Biofilm production of Pseudomonas spp. isolates from canine otitis in three different enrichment broths.

BACKGROUND: Pseudomonas spp. are commonly isolated from dogs with clinical otitis and have been shown to produce biofilm. There is a paucity of studies demonstrating biofilm growth in veterinary medicine. HYPOTHESIS/OBJECTIVES: To compare biofilm production of Pseudomonas spp. isolated from dogs with otitis using three different enrichment broths at two different time points. Speciation was performed. ANIMALS: One hundred isolates from 98 dogs with clinical otitis were assessed for biofilm production. METHODS AND MATERIALS: One hundred isolates were assessed for biofilm production using a microtitre plate assay. Biofilm production in Luria-Bertani Broth (LBB), Mueller-Hinton Broth (MHB) and Tryptic Soy Broth (TSB) were assessed after 18 and 24 h of incubation. RESULTS: At 18 h, biofilm production was demonstrated in 87% of LBB, 91% of TSB and 93% of MHB grown isolates. By 24 h, this was 92% of LBB, 96% of TSB and 99% of MHB isolates. Biofilm production was significantly increased after 24 h incubation compared to 18 h. A significant difference was noted in biofilm production between LBB and MHB (P = 0.0349), but not between LBB and TSB (P = 0.3727) or MHB and TSB (P = 0.3687) at 24 h incubation. Two isolates were speciated as P. fluorescens and 98 as P. aeruginosa. CONCLUSION AND CLINICAL IMPORTANCE: Not all enrichment broths were equivalent to one another and 24 h incubation was superior to 18 h. Biofilm production was high in this population of Pseudomonas spp. isolates.

The United Kingdom and Ireland association of forensic toxicologists forensic toxicology laboratory guidelines (2018).

In 2010, the United Kingdom and Ireland Association of Forensic Toxicologists (UKIAFT) created forensic toxicology laboratory guidelines. This represents a revision of those guidelines as a result of the changing toxicological and technical landscape.

The United Kingdom and Ireland Association of Forensic Toxicologists; establishing best practice for professional training & development in forensic toxicology.

The current status of forensic toxicology in the United Kingdom is discussed with an emphasis on professional training and development. Best practice is proposed using a blend of modular foundation knowledge training, continuing professional development, academic study, research & development and ongoing analytical practice. The need for establishing a professional career structure is also discussed along with a suggested example of a suitable model. The issues discussed in this paper are intended to provoke discussion within the forensic toxicology community, industry regulators and other government bodies responsible for the administration of justice.

Cannabinoid concentrations detected in fatal road traffic collision victims compared with a population of other postmortem cases.

BACKGROUND: Acute cannabis consumption nearly doubles the risk of motor vehicle collision resulting in injury or death. Limited data have been published regarding the concentrations of cannabinoids associated with fatal road traffic collisions (RTCs), and these have not previously been compared to a population of other postmortem cases. METHODS: We conducted analysis for cannabinoids [Δ(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC, 11-nor-THC-9-carboxylic acid, cannabidiol, and cannabinol], drugs, and alcohol on consecutive fatal RTC cases (100) and non-RTC cases (114) from coroners' jurisdictions in London and southeast England and compared the data. RESULTS: The incidence of cannabinoids detected in non-RTC and RTC cases was similar (25% vs 21%, P = 0.44), but THC was detected more frequently (90% vs 59%, P = 0.01) and at significantly higher concentrations in the cannabinoid-positive RTC cases than the non-RTC cases (P = 0.01). The distribution of non-RTC and RTC cases over 4 categories of THC concentration was significantly different (P = 0.004). There was no significant difference in the concentrations of other cannabinoids detected between the 2 groups. Cannabinoids were detected in more fatal RTC cases (21) than alcohol >80 mg/dL (17). Detection of other drugs was low compared to cannabis and alcohol. CONCLUSIONS: These first data on the concentrations of cannabinoids in the postmortem blood of fatal RTC victims compared with a population of other routine coroners' cases highlight the importance of specifically measuring THC concentrations in the blood to aid interpretation of postmortem cases where cannabis may be implicated.

Facilitating high-quality EMS through clear communication.

For the final article in this series about the RCVS' extramural studies (EMS) reforms, Sue Paterson, RCVS junior vice president, describes how a new database will act as a nexus between students, universities and placement providers to ensure that the right people get the right EMS placements. Two young vets who helped shape the proposals also discuss how they hope the new EMS policy will improve outcomes.

