Postmortem metabolomics: influence of time since death on the level of endogenous compounds in human femoral blood. Necessary to be considered in metabolome study planning?

INTRODUCTION: The (un)targeted analysis of endogenous compounds has gained interest in the field of forensic postmortem investigations. The blood metabolome is influenced by many factors, and postmortem specimens are considered particularly challenging due to unpredictable decomposition processes. OBJECTIVES: This study aimed to systematically investigate the influence of the time since death on endogenous compounds and its relevance in designing postmortem metabolome studies. METHODS: Femoral blood samples of 427 authentic postmortem cases, were collected at two time points after death (854 samples in total; t1: admission to the institute, 1.3-290 h; t2: autopsy, 11-478 h; median ∆t = 71 h). All samples were analyzed using an untargeted metabolome approach, and peak areas were determined for 38 compounds (acylcarnitines, amino acids, phospholipids, and others). Differences between t2 and t1 were assessed by Wilcoxon signed-ranked test (p < 0.05). Moreover, all samples (n = 854) were binned into time groups (6 h, 12 h, or 24 h intervals) and compared by Kruskal-Wallis/Dunn's multiple comparison tests (p < 0.05 each) to investigate the effect of the estimated time since death. RESULTS: Except for serine, threonine, and PC 34:1, all tested analytes revealed statistically significant changes between t1 and t2 (highest median increase 166%). Unpaired analysis of all 854 blood samples in-between groups indicated similar results. Significant differences were typically observed between blood samples collected within the first and later than 48 h after death, respectively. CONCLUSIONS: To improve the consistency of comprehensive data evaluation in postmortem metabolome studies, it seems advisable to only include specimens collected within the first 2 days after death.

A retrospective review of methylamphetamine detected in child deaths reported to the Victorian Coroner, Australia.

This study investigated methylamphetamine (MA) exposures in the deaths of children (≤ 12 years old) reported to the Coroner in the state of Victoria, Australia, between 2011 and 2020. Demographics, autopsy findings including the cause of death, self-reported prenatal or caregiver drug use, child protection services information, and toxicological findings were summarized by descriptive statistics. Validated methods of liquid chromatography-tandem mass spectrometry were used in the analysis of drugs. There were 50 child deaths with MA detected in blood, urine, and/or hair with 64% (n = 32) identified in 2018-2020. Most children were 1-365 days old (66%, n = 33) and the cause of death was unascertained in 62% (n = 31) of cases. MA was toxicologically confirmed in hair (94%, n = 47) significantly more than blood (18%, n = 9). Prenatal or caregiver drug use was self-reported in 44% (n = 22) and 42% (n = 21) of cases, respectively. Moreover, only 54% (n = 27) of deceased children were a child protection client at their time of death. These findings suggest the number of deceased children exposed to MA has increased over the past 10 years, which is consistent with the greater supply of crystal MA in the Australian community. Hair analysis provided additional means to identify cases that were unknown to child protection services and may have implications for other children in the same drug exposure environment.

Methylamphetamine toxicity and its involvement in death: A retrospective observational study of deaths reported to the Victorian Coroner, Australia.

A retrospective observational study of Victorian deaths involving MA between 2010 and 2019 was conducted to determine the prevalence and contribution of methylamphetamine (MA) toxicity to death in the absence of other factors. Demographics, autopsy findings, toxicology, and the cause of death were reviewed. Coronial cases were categorized into five groups: deaths due to MA toxicity in the absence of other factors (Group A1); deaths due to MA toxicity in the setting of other potentially contributing factors (Group A2); deaths due to MA toxicity in the setting of significant natural disease (Group B); deaths primarily due to multiple-drug toxicity (Group C); and deaths primarily due to natural causes (Group D). There were 506 deaths involving MA categorized into Group A1 (n = 1, 0.6%), Group A2 (n = 8, 1.6%), Group B (n = 28, 5.5%), Group C (n = 229, 45%), and Group D (n = 240, 47%). Significant natural disease was prevalent among deaths involving MA and mainly concerned forms of cardiovascular disease (n = 277, 55%). The MA concentration in the one death included in Group A1 was 2.1 mg/L. The median MA concentrations of Group A2 (1.6 mg/L) and Group B (0.5 mg/L) were significantly higher than Group C (0.2 mg/L) and Group D (0.2 mg/L). Additionally, many other toxicologically significant drugs were detected and mostly comprised of central nervous system depressants. Deaths due to MA toxicity in the absence of other factors were rare despite the greater availability of crystal MA in the Australian community. The study highlights the interpretative challenges of MA blood concentrations and the continuing harms of this drug in Australia.

