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Alprazolam is a psychoactive triazolo-benzodiazepine, commonly prescribed for the treatment of anxiety disorders and considered safe due to its high therapeutic index. It is frequently detected in polydrug-related deaths but there are very few cases of fatal intoxication by alprazolam not in combination with alcohol or other drugs. A fatal poisoning by alprazolam in a 60-years old male physician has been reported. At autopsy, no evidence of traumatic injuries or diseases was observed except for signs of bronchopneumonia and slight hypertrophy of the left ventricle. Alprazolam and its major metabolite (α-hydroxyalprazolam) were detected in biological samples through liquid chromatography-tandem mass spectrometry (LC-MS/MS). High concentrations of alprazolam and α-hydroxyalprazolam were found, respectively, in femoral blood 0.45 and 0.03 mg/L, urine 2.12 and 0.42 mg/L, bile 1.33 and 0.56 mg/L, liver 3.81 and 0.28 mg/L. Alprazolam alone was also detected in vitreous humour (0.19 mg/L) and in stomach content (0.34 mg/L). No conclusive information confirmed the hypothesis of 25 a suicidal ingestion of alprazolam. However, an alprazolam drug-related death was assessed based on the pre-existing cardiovascular and respiratory defects. The high levels of alprazolam several times greater than the therapeutic range contributed to death due to its respiratory and central nervous system-depressant effects.

The COVID-19 pandemic has consistently raised the number of drug seizures, in United States as in Europe. The COVID-19 pandemic has also changed the typology of seizures from “more traditional” drugs to New Psychoactive Substances (NPSs), depending on geographical area. In Europe, the most frequent NPSs are synthetic cannabinoids (SCs) and cathinones, nonetheless synthetic opioids and phenethylamines are widely used. The aim of the study is the detection of NPS and other substances of abuse available in the black market, by quali/quantitative methods in LC-MS/MS and GC-MS. From 2018 to 2021, 268 seizures occurred in total and were analyzed by the Forensic Toxicology Laboratory (FTL) of Naples (Italy). The distribution of analyzed seizures over the years is the following: 53 in 2018, 61 in 2019, 89 in 2020 and 65 during the first semester of 2021. Cannabis was the most detected drug both in hashish and marijuana seizures, followed by cocaine > heroine > prescribed drugs > ketamine–amphetamine MDMA. No NPSs were seized until June 2021, when NPSs were found in two different seizures: Case #1 showed a bar of Cannabis resin containing a low level of Δ9THC = 0.57% associated to SC AB-FUBINACA in 4.5%; Case #2 showed a vegetal resinous substance (Δ9THC = 0.27%) with SC 5F-APINACA (4.1%) associated with methadone (1.0%). The detection of NPSs is alarming evidence that can lead to an increase in the risk of overdose or other negative and unpredictable consequences, such as violent or self-harming behavior in unaware users of cannabis derivatives considered of “natural” origin.

An unusual case of poisoning by the ingestion of oleander leaves is reported. A 71 year old male laboratory technician committed suicide at home in this unusual manner. At the death scene a steel pan and other paraphernalia, used for the extraction of oleandrin and other cardiac glycosides from the leaves of the Nerium oleander plant were found.Toxicological investigations for oleandrin, oleandrigenin, neritaloside, and odoroside were performed by LC–MS/MS on all biological samples (peripheral blood, vitreous humor, urine, liver, gastric contents) and on the yellow infusion found at the death scene.In all samples, toxic levels of oleandrin were detected (blood 37.5 ng/mL, vitreous humor 12.6 ng/mL, urine 83.8 ng/mL, liver 205 ng/mg, gastric content 31.2 µg/mL, infusion 38.5 µg/mL). Qualitative results for oleandrigenin, neritaloside, and odoroside were obtained. Oleandrigenin was present in all tissue samples whereas neritaloside and odoroside were absent in the blood and vitreous humor but present in urine, liver, gastric content, and in the leaf brew.The purpose of this study was the identification of oleandrin and its congener oleandrigenin, detected in the vitreous humor. The blood/vitreous humor ratio was also calculated in order to assess of the likely time interval from ingestion to death. According to the toxicological results death was attributed to fatal arrhythmia due to oleander intoxication. The manner of death was classified as suicide through the ingestion of the infusion.

