Characteristics of analytically confirmed gamma-hydroxybutyrate (GHB) positive patients in the emergency department: presentation, poly-drug use, disposition and impact on intensive care resource utilisation.

BACKGROUND: Gamma-hydroxybutyrate is a potent central nervous system depressant with a narrow recreational dose window and analytical detection time. We describe data relating to intoxicated patients presenting to emergency departments across metropolitan Adelaide who tested positive for gamma-hydroxybutyrate. This work was part of the Emergency Department Admission Blood Psychoactive Testing study. METHODS: Over a 15-month period, patients presenting to four metropolitan emergency departments with symptoms of drug intoxication were enrolled in the study. The methodology involved the collection of demographic and clinical data and a de-identified blood sample which underwent comprehensive toxicological analysis. Gamma-hydroxybutyrate was determined using an acid-catalysed cyclisation followed by liquid-liquid extraction and gas chromatography-mass spectrometry. Data relating to samples positive for gamma-hydroxybutyrate were examined. RESULTS AND DISCUSSION: A total of 1120 patients were enrolled between March 2019 and May 2020, 309 of whom were positive for gamma-hydroxybutyrate (27.6%). Of these, 256 (83%) were also positive for metamfetamine (methamphetamine). The most common clinical observation in gamma-hydroxybutyrate-positive patients was central nervous system depression (89%). There was a significant relationship between gamma-hydroxybutyrate status and sex; although males outnumbered females in absolute terms, a higher proportion of females (32%) tested positive for gamma-hydroxybutyrate than males (25%, P = 0.0155). Blood gamma-hydroxybutyrate concentrations ranged from 10 to 651 mg/L (0.096-6.2 mmol/L) and increasing gamma-hydroxybutyrate concentration correlated with severe toxicity. The presence of gamma-hydroxybutyrate had a significant impact on the patient discharge destination: the majority (69.2%) of gamma-hydroxybutyrate-positive patients were managed and discharged from the emergency department or their attached short stay wards. A significantly higher proportion of gamma-hydroxybutyrate-positive patients were admitted to the intensive care unit (28.2%) compared with gamma-hydroxybutyrate-negative patients (12.7%, chi-squared = 36.85, P <0 .001). Gamma-hydroxybutyrate positive cases accounted for 45.8% of all study-related intensive care unit admissions. CONCLUSIONS: Gamma-hydroxybutyrate is commonly detected in illicit drug-related emergency department presentations and is detected disproportionately in the patient cohort who require intensive care unit level care.

Residual effects of intra-articular betamethasone and triamcinolone acetonide in an equine acute synovitis model.

BACKGROUND: Intra-articular (IA) corticosteroids are regularly used in equine athletes for the control of joint inflammation. OBJECTIVES: The goal of this study was to use an acute synovitis inflammation model to determine the residual effects of IA betamethasone and triamcinolone acetonide on various inflammatory parameters and lameness. STUDY DESIGN: Crossover randomised trial. METHODS: Five mixed-breed, 2-year-old horses were randomly allocated to an IA treatment of the radiocarpal joint with 9 mg of either betamethasone or triamcinolone acetonide. Two weeks following treatment, horses were injected with 1 µg of lipopolysaccharide (LPS) diluted in 1 ml of saline. Following LPS injection, horses were crossed-over and both sets of injections were repeated after a washout period. Blood samples were collected at multiple time points for mRNA analysis, as well as serum amyloid A (SAA) and cortisol determination. At each time point, lameness was also subjectively scored. Additional injections with saline-only or LPS-only (twice) were conducted as negative and positive controls, respectively. Two-way repeated measures analysis of variance was used to analyse all data. RESULTS: Corticosteroid-only treatments result in significant mRNA expression differences, as well as significant and prolonged cortisol suppression. Following LPS injection, there was a residual treatment effect with triamcinolone evidenced by a significant treatment effect on IL-6 and PTGS1 (cyclooxygenase-1), lameness, SAA and cortisol concentrations, while only IL-6 expression was affected by betamethasone. MAIN LIMITATIONS: The acute synovitis model used here results in significant inflammation and is not representative of the low-grade inflammation seen with typical joint disease and residual anti-inflammatory effects may be more profound in naturally occurring joint disease. CONCLUSIONS: Current regulatory guidelines may be insufficient if the concern is residual anti-inflammatory effects. Additionally, intra-articular corticosteroid administration is not without risk, as evidenced by a significant suppression of serum cortisol concentration and, as such, the benefits of their administration should be weighed against those risks.

Expression of select mRNA in Thoroughbreds with catastrophic racing injuries.