The role of voluntary and involuntary drug and alcohol consumption and premorbid mental health factors in drug-facilitated sexual assault.

In order to better understand risk factors associated with drug-facilitated sexual assault (DFSA), this study examined complainant-specific and contextual factors, as well as the toxicological profile of DFSA in Victoria, Australia. Clinical files and toxicological analysis results collected by the Victorian Institute of Forensic Medicine (VIFM) for all cases of alleged DFSA in Victoria that occurred between 1st January 2011 - 31st December 2013 were reviewed. Two hundred and four cases of alleged DFSA were identified; complainants were predominately female (93%), and their median age was 26 years (range = 18-54). Self-reported premorbid depression (21.1%) and drug and alcohol abuse (9.8%) were four and two times higher than 12-month prevalence rates in Australia, respectively. All assailants were male, half were known to the complainant and half of alleged assaults occurred in private residences. Most (93.6%) complainants reported voluntary consumption of psychoactive substances prior to the alleged DFSA. Alcohol was the most commonly self-reported substance consumed (n = 164; 64%) and concomitant use of alcohol, prescription and illicit drugs was also commonly self-reported (24%). There were 14 cases that produced a positive toxicology result where the complainant did not report voluntary consumption, which suggests these drugs may have been used covertly to facilitate sexual assault. The results of this study indicate that Females in their mid-20's who exhibit higher rates of mental health concerns represent a sub-group of the Australian population with increased vulnerability to DFSA, which typically occurs in a familiar setting in the context of voluntary alcohol and other substance use.

The effectiveness of decontamination procedures used in forensic hair analysis.

Hair is a mainstream specimen used in forensic toxicology to determine drug use and exposure. However, the interpretation of an analytical hair result can be complicated by the presence of external drug contamination. Decontamination procedures are included in hair analysis methods to remove external contamination, but the capacity of these washes to completely remove contamination for all drugs is controversial. It is evident that there is no consensus on the most effective decontamination procedure, nor can decontamination procedures consistently remove external drug contamination to less than reportable cut-offs for all analytes. ∆9-tetrahydrocannabinol deposited from cannabis smoke is mostly removed by organic solvents, whereas ionizable drugs are more effectively removed by an aqueous wash. Organizations such as the Society of Hair Testing recommend a hair decontamination procedure should include both an organic and aqueous washing step, which is in accordance with the reviewed literature. Studies involving a systematic evaluation of various solvents have shown that the most effective organic solvent was methanol and the most effective aqueous solvent contained sodium dodecyl sulfate detergent. If future systematic studies can demonstrate similar findings, a consensus on the most effective decontamination procedure for forensic hair analysis may be established.

Ultra-rapid targeted analysis of 40 drugs of abuse in oral fluid by LC-MS/MS using carbon-13 isotopes of methamphetamine and MDMA to reduce detector saturation.

The number of oral fluid samples collected by the road policing authority in Victoria, Australia, requiring confirmatory laboratory analysis for drugs proscribed under Victorian legislation (methamphetamine, MDMA and Δ9-tetrahydrocannabinol) has greatly increased in recent years, driving the need for improved analysis techniques to enable expedient results. The aim of this study was to develop an LC-MS/MS-based targeted oral fluid screening technique that covers a broad range of basic and neutral drugs of abuse that can satisfy increased caseload while monitoring other compounds of interest for epidemiological purposes. By combining small sample volume, simple extraction procedure, rapid LC-MS/MS analysis and automated data processing, 40 drugs of abuse including amphetamines, benzodiazepines, cocaine and major metabolites, opioids, cannabinoids and some designer stimulants were separated over 5 min (with an additional 0.5 min re-equilibration time). The analytes were detected using a Sciex® API 4500 Q-Trap LC-MS/MS system with positive ESI in MRM mode monitoring three transitions per analyte. The method was fully validated in accordance with international guidelines and also monitored carbon-13 isotopes of MDMA and MA to reduce detector saturation effects, allowing for confirmation of large concentrations of these compounds without the need for dilution or re-analysis. The described assay has been successfully used for analysis of oral fluid collected as part of law enforcement procedures at the roadside in Victoria, providing forensic results as well as epidemiological prevalence in the population tested. The fast and reliable detection of a broad range of drugs and subsequent automated data processing gives the opportunity for high throughput and fast turnaround times for forensic toxicology.