Background: A ligature mark is a common injury in cases of hanging or strangulation. Estimation of age and vitality of the ligature mark can be crucial for differentiating antemortem and postmortem wounds and to distinguish between simulated suicidal hanging or accidental strangulation to conceal a crime and not simulated events. The immunohistochemistry has been recommended by several Authors as a reliable tool to determine whether an injury was sustained during life or not. Unfortunately, no general agreement on the immunohistochemical markers to be used has been found among the scientific community. The aim of the study was to detect the type and function of the immunohistochemical markers useful in the assessment of the vitality and age of the ligature marks for routine diagnostics. Methods: Papers available on Pubmed, Scopus, and Web of Science were reviewed according to the PRISMA statement. Results: Only eight papers satisfied all the following inclusion criteria: full texts in English dealing with human ligature marks and immunohistochemistry published on impacted or indexed scientific journals. Conclusions: The assessment of the vitality of a ligature mark is still a challenging topic in forensic science. Under ideal conditions and in compliance with autopsy protocols, the diagnosis of death by hanging or strangulation on fresh bodies can be better supported by autopsy findings other than a ligature mark. The validation of immunohistochemical markers on large series could be of help in doubtful cases and differential diagnoses.

Chronic encapsulated intracerebral hematoma (CEIH) is a rare solid mass characterized by the presence of a fibrotic capsule that can present a variety of signs and symptoms due to the mass effect and hydrocephalus. It may be caused by post-traumatic or spontaneous bleeding as related to an adjacent aneurysm, angiomas or neoplasms. Differential diagnosis must be applied in order for it to mimic neoplasm or a vascular malformation. Several cases of CEIH have been reported but only a few of them have an intraventricular localization. A forensic autopsy of a 50-year-old male who died suddenly while driving is discussed. Gross analysis, histology and toxicology were performed and a CEIH of the right lateral ventricle was found in a case of acute coronary death.

Background: Haematological malignancies, such as lymphoma and leukaemia, can have a variety of clinical manifestations. The most frequent cause of death from haematological malignancies is multiple organ failure due to neoplastic organ infiltration and/or septic shock. Histiocytic sarcoma (HS) is a rare malignant nodal or extranodal tumour with histiocytic immunophenotype that originates from a lymphohematopoietic precursor. The patients with HS usually have a poor prognosis due to its aggressive clinical behaviour. Rare cases of undiagnosed sudden HS death have been described in the literature. Methods: A forensic autopsy of a 46-year-old white male who died at home suddenly and unexpectedly without warning conditions or known diseases. Gross analysis, histology and toxicology were also performed. Results: The diagnosis of HS of the ileum with secondary nodal and cardiac metastatization was made. Conclusions: A prompt diagnosis of HS in life is paramount because it can make a difference in prognostic outcomes.

Background: Diagnostic criteria for electrocution related death are still a challenge in forensic pathology and it seems that the electrical mark is the only reliable evidence. Methods: A comparison of histological and morphological findings of skin and internal organs from an autopsy series of electrocution deaths with those mostly reported in literature as representative for electrocution. Results: The morphological changes of heart, brain and other main internal organs are still unspecific. Organ’s damage observed in electrocution deaths shows a wide variability, not reliable for a certain diagnosis of electrocution. The electrical mark is still the golden standard for diagnosis of electrocution. Conclusions: In electrocution related deaths, pathological findings of the main internal organs are not enough evidence to support with certainty a post-mortem diagnosis that a victim suffered an electrical damage. Although the organ histological changes are undoubtedly the starting point for a better understanding of the fatal even, the diagnosis of death from electrical damage is still a dark and unsolved chapter. The electrical mark still represents a fundamental indicator above all in the medical-legal field, but the identification of pathognomonic elements and signs not limited to the skin alone could be a valid help in the future, especially in unclear cases.