Spreading the word for veterinary careers.

As she starts her presidential year at the helm of the RCVS, Sue Paterson chats to Arabella Gray about her aspirations for the veterinary profession and what she can bring to the table.

Toxicological Relevance of Pregabalin in Heroin Users: A Two-Year Postmortem Population Study.

Pregabalin (PGL) is a gabapentinoid used to treat epilepsy, neuropathic pain and generalized anxiety disorder. PGL is also misused by heroin users as it enhances the effects of heroin. While it is thought those who misuse PGL take it in amounts greater than the recommended therapeutic dose, it is unknown whether there is a significant difference between the amounts of PGL used by heroin users compared to non-heroin users. This study hypothesized that the PGL concentrations in postmortem (PM) samples taken from heroin users positive for PGL would be higher than those in non-heroin users. Between 1 January 2016 and 31 December 2016, a routine drug screen and a specific screen for PGL were carried out on femoral-vein bloods from 3,750 PM Coroners' cases. Of the cases screened, 354 were heroin users, of which 264 cases were negative for gabapentinoids and therefore used as the control-heroin-user group. PGL was positive in 229 cases, of which 69 were heroin users and 160 were non-heroin users. On comparing the PGL concentrations, statistically higher concentrations were observed in the heroin users compared to non-heroin users (P = 0.002). There was no correlation between the concentrations of PGL and morphine (from heroin) in the heroin users (P = 0.95), and the amount of heroin (morphine) consumed was not dependant on whether PGL was consumed or not (P = 0.98). The prevalence of anti-depressants, benzodiazepines, methadone and non-heroin-related opioids was seen to be significantly higher in heroin users that were positive for PGL than the control-heroin users (P = < 0.001 for all drugs). This study suggests that heroin users are using greater amounts of PGL compared to non-heroin users; however, the magnitude of the difference in use may not be sufficient to conclude that heroin users are at substantially greater risk of PGL toxicity compared to non-heroin users. Results indicate that heroin users who take PGL are more likely to use multiple depressant drugs, hence increasing the risk of multi-drug toxicity and death in this population.

Baclofen: To Screen or Not to Screen in Postmortem Blood?

Baclofen (BLF) has been prescribed in the UK since 1972 for the alleviation of spasticity. However, evidence suggests BLF is also recreationally misused. It has been associated with ethanol, gamma-hydroxybutyric acid (GHB), pregabalin (PGL) and gabapentin (GBP) use/abuse, and deaths have been reported. With current postmortem (PM) toxicological screening approaches, BLF is not routinely included in the general drugs screen and is only screened for if specifically mentioned in the case documents. The extent of BLF misuse is thus unclear. This study was carried out to determine the prevalence and concentrations of BLF in Coroners' toxicology, to investigate whether BLF misuse with ethanol, GHB, PGL and GBP is causing death and to determine the potential extent of the underreporting of BLF-associated deaths. Between 1 January 2016 and 31 December 2017, 3,750 PM femoral vein bloods were screened for BLF; all positive cases were quantified. Only 0.56% of samples screened positive for BLF, with concentration ranging from 0.08 to 102.00 µg/mL (median = 0.28). It was determined that if routine analysis without additional screening of BLF had been performed, 43% of BLF positives cases would have been missed. However, given the low incidence of detection, this only represents 0.25% of the cohort. Likely illicit use of BLF with GHB was seen in one case only. Death from the recreational use of BLF with PGL and GBP was not observed. Only two cases positive for BLF had an ethanol concentration of ≥50 mg%. Two cases of presumed intentional overdose of BLF were observed. This study highlights that although BLF abuse may be occurring, deaths are rare. It is therefore not cost- or time-effective to screen for BLF in all PM cases. With BLF currently being investigated for the treatment of alcoholism and withdrawal symptoms of illicit drug use, BLF-related deaths may rise in the future.

Misuse and Mortality Related to Gabapentin and Pregabalin are Being Under-Estimated: A Two-Year Post-Mortem Population Study.