The postmortem redistribution of iso-α-acids in postmortem specimens.

Iso-α-acids (IAA) and reduced IAA can be used as beer-specific ingredient congeners to confirm beer consumption when detected in blood and other specimens using a UHPLC-MS/MS method. Recent analysis of postmortem casework demonstrated a high prevalence of beer consumption and the possibility of providing the source of alcohol in forensic casework. Research outlined in this manuscript has examined the degree to which the interval after death and quality of blood affects the concentration of IAA in postmortem cases. Postmortem whole blood and serum were analyzed in cases where natural or reduced IAA groups were detected. The trans-IAA, cis-IAA, and tetrahydro-IAA (TIAA) groups were subject to postmortem redistribution, although only weakly associated with the length of time from death to collection of specimens. Serum had threefold higher concentrations than blood for trans-IAA, cis-IAA, and TIAA. These studies confirm that although postmortem concentrations cannot be easily compared to concentrations found in living persons the presented findings do provide some understanding to assist in interpretation where the confirmation of beer consumption is required in forensic casework.

The analysis of methylamphetamine and para-hydroxy-methylamphetamine in post-mortem hair samples using liquid chromatography-tandem mass spectrometry.

External contamination is a well-recognized limitation of hair analysis for drugs of abuse like methylamphetamine (MA), and there are no guidelines regarding the analysis of specific metabolites of MA to assist interpretation. We developed an analytical method to detect MA, amphetamine (AMP), and para-hydroxy-methylamphetamine (p-OH-MA) in hair and present their concentrations among a cohort of deceased persons positive for MA in blood (n = 63). Hair samples (≤ 3 cm) were washed with dichloromethane and water prior to extraction using a methanolic micro-pulverization. The reconstituted hair extracts were separated on a UCT Selectra® Aqueous C18 HPLC Column (100 × 2.1 mm, 3 µm) by gradient elution and detected using a Sciex Triple Quad 6500+ system. Validation was satisfactory, and the lower limits of quantitation were 0.01 ng/mg for MA and AMP and 0.001 ng/mg for p-OH-MA. The median hair concentrations of MA, AMP, and p-OH-MA were 13 ng/mg (range = 0.015-49; n = 51), 1.1 ng/mg (range = 0.018-44; n = 60), and 0.020 ng/mg (range = 0.0012-0.38, n = 62), respectively. These concentrations in hair were strongly positively correlated (r = .7202 to .8641, p < .001), suggesting similar modes of incorporation. Moreover, the wash/hair ratios were indicative of external contamination, especially among the soiled group of hair samples. Therefore, further studies are necessary to determine concentrations of p-OH-MA in living MA users and confirm if this metabolite constitutes a potential marker of MA consumption.

Identification of clobromazolam in Australian emergency department intoxications using data-independent high-resolution mass spectrometry and the HighResNPS.com database.

