The Hippocratic Risk: Epidemiology of Suicide in a Sample of Medical Undergraduates.

In spite of the growing awareness on medical students' mental health in recent years, epidemiologic literature about the topic is scarce, particularly for what concerns suicide. Here, we collected cases of suicides among medical students enrolled at a local University in Italy and compared it to the general population of the same age group. Our sample was collected using records of suicides in the city of Pavia, Italy between the years of 2014 and 2019. This record was cross-referenced with enrolment numbers to the Medical Faculty of the city in order to select cases that involved medical students A time-window of 6 years was chosen for the evaluation. The odds ratio of suicide in medical students in comparison to the general population was of 14.58 (p value at the exact Poisson test <0.001). We then differentiated between native Italians and students from abroad. In this case, an odds ratio of 6.81 was observed (p value at the exact Poisson test 0.011). Our estimated suicide rates exceeded what was previously reported for the general population. We call for a closer attention on mental health in medical students, and an increased effort in collecting objective data on the topic.

The roles of medical examiners in the COVID-19 era: a comparison between the United States and Italy.

Italy and the United States are two of the countries most affected by SARS-CoV-2 (COVID-19), with more than 240,760 confirmed cases in Italy and 2,699,658 in the United States (as of July 2, 2020). The current COVID-19 pandemic has led to substantial changes in many fields of medicine, specifically in the forensic discipline. Medicolegal activities related to conducting autopsies have been largely affected by the COVID-19 pandemic. Postmortem examinations are generally discouraged by government regulations due to the risk of spreading the disease further through the handling and dissection of bodies from patients who succumbed to COVID-19 infection. There is a paucity of data regarding the persistence of SARS-CoV-2 in bodies, as well as concerning the reliability of swabbing methods in human remains. On the other hand, the autopsy is an essential tool to provide necessary information about the pathophysiology of the disease that presents useful clinical and epidemiological insights. On this basis, we aim to address issues concerning general medical examiner/coroner organization, comparing the Italian and American systems. We also discuss the pivotal roles of forensic pathologists in informing infectious disease surveillance. Finally, we focus on the impact of COVID-19 emergency on medicolegal practices in Italy and the United States, as well as the responses of the forensic scientific community to the emerging concerns related to the pandemic. We believe that stronger efforts by authorities are necessary to facilitate completing postmortem examinations, as data derived from such assessments are expected to be paramount to improving patient management and disease prevention.

Determination of Antidepressants and Antipsychotics in Dried Blood Spots (DBSs) Collected from Post-Mortem Samples and Evaluation of the Stability over a Three-Month Period.

An LC-MS/MS method for the identification and quantification of antidepressants and antipsychotics was developed on dried blood spots (DBSs). Moreover, analyte stability on DBSs within a 3-month period was monitored. Aliquots of 85 µL of blood from autopsy cases were pipetted onto DBS cards, which were dried and stored at room temperature. DBSs were analyzed in triplicate immediately, within the following 3 weeks, and after 3 months. For each analysis, a whole blood stain was extracted in phosphate buffer and purified using Solid Phase Extraction (SPE) cartridges in order to avoid matrix effects and injected in the LC-MS/MS system. Thirty-nine molecules were screened. Limits of detection (LODs) ranged between 0.1 and 3.2 ng/mL (g) and 0.1 and 5.2 ng/mL (g) for antidepressants and antipsychotics, respectively. Limits of quantification (LOQs) varied from 5 to 10.0 ng/mL for both. Sixteen cases among the 60 analyzed resulted positive for 17 different analytes; for 14 of these the method was fully validated. A general good agreement between the concentrations on DBSs and those measured in conventional blood samples (collected concurrently and stored at -20 °C) was observed. The degradation/enhancement percentage for most of the substances was lower than 20% within the 3-month period. Our results, obtained from real post-mortem cases, suggest that DBSs can be used for routine sample storage.

Death of a seven-month-old child in a washing machine: a case report.

The authors present a case which brings out a unique modality of child homicide by placing the baby in a washing machine and turning it on. The murder was perpetrated by the baby's mother, who suffered from a serious depressive disorder. A postmortem RX and then a forensic autopsy were performed, followed by histologic examinations and toxicology. On the basis of the results of the autopsy, as well as the histology and the negative toxicological data, the cause of death was identified as acute asphyxia. This diagnosis was rendered in light of the absence of other causes of death, as well as the presence of typical signs of asphyxia, such as epicardial and pleural petechiae and, above all, the microscopic examinations, which pointed out a massive acute pulmonary emphysema. Regarding the cause of the asphyxia, at least two mechanisms can be identified: drowning and smothering. In addition, the histology of the brain revealed some findings that can be regarded as a consequence of the barotrauma due to the centrifugal force applied by the rotating drum of the washing machine. Another remarkable aspect is that we are dealing with a mentally-ill assailant. In fact, the baby's mother, after a psychiatric examination, was confirmed to be suffering from a mental illness-a severe depressive disorder-and so she was adjudicated not-guilty-by-reason-of-insanity. This case warrants attention because of its uniqueness and complexity and, above all, its usefulness in the understanding of the pathophysiology of this particular manner of death.