BACKGROUND: The ability to identify horses at risk for catastrophic injuries continues to be a pressing issue for the racing industry, especially given recent events in North America. OBJECTIVES: Since most catastrophic injuries occur in areas of existing pathology and this pathology is likely to elicit an inflammatory response, it was hypothesised that analysis of messenger RNA (mRNA) expression would detect significant changes in select genes in horses at risk for a catastrophic injury. STUDY DESIGN: Prospective cohort study. METHODS: Five racing jurisdictions across the United States participated in this study. A total of 686 Tempus® RNA Blood Tube samples were collected for mRNA analysis from 107 catastrophically injured horses, as well as from noninjured horses sampled either prerace (n = 374) or postrace (n = 205). A subset of horses (n = 37) were sampled both prerace and postrace for analysis of expression changes during the postrace period. RESULTS: Of 21 genes analysed via RT-qPCR, the expression of 12 genes (ALOX5AP, CD14, IL-10, IL-1ß, IL-6, IL-8, MMP1, PTGS2, TLR4, TNFα, TNFSF13B and VEGFA) changed significantly within 45 minutes after a race and were excluded. Of the remaining nine genes (BMP-2, IGF-1, IL1RN, MMP2, MMP9, Osteoprotegrin, RANKL, SAA1 and TGFß), three genes (IGF-1, IL1RN and MMP2) were found to be significantly different between catastrophically injured and noninjured horses using multiple logistic regression modelling. Receiver operating characteristic analysis of models, which included mRNA expression, demonstrated sensitivities from 76%-82% (95% CI: 67%-93%) and specificities from 84%-88% (95% CI: 71%-94%) at the Youden Index. MAIN LIMITATIONS: Samples were collected as soon as possible postinjury (within 30 minutes). CONCLUSIONS: Analysis of mRNA expression of specific genes in the future may be considered as an economical, accessible and noninvasive means by which horses at risk for catastrophic injury can be identified.

Development and Use of an Enzyme-Linked Immunosorbent Assay to Determine Temporal Exposure Patterns to Putative Agents of Nocardioform Placentitis.

Cases of nocardioform placentitis are characterized by focal, mucoid placentitis resulting in late-term abortion, premature birth, or small, full-term foals, occur sporadically, and are most commonly associated with Crossiella equi and Amycolatopsis spp. infection. The goal of this project was to develop an enzyme-linked immunosorbent assay (ELISA) for quantifying antibodies against Crossiella equi and Amycolatopsis spp. and utilize the ELISA to determine when exposure occurs. Serum samples collected during the 2020 foaling season from Crossiella equi (n = 8) and Amycolatopsis spp. (n = 32) infected mares, as well as nonaffected mares (n = 51 mares), were used to develop and optimize bacteria-specific ELISAs. Following development of the ELISAs, banked serum samples from a single, central Kentucky Thoroughbred farm collected during 2012 to 2013 (n = 104 mares) and 2013-14 (n = 82 mares) were analyzed. Differences in various groups were analyzed using one-way analysis of variance (ANOVA). Crossiella equi-infected mares had significantly higher ELISA unit (EU) values on the Crossiella equi ELISA near parturition when compared to the other two groups (P < .001). Using the Amycolatopsis spp. ELISA, EU values were not significantly different between Amycolatopsis spp. infected and non-affected mares, suggesting this ELISA is not specific for Amycolatopsis spp. During 2013 to 2014, there were significant increases in EU values between June and late September for the Crossiella equi ELISA, suggesting exposure in the summer and early fall months. Data from the Crossiella equi ELISA may help provide a better understanding of the epidemiology of nocardioform placentitis, guide the development of a successful experimental challenge model, and allow for further refinement of these ELISAs.

Equine Peripheral Gene Expression Changes in Response to Dose-Dependent Lipopolysaccharide-Induced Synovitis.

The use of lipopolysaccharide to induce a localized source of inflammation (acute synovitis) and allow for monitoring of changes in systemic mRNA expression has been recently reported. Here, the goal was to maintain a significant systemic mRNA response while limiting the severity of lameness such that this model can be used to examine the effects of various anti-inflammatory treatment modalities on mRNA expression. Three mixed breeds, four-year-old geldings were utilized for this study. One milliliter of phosphate-buffered saline containing 1,000 ng or less of lipopolysaccharide from E. coli O111:B4 was aseptically injected into alternating radiocarpal joints following washout periods. Blood for complete blood cell count, serum amyloid A concentration, and mRNA analysis via RT-qPCR for 23 different genes were collected before each injection, as well as at multiple times post-injection. Lameness severity was also graded at each time point. Two-way, repeated measures analysis of variance was used for statistical analysis (P < .05). Results largely replicated those previously reported, with multiple genes exhibiting significant expression changes during the acute inflammatory period (including increases in CD14, TLR4, IL-1ß, IL1RN, MMP1, and MMP9 expression) while some demonstrated dose-dependent changes; significant increases in complete blood cell count parameters and serum amyloid A concentrations were also noted. Attempts to temper the severity of lameness were not successful as nonweight bearing lameness was noted at doses of 10ng or higher, while a dose of 1ng elicited neither a detectable lameness nor a significant change in mRNA expression.