The proliferation of novel psychoactive substances (NPSs) continues to challenge toxicology laboratories. In particular, the United Nations Office on Drugs and Crime considers designer benzodiazepines to be a current primary threat among all NPSs. Herein, we report detection of a new emerging designer benzodiazepine, clobromazolam, using high-resolution mass spectrometry and untargeted data acquisition in combination with a "suspect screening" method built from the crowd-sourced HighResNPS.com database. Our laboratory first detected clobromazolam in emergency department presenting intoxications included within the Emerging Drugs Network of Australia-Victoria project in the state of Victoria, Australia, from April 2022 to March 2023. Clobromazolam was the most frequent designer benzodiazepine detected in this cohort (100/993 cases, 10%). No patients reported intentional administration of clobromazolam, although over half reported exposure to alprazolam, which was detected in only 7% of cases. Polydrug use was prevalent (98%), with phenazepam (45%), methylamphetamine (71%) and other benzodiazepines (60%) most frequently co-detected. This is the first case series published in the literature concerning clobromazolam in clinical patients. The identification of clobromazolam in patients presenting to emergency departments in Victoria demonstrates how high-resolution mass spectrometry coupled with the HighResNPS.com database can be a valuable tool to assist toxicology laboratories in keeping abreast of emerging psychoactive drug use.

The rapid identification and quantification of iso-α-acids and reduced iso-α-acids in blood using UHPLC-MS/MS: validation of a novel marker for beer consumption.

A method for the detection of iso-α-acid (IAA) type ingredient congeners that are derived from the hop plant (Humulus lupulus L.) was developed to detect recent consumption of beer in blood. Three structurally similar but chemically altered IAA, also used as beer-specific ingredients, are known as "reduced IAA", consisting of the rho-, tetrahydro-, and hexahydro-IAA were also targeted. The use of a simple protein precipitation extraction and ultrahigh-performance liquid chromatography system coupled with a tandem mass spectrometer system enabled detection of these analytes in both antemortem and postmortem blood. Extracts were injected onto a C18 solid-core column under gradient elution to achieve separation of isobaric analogs and isomers within a 10-min run time. Electrospray ionization in negative multiple reaction monitoring mode was used to monitor three transitions for each of the analytes that were ultimately grouped together to form a calibration curve for quantification of each of the four IAA groups. The method was fully validated according to international guidelines that included extraction efficiency, matrix effects, process efficiency, ion suppression/enhancement of co-eluting analytes, selectivity, crosstalk, accuracy and precision, stabilities, and lower limits of quantification. Finally, applicability of the method described was demonstrated by the detection of IAA ingredient congeners in the blood of a volunteer following the consumption of a relatively small amount of beer in a pilot study.

Alcohol congener analysis and the source of alcohol: a review.

For many decades traditional alcohol congener analysis has provided the concentrations of fermentation by-product congeners found in blood, to ascertain if the claims of an individual regarding the alcoholic beverage(s) they have consumed were feasible, assisting in cases where after-drinking is involved. However, this technique does not provide information on the exact alcoholic beverage(s) consumed. More recently, ingredient biomarker congeners specific to certain alcoholic beverages have been detected in blood, making it possible to identify the particular alcoholic beverage consumed and therefore the source of alcohol (albeit only for a limited number of beverages). This novel approach may reduce current limitations that exist with traditional methods of detecting fermentation by-product congeners, which restrict the use of alcohol congener analysis internationally and for other medico-legal scenarios. This review examines the forensic application of alcohol congener analysis in determining the source of alcohol and other techniques.

Changing trends and characteristics of one punch deaths in Australia (2012-2018).

One-punch assaults also known as 'coward punches', are characterised by a single severe blow to the head causing the victim to lose consciousness, resulting in a secondary impact between the head and surrounding environment. Such impacts may result in brain injury leading to fatality or permanent neurological impairment. In a previous publication, there were 90 one punch deaths around Australia between 2000 and 2012, mostly involving young men drinking alcohol at a licensed venue at the weekend. This prompted a surge of public education and awareness campaigns around Australia, in addition to regulatory and legislative changes aimed at curbing social violence. This retrospective descriptive study aimed to examine one punch deaths since 2012 in Australia to determine if there has been a decrease in deaths, and whether the demographics and circumstances of these deaths have changed. A search of the National Coronial Information System was undertaken for all closed coronial cases between 1 January 2012 and 31 December 2018. Additional information was collected from medicolegal reports including toxicology, pathology and coronial findings. There were 80 one punch fatalities in Australia, almost exclusively involving males. The median age was 43.5 (range 18-71) years and there was a decreasing trend in the number of deaths annually. Most fatal assaults occurred in the state of New South Wales (28.8%) followed by Queensland (23.8%), and in metropolitan locations (64.6%) rather than regional areas (35.4%). Alcohol was the most commonly detected drug, found in 47 cases of the 71 cases where toxicology results were available (66%), with a median concentration of 0.14 and 0.19 g/100 mL in antemortem and postmortem samples, respectively (range 0.005-0.32 g/100 mL). Five deaths reported methylamphetamine, with THC detected in 21.1% of cases. Assaults more commonly occurred on a footpath or roadside (41.3%), followed by a home or dwelling (32.5%). 8.8% of assaults occurred inside hotels, bars or other licenced venues. Most transpired on a weekday, which differed from the pre-2012 period when these assaults occurred mainly on the weekend. While some trends are positive, there has been a shift in the victim demographic as well as the typical environment for fatal one punch assaults, highlighting the importance of public health surveillance in providing a current evidence base to inform policy and practice.

