy (SLIM) live cell imaging studies are carried out to quantify cellular motilities for each cell type, substrate, and surface treatment of interest. 3D scaffold mechanics is investigated using atomic force microscopy (AFM), while their absorption kinetics are determined by confocal fluorescence microscopy (CFM) for a series of hydrated hydrogel films prepared from prepolymers with different homopolymer-to-monomer (Mr ) ratios. The observations reveal that the inks with higher Mr values yield relatively more open-mesh gels due to a lower degree of entanglement. The biocompatibility of printed hydrogel scaffolds can be controlled by both PLL content and hydrogel mesh properties.