Snakebite envenoming (SBE) is a public health issue in developing countries. The estimated annual global incidence of snakebites is about 5.4 million snakebites per year, resulting from 1.8 to 2.7 million cases of SBE and from 81,000 to 138,000 deaths with 400,000 survivors suffering permanent physical and psychological disabilities. There are more than 3000 species of snakes around the world: 600 are venomous and over 200 are considered to be medically important because of their clinical effects. The severity of SBE depends on several factors among which bite localization, snake’s size, condition of glands and teeth, bite angle and bite duration, the microflora of the snake’s mouth and victim’s skin, age of the victim, weight, health status, and victim’s activity after a bite. Snake venoms are mixtures of protein families, and each of these families contains many different toxins or toxin isoforms. Based on their effects, snake venoms can be classified as hemotoxic, neurotoxic, or cytotoxic and they can all act together involving multiple tissues and organs. When the bite is fatal, the mechanism of death is primarily related to the paralysis of respiratory muscles, which causes asphyxia and hypoxic-ischemic encephalopathy, but also anaphylactic shock, hemorrhagic shock, cardiomyopathy, acute tubular necrosis (ATN). The purpose of this literature review is to evaluate epidemiological and post-mortem examination findings in fatal SBEs in order to better understand the pathophysiological mechanisms, thus helping pathologists in defining the correct diagnosis.

Drug-facilitated sexual assault (DFSA) cases are pretty common in forensic toxicology. In this case report, a 56-year-old female tourist claimed to have been sexually assaulted by five men after having had a drug-spiked alcoholic drink. Urine samples were collected at 38, 44 and 45 h after the alleged rape. After 7 months, hair strands (28 cm in length) were also sampled to perform the segmental hair testing. The urine samples and decontaminated hair segments were tested for different groups of basic, acidic and neutral substances (γ-hydroxybutyrate or GHB, Z-drugs, barbiturates, benzodiazepines, hypnotics, antipsychotics and drugs of abuse). Gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry methods were applied for the qualitative and quantitative analyses. Toxicological analyses performed on the urine samples showed inconclusive findings. Zolpidem, flunitrazepam and oxazepam were detected in the hair segments corresponding to the time frame of the alleged assault. The endogenous levels of GHB were detected along the hair shaft. No drugs were detected in the proximal and distal hair segments or in washing solutions. This DFSA case demonstrated that the segmental toxicological analysis of hair, even when performed 7 months after the sexual assault, can provide evidence consistent with a single exposure to psychoactive drugs, at the time of the offense.

Introduction: The human post-mortem microbiome (HPM) plays a major role in the decomposition process. Successional changes in post-mortem bacterial communities have been recently demonstrated using high throughput metagenomic sequencing techniques, showing great potential as a post-mortem interval (PMI) predictor. The aim of this study is to verify the application of the mass spectrometry technique, better known as MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry), as a cheap and quick method for microbe taxonomic identification and for studying the PM microbiome. Methods: The study was carried out on 18 human bodies, ranging from 4 months to 82 years old and with a PMI range from 24 h up to 15 days. The storage time interval in the coolers was included in the final PMI estimates. Using the PMI, the sample study was divided into three main groups: seven cases with a PMI < 72 h; six cases with a PMI of 72–168 h and five cases with a PMI > 168 h. For each body, microbiological swabs were sampled from five external anatomical sites (eyes, ears, nose, mouth, and rectum) and four internal organs (brain, spleen, liver, and heart). Results: The HPM became increasingly different from the starting communities over time in the internal organs as well as at skin sites; the HPM microbiome was mostly dominated by Firmicutes and Proteobacteria phyla; and a PM microbial turnover existed during decomposition, evolving with the PMI. Conclusions: MALDI-TOF is a promising method for PMI estimation, given its sample handling, good reproducibility, and high speed and throughput. Although several intrinsic and extrinsic factors can affect the structure of the HPM, MALDI-TOF can detect the overall microbial community turnover of most prevalent phyla during decomposition. Limitations are mainly related to its sensitivity due to the culture-dependent method and bias in the identification of new isolates.