Quantitative analysis of tetrahydrocannabinol isomers and other toxicologically relevant drugs in blood.

A multi-analyte liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described, involving the separation of delta-9-tetrahydrocannabinol (delta-9-THC) and delta-8-THC in addition to other commonly encountered drugs and metabolites. Briefly, sample preparation involved an alkaline liquid-liquid extraction (methyl tert-butyl ether) of blood (100 µl). The solvent layer was transferred, evaporated to dryness, reconstituted, and samples then separated on an Agilent Poroshell 120 EC-C18 100 Å (50 mm × 3.0 mm, 2.7 µm) analytical column using a multi-step gradient elution of 50 mM ammonium formate in water (pH 3.5) and 0.1% formic acid in methanol over 14 min. A SCIEX Triple Quad 6500+ system operating in scheduled multiple reaction monitoring and positive electrospray ionization was used for detection. There were no interferences, and matrix effects were generally acceptable (±20% of neat response). Linearity was achieved within the calibration range, including methylamphetamine (MA) (10-1000 ng/ml), 3,4-methylenedioxy-N-methylamphetamine (MDMA) (10-1,000 ng/ml), cocaine (10-1000 ng/ml), and two THC isomers (1-100 ng/ml). Accuracies of MA, MDMA, cocaine, and two THC isomers were 3.6 to 8.9%, -1.2 to 4%, -5.3 to 5.8%, and -11 to 14%, respectively; while precision estimates of the same were 1.6 to 5.4%, 1.7 to 5.3%, 1.2 to 4.5%, and 2 to 10%, respectively. Autosampler stability and dilution integrity were within acceptable limits, and no carryover was detected at the limit of detection. This validated LC-MS/MS method made the routine identification of both delta-9-THC and delta-8-THC in blood possible.

Fatal Intoxications from a Combination of 4-Fluoroamphetamine and 25C-NBOMe.

Six fatalities have occurred from the ingestion of a combination of new psychoactive substances (NPSs), 4-fluoroamphetamine (4FA) and 2-(4-chloro-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25C-NBOMe) over a 9-month period. Four of these fatalities (one older female and three young males) were from direct adverse effects of drugs, and one each from a fall while being intoxicated and during restraint. All cases were subject to full postmortem examinations that included collection of femoral blood. The four drug-caused fatalities had postmortem blood concentrations for 4FA and 25C-NBOMe of 330-682 ng/L (median 417) and 1.4-12 ng/mL (median 4.3), respectively. The other two cases (both young males) where death was considered to have been caused indirectly by drug intoxication had 4FA and 25C-NBOMe postmortem concentrations of 21 and 123 ng/mL, and 1.8 and 4.5 ng/mL, respectively. None of these cases showed concentrations of drugs that suggested use of high recreational doses. In one drug-caused death, capsules and a brown powder obtained from the scene were found to contain a mixture of these two NPSs. With the exception of one drug-caused death, other drugs were detected; however, the effects of the two NPSs together were regarded as the primary triggers for the deaths. There were no consistent symptoms or pathology in these cases; however, agitation/aggression was observed in two cases prior to their collapse, with seizures in possibly three cases. Pulmonary and/or cerebral edema was noted in three cases. Potentially significant natural disease (a mildly enlarged heart) was only observed in one drug-caused case. These cases illustrate a possible increased risk of sudden death with this combination of drugs, both of which can elevate serotonin concentrations as well as act as strong stimulants. These cases also illustrate the difficulty in detecting NPS in cases where no prior information is available that might suggest their use.