Due to the rise in their misuse and associated mortality, the UK government is reclassifying gabapentin (GBP) and pregabalin (PGL) to Class C controlled drugs from April 2019. However, it is impossible to gauge the extent of their use with current post-mortem toxicological screening, where GBP and PGL are only screened for if they are mentioned in the case documents. This study determines the prevalence of GBP and PGL, the potential extent of their under-reporting and poly-drug use in a post-mortem population. Between 1 January 2016 and 31 December 2017, 3,750 deceased from Coroners' cases in London and South East England underwent a routine drugs screen and a specific screen for GBP and PGL. The prevalence of both drugs was determined in the cohort and the subcategories of heroin users and non-heroin-users. The prevalence of both drugs was compared to tramadol (Class C drug). Case documents were reviewed to investigate the under-reporting of GBP and PGL and poly-drug use. Of 3,750 samples analyzed, 118 (3.1%) were positive for GBP, 229 (6.1%) for PGL and 120 (3.2%) were positive for tramadol. If routine analysis without additional screening of GBP and PGL had been performed in this cohort, GBP would have been under-reported by 57.6% (P < 0.0001) and PGL by 53.7% (P < 0.0001) in deaths. The most common drug group observed with GBP and PGL was non-heroin-related opioids at 60.2% and 64.6%, respectively. In total 354 deceased (9.4%) were heroin users. GBP was positive in 23 (6.5%) of these cases and PGL was positive in 69 (19.5%). The prevalence of PGL in heroin users (19.5%) was 4.1 times greater than in non-heroin users (4.7%) (P < 0.0001). GBP and PGL are being significantly under reported in fatalities. Both drugs are extensively used with opioids. The prevalence of PGL in heroin users is highly significant.

Simultaneous quantification of opiates, amphetamines, cocaine and metabolites and diazepam and metabolite in a single hair sample using GC-MS.

A method is described for the simultaneous identification and quantification of opiates, amphetamines, cocainics, diazepam and nordiazepam from one hair extract (typically 10-50mg hair). After decontamination by washing with shampoo, dichloromethane, isopropanol and acetone, drugs were extracted using 0.1M HCl followed by SPE clean-up using mixed-mode extraction cartridges. The SPE extracts were submitted to a two-step derivatisation using MBTFA and MSTFA+1% TCMS and analysis was performed by GC-MS using both SIM and scan modes. Four deuterated standards were used to monitor 14 compounds. The limit of quantification was the total drug detected from the sample. This was 5 ng for amphetamines and 10 ng for remaining drugs which is equivalent to 0.1 and 0.2 ng/mg from a 50mg sample. Standard curves for the range 5-400 ng total drug concentration for all drugs had regression coefficients greater than 0.98. An authentic hair sample was used to validate the method and gave R.S.D.s <25% for both inter and intra-day reproducibility. The results of the analysis of hair taken from four patients attending a drug treatment clinic and six hair samples including head hair, pubic hair, axial hair and beard taken at post-mortem are presented.

An observed rise in γ-hydroxybutyrate-associated deaths in London: Evidence to suggest a possible link with concomitant rise in chemsex.

BACKGROUND: Gamma-hydroxybutyrate (GHB) is the drug most linked to acute harm out of those used in chemsex, the incidence of which is reported to be increasing. However, there have been few systematic studies of the harms associated with GHB use. We investigated GHB-associated deaths from London coroners' jurisdictions between 2011 and 2015. METHODS: Blood and urine samples were collected by pathologists and submitted for toxicological analysis at the request of coroners. Data from the Toxicology Unit, Imperial College London was retrospectively analysed. This comprised of 6633 cases from seven out of eight coroners' jurisdictions in London that underwent toxicological analysis between January 2011 and December 2015. RESULTS: A total of 61 GHB-associated deaths (0.92% of total cases), 184 cocaine-associated deaths (2.8% of total cases) and 83 MDMA-associated deaths (1.3% of total cases) were identified. There was a 119% increase in the proportion of GHB-associated deaths detected in 2015 compared to 2014. Over the same time period there was a 25% increase in cocaine-associated deaths and a 10% decrease in MDMA-associated deaths. CONCLUSIONS: Our data suggest that GHB-associated deaths are increasing in London, and that this is likely at least in part due to increasing use of GHB for chemsex. Further studies on the use of GHB are urgently required to understand the extent of its use, whether this is as prevalent in other major urban areas in the UK, and the full extent of the harms it causes.

Validated Method for the Screening and Quantification of Baclofen, Gabapentin and Pregabalin in Human Post-Mortem Whole Blood Using Protein Precipitation and Liquid Chromatography-Tandem Mass Spectrometry.