A recent update of the Italian Road Traffic Law (RTL 41/2016), established severe penal sanctions when drivers, driving under the influence of alcohol (DUI) or drugs (DUID), are involved in road accident that results in death or injuries. A study was carried out to assess the trends of consumption of alcohol, illicit drugs or pharmaceutical among injured drivers suspected for DUI or DUID from 2009 to 2016 in the region of Campania (Italy). Confirmation toxicological analyses were performed on 780 blood samples and 1017 urine samples collected from 1797 injured drivers. These drivers all tested positive for alcohol or drug use through immunoassay screening applied at hospital emergency units and their biological samples transferred to the Forensic Reference Laboratory (FRL) for confirmation analysis. The GC/HS-FID methodology was used to test Blood Alcohol Concentration (BAC). Qualitative and quantitative analyses for drugs were performed using the GC/MS or LC-MS/MS methodology. The BAC >0.5g/L was confirmed in 91.5% of drivers suspected for DUI cases and in 93% of DUID respectively. In DUI cases, results show an increasing incidence of road accidents involving drivers with BAC above 1.5g/L while at concentrations above 0.8g/L alcohol and drugs are both used. Among the suspected DUID cases, the intake of alcohol in association with drugs has consistently increased over time and positive results on blood samples was confirmed for multiple drugs (20%) or cannabis and cocaine alone (18%) followed by benzodiazepines (6%) and methadone (3.5%) respectively. The majority of injured drivers suspected for DUID (1017 cases) did not authorize blood sampling, therefore only urine was analyzed showing the prevalent use of cannabis, followed by multiple drug>cocaine>benzodiazepines>opiates. Among 1797 drivers, suspected at screening for DUI or DUID, 15.4% of cases (64 blood and 213 urine samples) were not confirmed by GC/HS, GC/MS or LC-MS/MS analysis. In forensic toxicological investigations, it is mandatory to satisfy the best quality standards, which is not achievable if immunochemical screening is only performed on urine. Therefore, only confirmed positive results of alcohol or drugs on blood samples can represent conclusive evidence to demonstrate the DUI or DUID related offences. An improvement of the protocols currently applied in Italy for the assessment of DUI or DUID crimes is needed and the confirmation analysis on blood should be considered mandatory.

Different immunotherapeutic approaches are in the pipeline for the treatment of drug dependence. "Drug vaccines" aim to induce the immune system to produce antibodies that bind to drugs and prevent them from inducing rewarding effects in the brain. Drugs of abuse currently being tested using these new approaches are opioids, nicotine, cocaine, and methamphetamine. In human clinical trials, "cocaine and nicotine vaccines" have been shown to induce sufficient antibody levels while producing few side effects. Studies in humans, determining how these vaccines interact in combination with their target drug, are underway. However, although vaccines can become a reasonable treatment option for drugs of abuse, there are several disadvantages that must be considered. These include i) great individual variability in the formation of antibodies, ii) the lack of protection against a structurally dissimilar drug that produces the same effects as the drug of choice, and iii) the lack of an effect on the drug desire that may predispose an addict to relapse. In addition, a comprehensive overview of several crucial ethical issues has not yet been widely discussed in order to have not only a biological approach to immunotherapy of addiction. Overall, immunotherapy offers a range of possible treatment options: the pharmacological treatment of addiction, the treatment of overdoses, the prevention of toxicity to the brain or the heart, and the protection of the fetus during pregnancy. So far, the results obtained from a small-scale experiment using vaccines against cocaine and nicotine suggest that a number of important technical challenges still need to be overcome before such vaccines can be approved for clinical use.