A risk-based approach to community illicit drug toxicosurveillance: operationalisation of the Emerging Drugs Network of Australia - Victoria (EDNAV) project.

INTRODUCTION: The Emerging Drugs Network of Australia - Victoria (EDNAV) project is a newly established toxicosurveillance network that collates clinical and toxicological data from patients presenting to emergency departments with illicit drug related toxicity in a centralised clinical registry. Data are obtained from a network of sixteen public hospital emergency departments across Victoria, Australia (13 metropolitan and three regional). Comprehensive toxicological analysis of a purposive sample of 22 patients is conducted each week, with reporting of results to key alcohol and other drug stakeholders. This paper describes the overarching framework and risk-based approach developed within Victoria to assess drug intelligence from EDNAV toxicosurveillance. METHODS: Risk management principles from other spheres of public health surveillance and healthcare clinical governance have been adapted to the EDNAV framework with the aim of facilitating a consistent and evidence-based approach to assessing weekly drug intelligence. The EDNAV Risk Register was reviewed over the first two years of EDNAV project operation (September 2020 - August 2022), with examples of eight risk assessments detailed to demonstrate the process from signal detection to public health intervention. RESULTS: A total of 1112 patient presentations were documented in the EDNAV Clinical Registry, with 95 signals of concern entered into the EDNAV Risk Register over the two-year study period. The eight examples examined in further detail included suspected drug adulteration (novel opioid adulterated heroin, para-methoxymethamphetamine adulterated 3,4-methylenedioxymethamphetamine (MDMA)), drug substitution (25B-NBOH sold as lysergic acid diethylamide, five benzodiazepine-type new psychoactive substances in a single tablet, protonitazene sold as ketamine), new drug detection (N,N-dimethylpentylone), contamination (unreported acetylfentanyl) and a fatality subsequent to MDMA use. A total of four public Drug Alerts were issued over this period. CONCLUSIONS: Continued toxicosurveillance efforts are paramount to characterising the changing landscape of illicit drug use. This work demonstrates a functional model for risk assessment of illicit drug toxicosurveillance, underpinned by analytical confirmation and evidence-based decision-making.

The Emerging Drugs Network of Australia - Victoria Clinical Registry: A state-wide illicit substance surveillance and alert network.

OBJECTIVES: With an increasingly dynamic global illicit drug market, including the emergence of novel psychoactive substances, many jurisdictions have moved to establish toxicosurveillance systems to enable timely detection of harmful substances in the community. This paper describes the methodology for the Emerging Drugs Network of Australia - Victoria (EDNAV) project, a clinical registry focused on the collection of high-quality clinical and analytical data from ED presentations involving illicit drug intoxications. Drug intelligence collected from the project is utilised by local health authorities with the aim to identify patterns of drug use and emerging drugs of concern. METHODS: The project involves 10 public hospital EDs in Victoria, Australia. Patients 16 years and over, presenting to a network ED with a suspected illicit drug-related toxicity and a requirement for venepuncture are eligible for inclusion in the study under a waiver of consent. Clinical and demographic parameters are documented by site-based clinicians and comprehensive toxicological analysis is conducted on patient blood samples via specialised forensic services. All data are then deidentified and compiled in a project specific database. RESULTS: Cases are discussed in weekly multidisciplinary team meetings, with a view to identify potentially harmful substances circulating in the community. High-risk signals are escalated to key stakeholders to produce timely and proportionate public health alerts with a focus on harm minimisation. CONCLUSIONS: The EDNAV project represents the first centralised system providing near real-time monitoring of community drug use in Victoria and is fundamental in facilitating evidence-based public health intervention.

Diagnostic accuracy for self-reported methamphetamine use versus oral fluid test as the reference standard in a methamphetamine-dependent intervention trial population.