Effect of Dose and Fasting on Oral Sugar Test Responses in Insulin Dysregulated Horses.

The oral sugar test (OST) is frequently used to identify insulin dysregulated (ID) equines. The effect of fasting and varying sugar dose for the OST has been investigated in the pony but little work has been done in the horse. This study aimed to investigate (1) an OST response with access to forage continued until the time of the OST or prevented for 3 hours prior to the OST and (2) responses of ID and non-insulin dysregulated (NID) horses to two different OST doses. Twenty-one mixed-breed horses (14.8 ± 3.2 years; 574.3 ± 83.3 kg) were used in two randomized crossover studies. Seven ID and seven NID horses were used in study A, and eight ID and eight NID in study B. Study A horses underwent an OST (0.15 mL/kg BW) either after a fast (FA) or directly off pasture (FE). Study B horses received either a low (LD; 0.15 mL/kg BW) or high dose (HD; 0.45 mL/kg BW) OST on one occasion each. Blood was collected at basal (T0), and post-60 minute (T60) for later determination of insulin (RIA). Data were analyzed via ANOVA with repeated measures. ID horses had significantly (P < 0.05) greater insulin responses than NID for all OSTs. There was no statistical difference between LD versus HD mean insulin concentrations (T0, T60, delta insulin) for either ID or NID horses. ID had higher T0 (P < 0.01) for FE compared to FA; however, FE and FA did not significantly affect T60 or delta insulins (DI) concentrations.

Establishing the protocols for the South Australian Emergency Department Admission Blood Psychoactive Testing (EDABPT) programme for drug surveillance.

OBJECTIVE: ED presentations because of illicit use of psychotropic drugs and pharmaceuticals result in significant medical harm and resource consumption. Patient assessment is complicated by the regular emergence of new psychoactive substances, difficulties associated with their identification and a lack of information about their effects. Here we report the protocol for the Emergency Department Admission Blood Psychoactive Testing (EDABPT) programme, an observational study utilising clinical data capture and definitive drug identification to assess the medical impact and patterns of illicit drug use in the community, and their geographic and temporal fluctuations. The study provides data to an early warning system targeting an improved public health response to emerging drugs of concern. METHODS: Enrolment of adult patients presenting with suspected illicit drug use occurs at four major EDs in a single urban setting. Clinical and demographic data are collected by treating clinicians. Blood samples are collected at presentation and frozen on site prior to transport to a specialised forensic facility for comprehensive toxicological screening. RESULTS: Results are fed back to clinicians and disseminated more broadly via an existing local early warning system. Targeted warnings and public health releases are instigated where heightened risk or harm is identified. CONCLUSION: The study pairs city-wide patient enrolment with analytically confirmed toxicology results to allow broad sampling and identification of illicit drugs causing medical harm. It provides a mechanism for the identification of new agents as they emerge in the community, delivers a relevant and reliable source of information for public health agencies and clinicians and supplements existing local early warning mechanisms.

Comparison of Nutrient Estimates Based on Food Volume versus Weight: Implications for Dietary Assessment Methods.

Novel technology-based dietary assessment methods use volume estimates of foods to assess dietary intake. However, the nutrient content of standard databases is based on food weight. The goal of this study is to evaluate the accuracy of the United States Department of Agriculture National Nutrient Database for Standard Reference (USDA-SR) estimates of volume and the corresponding macronutrient content of the foods. The weights of 35 individual food volumes were measured (on trial) and compared to the USDA-SR-determined weight for the food volume. Macronutrient content corresponding to the trial weight and the USDA-SR weight for the food volume (USDA) were determined using the USDA-SR, and the differences were calculated. There were statistically significant differences between the USDA and trial weights for 80% of foods measured. Calorie estimates by USDA weight were significantly lower than that of trial weight for 54% of foods but were significantly greater for 26% of foods. Differences in macronutrient estimates by trial and USDA weight varied by food type. These findings suggest that nutrient databases based on food weight may not provide accurate estimates of dietary intake when assessed using food volumes. Further development of image-assisted dietary assessment methods which measure food volumes will necessitate evaluation of the accuracy of the processes used to convert weight to volume in nutrient databases.

The Increasing Use and Abuse of Tapentadol and Its Incorporation Into a Validated Quantitative Method.