The aim of the study is the evaluation of incidence of mortality, directly or indirectly linked to drug abuse in one of the most populated Italian region (Campania). Trends on psychotropic substances intake and prevalence of drug-related deaths (DRDs) are assessed over an eleven-year observation period from 2008 to 2018. Data from toxicological investigations applied, for forensic purpose, on post-mortem blood sampled from 640 suspected DRDs (267 in the years 2008– 2012 and 373 in the years 2013 – 2018) has been revised. A Systematic Toxicology Analysis (STA) by validated GC/MS or LC-MS/MS methods for licit drugs (benzodiazepines, antidepressants/antipsychotics, hypnotics) and illicit drugs (amphetamine and analogous, cocaine, opiate, methadone, barbiturate, buprenorphine, cannabis and new psychoactive substances) was applied. A conventional methodology (GC/HS-FID) was used to test the blood alcohol concentration (BAC). Toxicological results were positive in 403 cases out of 640 autopsies totally performed: 202 DRDs out of 267 deaths were identified during the years 2008-12 and 201 DRDs out of 373 during the 2013-2018 period. Among the 403 DRDs, more than 90% of cases were males aged between 41-50 years. A remarkable increase in the number of alcohol related deaths (42 cases) was observed in the years from 2013 to 2018 compared with the previous one. Most of these cases showed a BAC in the range 1.5 – 4 g/L, compared to the only 3 cases occurred in 2008-2012 years. During the eleven-year observation period, the poly-drug use has been recognized as responsible for 258 deaths (64%) out of 403 cases in total. The association of cocaine and morphine was predominant in the years 2008-2012 while in the years 2013-2018 pharmaceuticals (benzodiazepines, antidepressants etc.), alcohol and illicit drugs (cocaine and morphine), alone or in combination, were the substances mainly detected. Methadone was detected more frequently in associations with other drugs (mainly pharmaceuticals) or ethyl alcohol but it was also found alone in 3 DRDs only. Surprisingly in few DRDs cannabis metabolites were found neither a single fatal poisoning by amphetamines and new psychoactive substances (NPS) was recorded. During the eleven-year observation period, an increase in DRDs involving ethyl alcohol intake has been observed. A relevant variability in the prevalence among the licit/illicit drug use has been also found, mainly represented by the poly-drug intake respect to the abuse of a single drug. Data collected demonstrate that it is crucial a continuous updating about the territorial trends of the drug abuse in order to develop a Community Data Bank, for planning strategies of prevention.

The study reports the environmental, toxicological and histopathological forensic investigations applied on three victims of accidental death (father, mother and son), due to the fall in a volcanic pothole, during the touristic visit of the “Solfatara park”, near Naples (Italy). At autopsy greenish skin discolouration was observed and all bodies showed the classical signs of asphyxial deaths, such as cyanosis and hemorrhagic pulmonary edema. Focal micro-hemorrhages were found in the brain at intracranial and subpial levels. The hemogasanalysis and spectrophotometric test on blood for Methemoglobin (MetHb), Carboxyhemoglobin (HbCO) and Sulfhemoglobin (SHb) showed pCO2, SHb and MetHb above the physiological levels. On biological specimens, toxicological analyses performed by GC/MS revealed high concentrations of hydrogen sulfide (H2S) and of thiosulfate (TS), its main metabolite. The monitoring of toxic gases on the death scene showed an unsafe environment, into the pothole, able to cause the sudden loss of consciousness of the victims with subsequent asphyxiation (knockdown effect). In particular, at the bottom of the hole, the maximum levels of H2S and carbon dioxide (CO2) were 2200 ppm and 98% respectively. For the family members, the cause of the death was assessed as acute poisoning by H2S and CO2. The fatalities, happened in quick succession as for a domino effect, were pretty similar to the asphyxial deaths by confined spaces, frequently observed in occupational setting. Fatalities secondary to accidental volcanic gases inhalation, such as H2S and CO2 in geothermal areas, have been already described but often without a forensic approach. To the best of our knowledge this is the first case that reports the accidental poisoning by volcanic gases involving three people, with different caracteristic of age and sex, allowing the correlation between toxicological and pathological results with the true levels of asphyxiating gas, measured on the death scene.

Two cases of poisoning involving two physicians (an anesthesiologist and a cardiologist) are herein presented. in both cases, autopsy and toxicological analyses were requested for forensic purposes. a systematic toxicological analysis by gas chromatography-mass spectrometry or liquid chromatography with tandem mass spectrometry was applied to biological specimens collected at autopsy and on paraphernalia (vials, residues within bottles, used syringes, and needles) seized at the scene of death. Each positive result obtained by systematic toxicological analysis was confirmed by a specific quantitative gas chromatography – mass spectrometry analysis. In the first case, fentanyl was revealed in the bottles and in all biological specimens in concentrations above the pharmacological levels. in the second case, biological samples were positive for phenobarbital, morphine, and codeine, but only phenobarbital was present at lethal concentrations. Morphine, codeine, papaverine, noscapine, and phenobarbital were identified on the syringe. the toxicological results in the present cases were compared with data reported in other cases of suicides involving fentanyl and phenobarbital. risk factors linked to suicide in physicians and the general population were also discussed.