AIMS: Treatment of methamphetamine dependence requires monitoring of recent use or abstinence. Self-report is commonly used for routine monitoring, but the accuracy of self-report is not established. For the treating clinician, the key accuracy statistic is the negative predictive value (NPV). The study aim was to estimate the NPV of self-reported non-use of methamphetamine compared with an oral fluid reference standard. DESIGN, SETTING AND PARTICIPANTS: This study was a secondary (subgroup) analysis from a randomized controlled pharmacotherapy trial. Three Australian outpatient addiction services took part. Particpants were 139 people dependent on methamphetamine. MEASUREMENTS: Weekly oral fluid samples over 12 weeks to determine methamphetamine (and amphetamine) concentrations were used as the reference standard. Self-report of any methamphetamine use in the previous 7 days by the time-line follow-back method was the index test. Standard diagnostic accuracy statistics were calculated for all available paired episodes (n = 1134). Three NPV values were calculated: unadjusted NPV and NPV adjusted for clustering of observations through logistic regression and generalized estimating equation (GEE). We also calculated the NPVs for a range of prevalence rates of methamphetamine use, for the calculated levels of sensitivity and specificity. FINDINGS: Sensitivity was 96.4% [95% confidence interval (CI) = 95-97.5], specificity was 63.7% (95% CI = 57.3-69.8) and positive predictive value (PPV) was 90.8% (95% CI = 88.8-92.6). The unadjusted NPV was 82.7% (95% CI = 76.5-87.9), adjusted NPV by logistic regression 82.7% (95% CI = 73.9-91.5) and GEE 76.8% (95% CI = 66.8-86.8). At a methamphetamine use prevalence of 5%, the estimated NPV would be 99.7% (95% CI = 99.6-99.9) and at 95% prevalence, 48.2% (95% CI = 39.6-57.0). CONCLUSIONS: Self-report of no recent methamphetamine use appears to be sufficiently accurate to be clinically useful at the expected prevalence rates of methamphetamine use in clinical treatment settings. If generalizable to clinical settings, where these tests are routinely conducted, this may permit a reduction in the frequency and cost of oral fluid assays.

Characteristics and time course of benzodiazepine-type new psychoactive substance detections in Australia: results from the Emerging Drugs Network of Australia - Victoria project 2020-2022.

INTRODUCTION: The emergence of benzodiazepine-type new psychoactive substances (NPSs) are a growing international public health concern, with increasing detections in drug seizures and clinical and coronial casework. This study describes the patterns and nature of benzodiazepine-type NPS detections extracted from the Emerging Drugs Network of Australia - Victoria (EDNAV) project, to better characterise benzodiazepine-type NPS exposures within an Australian context. METHODS: EDNAV is a state-wide illicit drug toxicosurveillance project collecting data from patients presenting to an emergency department with illicit drug-related toxicity. Patient blood samples were screened for illicit, pharmaceutical and NPSs utilising liquid chromatography-tandem mass spectrometry. Demographic, clinical, and analytical data was extracted from the centralised registry for cases with an analytical confirmation of a benzodiazepine-type NPS(s) between September 2020-August 2022. RESULTS: A benzodiazepine-type NPS was detected in 16.5 % of the EDNAV cohort (n = 183/1112). Benzodiazepine-type NPS positive patients were predominately male (69.4 %, n = 127), with a median age of 24 (range 16-68) years. Twelve different benzodiazepine-type NPSs were detected over the two-year period, most commonly clonazolam (n = 82, 44.8 %), etizolam (n = 62, 33.9 %), clobromazolam (n = 43, 23.5 %), flualprazolam (n = 42, 23.0 %), and phenazepam (n = 31, 16.9 %). Two or more benzodiazepine-type NPSs were detected in 47.0 % of benzodiazepine-type NPS positive patients. No patient referenced the use of a benzodiazepine-type NPS by name or reported the possibility of heterogenous product content. CONCLUSION: Non-prescription benzodiazepine use may be an emerging concern in Australia, particularly amongst young males. The large variety of benzodiazepine-type NPS combinations suggest that consumers may not be aware of product heterogeneity upon purchase or use. Continued monitoring efforts are paramount to inform harm reduction opportunities.