Tapentadol is a centrally acting synthetic analgesic which is prescribed for the treatment of a range of chronic pain conditions. Its use in treating various pain conditions is increasing and, as with other opioids, it has the potential to be abused. We describe a three-stage incorporation of tapentadol into validated screening and quantitative methods through: (i) addition of retention time/mass spectral data to a database, (ii) qualitative validation and (iii) quantitative validation. This represents an efficient and flexible approach to the incorporation of new compounds of interest to existing screening methods. Tapentadol was analyzed in blood and serum samples using alkaline liquid-liquid extraction with identification and quantitation by liquid chromatography/time-of-flight mass spectrometry. In a series of six post-mortem cases where tapentadol was detected but was not a primary causative factor in death, blood concentrations ranged from 0.01 to 1.0 mg/L. In two cases where tapentadol was a significant contributor to death, post-mortem blood concentrations were 1.7 and 3.9 mg/L. In one of these fatal cases, ante-mortem blood and serum were also analyzed. The tapentadol concentration in the post-mortem blood was 30% higher than in the ante-mortem blood after a post-mortem interval of 13 days, indicating some potential for post-mortem redistribution. The measured ante-mortem blood:serum ratio was 1.7, and is the first such ratio to be reported. Other drugs were detected in almost all cases, with the majority being prescription analgesics, sedatives and antidepressants. The number of cases in which tapentadol has been detected has increased in recent years, highlighting the importance of screening for this drug in forensic toxicological laboratories.

A Case Study Involving U-47700, Diclazepam and Flubromazepam-Application of Retrospective Analysis of HRMS Data.

The number of new psychoactive substances (NPS) available is constantly increasing, making it difficult for toxicology laboratories to keep screening methods up to date. Full scan high-resolution mass spectrometry (HRMS) is a versatile technique which allows for progressive updating of spectral databases to increase the scope of screening. It also allows for retrospective screening of data-specifically, reprocessing of data files using an updated spectral database without the need for re-extraction or reanalysis.The coronial case reported here illustrates the application of retrospective processing of HRMS data in the detection of emerging NPS. A 28-year-old male with a history of illicit drug use was found deceased at home. Initial routine screening of the post-mortem peripheral blood identified only methylamphetamine, amphetamine and trace amounts of lorazepam. A compound with an accurate mass and isotope ratio consistent with the opioid AH-7921 was also detected in the liquid chromatography (LC)-HRMS screen; however; the retention time and mass spectrum did not match the library. Further investigation confirmed the compound to be U-47700, another opioid and structural isomer of AH-7921. Several months later, after additional NPS had been added to the in-house HRMS database, retrospective screening of the HRMS data was performed, revealing the presence of designer benzodiazepines, diclazepam and flubromazepam as well as the psychedelic drug 2,5-dimethoxy-4-chloroamphetamine (DOC). Quantitative analysis gave the following results in peripheral post-mortem blood: U-47700 (330 µg/L), diclazepam (70 µg/L), flubromazepam (10 µg/L), methylamphetamine (290 µg/L) and amphetamine (150 µg/L) (DOC not quantitated). These substances, along with lorazepam and etizolam, were also confirmed in the post-mortem urine and an investigation into blood and urinary metabolites was carried out. All analyses were performed using the same LC-quadrupole-time of flight method. The cause of death was aspiration (of gastric content into airways and lungs) due to mixed drug toxicity.

A Validated Method for the Screening of 320 Forensically Significant Compounds in Blood by LC/QTOF, with Simultaneous Quantification of Selected Compounds.

A broad drug screening method for toxicologically significant drugs and metabolites in whole blood using liquid chromatography time-of-flight mass spectrometry (LC/QTOF) was developed and comprehensively validated. The method qualitatively screens for 320 compounds while simultaneously quantifying 39. Compounds were extracted from the blood using alkaline liquid/liquid extraction and chromatographic separation was achieved in 12 min. The QTOF was operated using positive mode electrospray ionization using data dependent acquisition. Qualitative validation was performed for all 320 compounds, and included selectivity, recovery, limit of detection, matrix effects, carryover and extract stability. The limits of detection were in the low to sub ng/mL range for the majority of compounds. Full quantitative validation was performed for 39 compounds and accuracy and precision were within 15 and 18%, respectively. The qualitative data processing method uses an in-house retention time, accurate mass and MSMS spectral database, which can be easily updated with new compounds of interest as they emerge onto the market, without affecting method performance. The use of a non-targeted data acquisition method coupled with targeted data processing has proven to be a highly versatile, efficient and robust approach to screening, well suited to meet the needs of the modern toxicology laboratory involved in systematic toxicological analysis.