Soil microbial activities and chemical properties related to the N cycle were investigated in function of variations in plant cover type in a Mediterranean area of Southern Italy. Several biological (microbial biomass, soil respiration, protease activity, degradation of 7 amino acids, net N mineralization, net nitrification, potential net nitrification and denitrification) and chemical properties (water content, pH, organic C, total and mineral N, C/N ratio) were determined in soil samples collected in spring and early autumn under three plant cover types (high maquis, low shrub community and meadow) epitomizing distinct successional stages following disturbance. Water content, organic C and C/N ratio were higher in soil from high maquis than in soils from the other plant cover types; in contrast, no difference was generally observed between the three soil types as for total and mineral N content. Likewise, no clear-cut difference was recorded for most of the tested biological parameters if these were expressed per unit of soil dry weight, with the exception of protease activity and soil respiration, which were significantly higher in the high maquis. However, when biological data were expressed per unit of organic C, meadow soil showed higher values of microbial biomass, asparagine degradation (only in spring), net mineralization and net nitrification (only in autumn) compared to the other soils. Statistical analysis pointed to seasonal fluctuation of water availability as an important limiting factor for microbial biomass and activity, with the exceptions of net N mineralization, net nitrification, glutamine and glutamic acid degradation activities which appeared to be mainly driven by organic matter content in soils. Overall, the results suggest that, in the investigated Mediterranean environment, variations in plant community ranging from meadow to high maquis produce little changes in soil biological activities linked to the N cycle, these being primarily affected by the availability of water and soil organic matter during the annual cycle. © 2009 Elsevier B.V. All rights reserved.

Introduction: The Italian legislation on workplace drug testing (WDT), applied to “at-risk workers” since October 2008, calls for mandatory procedures about screening tests on urine performed by occupational health specialists and confirmation tests by gas chromatography/mass spectrometry performed by forensic laboratories. Aim: Evaluating the impact of the current law on WDT on trends of drug use in workers and discuss some ethical implications and professional responsibility connected to the management of the toxicological controls. Materials and Methods: The toxicological analyses performed from 2001 to 2014 on 73,656 workers (aged between 19 and 65 years; 92% males and 8% females), mainly employed in transports were selected for the present study. Confirmation analysis has been requested on urine samples (n = 3934) previously identified as positive for one or more drugs by immunoassay screening tests for opiates, methadone, cannabinoids, cocaine, buprenorphine, amphetamines, and analogues. Results: Relevant differences were observed, both in the incidence and in the type of drugs used, before and after the mandatory law application. At screening the positive urine were n = 2455 during 2001-2008 and n = 1479 since 2009. Among those of this second period, only 14.6% were confirmed as true positive versus the 74% of urine confirmed during 2001-2008. Moreover, the polydrug use, since 2009, decrease from 32% to 8% of cases. Cannabis and cocaine were most frequently used, alone or together with other drugs. The use of opiates, methadone and amphetamines has been observed for

Nitrification is a key biological process for the control of soil NO 3- availability and N losses from terrestrial ecosystems. The study investigates the causes for the absence of net nitrification activity in the soil of a Mediterranean monospecific woodland of Arbutus unedo, focusing in particular on the possible role of chemicals produced by this plant. The mineral N pool, net rates of mineralization and nitrification were measured in the soil top 10 cm over 18 months. Raw extracts of leaves and roots of Arbutus unedo and soil underneath Arbutus plant canopy were purified using chromatographic techniques and the structure of chemicals was defined using spectroscopic and spectrometric methods. Leaf extracts (raw, aqueous and organic fractions) were tested for their toxicity on net nitrification, using a test soil. Field and laboratory incubations showed soil NO3- concentration below the detection limit over the whole study period, despite the significant NH4+ availability. Toxicity tests indicated that more than 400 μg of extract g-1 dry soil were needed to have more than 50% reduction of net NO3- production. Gallocatechin and catechin were among the most abundant chemicals in the extracts of leaves, roots and soil. Their soil concentration was significantly higher than the annual calculated input via leaf litter, and it was in the range of toxic concentrations, as deduced from the dose-response curve of the toxicity test. Data support the hypothesis that plant produced chemicals might be involved in the limited net nitrate production in this Mediterranean woodland. © 2008 Springer Science+Business Media B.V.