Monitoring for fentanyl within Australian supervised injecting facilities: Findings from feasibility testing of novel methods and collaborative workshops.

BACKGROUND: Australia is yet to see widespread fentanyl-contaminated heroin, despite the established presence of fentanyl in other countries. International mortality trends alongside a local cluster of fentanyl-related deaths prompted interest in developing methods to monitor for fentanyl and other potentially harmful novel psychoactive substances (NPS) in Australia. METHODS: We tested novel methods to monitor for fentanyl and other NPS. From 2017-2021, clients from supervised injecting facilities (SIFs) in Melbourne and Sydney, Australia, contributed urine screens (UDS) with BTNX Rapid Response™ fentanyl test strips (FTS) paired with surveys, and injecting equipment associated with opioid overdoses for laboratory analysis. A single site piloted drug checking using FTS with laboratory confirmation. Two workshops were conducted with SIF staff, content experts and people with lived experience to determine how results can inform practices within SIFs. RESULTS: Of the 911 UDS with FTS conducted, less than 1% (n=8) yielded positive results that were not explained by self-reported pharmaceutical fentanyl use, with two laboratory confirmed fentanyl positive results. Injecting equipment from 59 overdoses was tested and neither fentanyl nor other NPS were identified. Drug checking with FTS (n=34) indicated the presence of fentanyl on three tests. Two specimens were subsequently sent for laboratory testing and classified as false positives as the presence of fentanyl was not confirmed. Workshop participants (n=21) felt routine monitoring with FTS currently had limited value. A process for using pre-defined signals to trigger surveillance was developed. CONCLUSION: The high false positive rates with FTS, relative to the small number of positive results and potential for them to undermine confidence in FTS emphasised the need for confirmatory testing. The role of routine surveillance was unclear within the current low-fentanyl context, however, a process was developed to upscale testing should signals of increased fentanyl prevalence in the Australian heroin market emerge.

A cluster of acute thebaine poisonings from non-food grade poppy seeds in the Australian food supply.

INTRODUCTION: Poppy seed tea is used for its opioid effects and contains multiple opium alkaloids, including morphine, codeine, papaverine, and thebaine. Animal studies indicate thebaine has strychnine-like properties, but there is limited literature describing human thebaine poisoning. We describe a cluster of acute thebaine poisoning in people ingesting tea made using poppy seeds with high thebaine content that entered the Australian food supply chain. METHODS: This is an observational study of patients poisoned after drinking poppy seed tea. Cases were identified by three prospective toxicovigilance systems: the Emerging Drug Network of Australia collaboration, the New South Wales Prescription, Recreational and Illicit Substance Evaluation program, and the Emerging Drugs Network of Australia Victoria study. We report characteristics of clinical toxicity in cases with reported ingestion of poppy seed tea and analytical confirmation of thebaine exposure. RESULTS: Forty cases presenting with multi-system toxicity following poppy seed tea ingestion were identified across seven Australian states/territories from November 2022 to January 2023. Blood testing in 23 cases confirmed high thebaine concentrations. All 23 were male (median age 35, range 16-71 years). All patients experienced muscle spasms. Rigidity was described in nine, convulsions in six, while rhabdomyolysis, acute kidney injury, and metabolic acidosis occurred in five patients. There were two cardiac arrests. The thebaine median admission blood concentration was 1.6 mg/L, with a range of 0.1-5.6 mg/L, and was the dominant opium alkaloid in all samples. Convulsions, acute kidney injury, metabolic acidosis, and cardiac arrest were associated with increasing median thebaine concentrations. Four patients were managed in the Intensive Care Unit, with two receiving continuous kidney replacement therapy (one also received intermittent haemodialysis) for kidney injury. There was one death. CONCLUSIONS: Thebaine toxicity, like strychnine poisoning, resulted in neuromuscular excitation characterized by muscle spasm, rigidity, and convulsions. Severe toxicity, including acute kidney injury, metabolic acidosis, and cardiac arrest, appears dose-dependent.