There has been a rapid increase in the number of prescriptions for baclofen (BLF), gabapentin (GBP) and pregabalin (PGL) in the UK since their introduction to therapy. Recent studies across the European Union and USA have shown the illicit abuse potential of these drugs and deaths have been observed. A simple, reliable and fully validated method was developed for the screening and quantification of BLF, GBP and PGL in human post-mortem (PM) blood. The analytes and their deuterated analogs as internal standard were extracted from blood using a single addition acetonitrile protein precipitation reaction followed by analysis using liquid chromatography-tandem mass spectrometry (LC-MS-MS) with triggered dynamic multiple reaction monitoring mode for simultaneous confirmation and quantification. The assay was linear from 0.05 to 1.00 µg/mL for BLF and 0.5 to 50.0 µg/mL for GBP and PGL, respectively with r2 > 0.999 (n = 9) for all analytes. Intra-day and inter-day imprecisions (n = 80) were calculated using one-way ANOVA; no significant difference (P > 0.99) was observed for all analytes over 8 non-consecutive days. The average recovery for all analytes was >98.9%. The limits of detection and quantification were both 0.05 µg/mL for BLF, and 0.5 µg/mL for GBP and PGL. The method was highly selective with no interference from endogenous compounds or from 54 drugs commonly encountered in PM toxicology. To prove method applicability, 17 PM blood samples submitted for analysis were successfully analyzed. The concentration range observed in PM blood for BLF was 0.08-102.00 µg/mL (median = 0.25 µg/mL), for GBP 1.0-134.0 µg/mL (median = 49.0 µg/mL) and 2.0-540.0 µg/mL (median = 42.0 µg/mL) for PGL.

Comparison of the various opiate alkaloid contaminants and their metabolites found in illicit heroin with 6-monoacetyl morphine as indicators of heroin ingestion.

In this study the use of the various opiate alkaloid contaminants as potential markers for illicit heroin ingestion were investigated. Urine samples (n = 227) taken from prisoners for routine drug screen, which were positive for opiates by immunoassay screening, were analyzed for contaminants in illicit heroin. A previously described method was used for the analysis; urines were extracted using mixed-mode solid-phase extraction; the extracts were derivatized using N-methyl-bistrifluoroacetamide and N-methyl-N-trimethylsilyltrifluoroactamide/trimethylchlorosilane. The derivatized extracts were subjected to electron impact gas chromatography-mass spectrometry. The extracts were injected in full scan mode followed by selected ion monitoring mode for target opiate alkaloids found as contaminants in illicit heroin. The opiate alkaloids and their metabolites specifically targeted included meconine, desmethylmeconine, hydrocotarnine, acetylcodeine, codeine, morphine, 6-monacetylmorphine (6-mam), papaverine, hydroxypapaverine, and dihydroxypapaverine. Of the 227 samples positive for opiates by immunoassay, using a cut-off of 300 ng/mL, 199 were confirmed positive for morphine and using a cut-off of 10 ng/mL, 28 were confirmed positive for 6-mam. Using the screening method described in the study, the following numbers of positives were found: 199 for morphine, 103 for codeine, 5 for meconine, 46 for desmethylmeconine, 18 for 6-mam, 136 for hydroxypapaverine, and 139 for dihydroxypapaverine. Acetylcodeine, hydrocotarnine, and papaverine were not detected in any of the samples. The results of this study show that analysis for papaverine metabolites is more sensitive than 6-mam as a way of demonstrating illicit heroin use.

Validated Method for the Quantification of Baclofen in Human Plasma Using Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry.

A highly sensitive and fully validated method was developed for the quantification of baclofen in human plasma. After adjusting the pH of the plasma samples using a phosphate buffer solution (pH 4), baclofen was purified using mixed mode (C8/cation exchange) solid-phase extraction (SPE) cartridges. Endogenous water-soluble compounds and lipids were removed from the cartridges before the samples were eluted and concentrated. The samples were analyzed using triple-quadrupole liquid chromatography-tandem mass spectrometry (LC-MS-MS) with triggered dynamic multiple reaction monitoring mode for simultaneous quantification and confirmation. The assay was linear from 25 to 1,000 ng/mL (r(2) > 0.999; n = 6). Intraday (n = 6) and interday (n = 15) imprecisions (% relative standard deviation) were <5%, and the average recovery was 30%. The limit of detection of the method was 5 ng/mL, and the limit of quantification was 25 ng/mL. Plasma samples from healthy male volunteers (n = 9, median age: 22) given two single oral doses of baclofen (10 and 60 mg) on nonconsecutive days were analyzed to demonstrate method applicability.