A Fatal Case of Coin Battery Ingestion in an 18-Month-Old Child: Case Report and Literature Review.

The ingestion of extraneous substances is quite common in clinical practice; it usually befalls in the pediatric age, mostly between 6 months and 6 years. In most cases, complications do not emerge, and the prognosis is considered favorable. However, when a case of battery ingestion occurs, serious adverse events may develop. The ingestion of these components is a potential life-threatening event for children.In this article, we report the case of an 18-month-old child who died from hemorrhagic shock due to an aortoesophageal fistula caused by a 20 mm lithium button battery lodged in the esophagus.The child presented vomiting blood, and laboratory results revealed a severe anemization, which later led to death.The autopsy showed a coin battery located in the middle third of the esophagus as well as a transmural erosion of the esophageal wall with fistulization into the aortic wall. The histological examination revealed a severe necrosis of the esophageal and aortic walls in line with the junction between the aortic arch and the descending part.

Distribution of Fluvoxamine and Identification of the Main Metabolite in a Fatal Intoxication.

Fluvoxamine is a selective serotonin reuptake inhibitor, with a half-life of about 30 hours, that is commonly prescribed in the treatment of depression and obsessive and compulsive disorders. Though its more favorable adverse effect profile in comparison to tricyclic antidepressants, overdosages could lead to severe central nervous system depression. We hereby report the case of a 48-year-old woman with psychiatric disorders, who died in the Protected Community where she lived. An autopsy, during which multiorgan congestion and aspiration of gastric content were found, was performed 9 days after the death. Femoral and cardiac blood, urine and bile were collected for toxicological analysis. GC-MS, LC-MS-MS and LC-HRMS screenings were performed on blood samples. The analysis allowed to identify the following drugs: fluvoxamine, clotiapine, 7-aminoclonazepam, propranolol, gabapentin and haloperidol. Quantification of the detected drugs in blood was performed by means of a validated LC-MS-MS analytical procedure, and the following results were achieved: fluvoxamine (2.20 mg/L), gabapentin (41.00 mg/L), 7-aminoclonazepam (0.24 mg/L), clotiapine (0.07 mg/L), haloperidol (<0.01 mg/L) and propranolol (0.24 mg/L). Fluvoxamine concentration in blood exceeded ~10 times the upper limit of therapeutic blood levels (0.23 mg/L). Contributory causes of death, such as due to multiple drug use, however, cannot be excluded. The distribution of fluvoxamine in all biological fluids was evaluated and a postmortem redistribution effect was observed (C/P blood ratio: 1.86). Fluvoxamine acid metabolite was identified in urine, bile and in cardiac blood, through an LC-QTOF analytical procedure.

Neuropathological Alzheimer's Disease Lesions in Nasu-Hakola Disease with TREM2 Mutation: Atypical Distribution of Neurofibrillary Changes.

Nasu-Hakola disease is a rare autosomal recessive disorder associated to mutations in TREM2 and DAP12 genes, neuropathologically characterized by leukoencephalopathy with axonal spheroids. We report the neuropathologic findings of a 51-year-old female with a homozygous mutation (Q33X) of TREM2 gene. Beside severe cerebral atrophy and hallmarks of Nasu-Hakola disease, significant Alzheimer's disease lesions were present. Neurofibrillary changes showed an atypical topographic distribution being severe at spots in the neocortex while sparing the mesial temporal structures. Our finding suggests that TREM2 genetic defects may favor Alzheimer's disease pathology with neurofibrillary changes not following the hierarchical staging of cortical involvement identified by Braak.

A comparison between two different dried blood substrates in determination of psychoactive substances in postmortem samples.

Purpose: Whatman™ 903 cards represent a valid type of support for collection, storage, and analysis of dried blood spots (DBS). Whatman™ FTA (Flinders Technology Associates) are a type of cards soaked in chemicals that cause denaturation of proteins, while preserving DNA and ensuring the safe handling of DBS; to date, these cards are still rarely employed in forensic toxicology. The aim of this study was to analyze several psychoactive substances on not-dried blood on the two different cards and to compare the qualitative and quantitative results. Methods: Twenty cardiac postmortem blood samples were collected and deposed on Whatman™ 903 and Whatman™ FTA cards. Spots and not-dried blood were analyzed following our validated and previously published liquid chromatography-mass spectrometry methods. Results: We were able to identify: eight drugs of abuse and their metabolites (15 cases), five benzodiazepines and their metabolites (3 cases), six antidepressants (6 cases) and two antipsychotics (3 cases). We observed a perfect qualitative correspondence and a general good quantitative correlation between data obtained from not-dried blood and the two different DBS cards, except for alprazolam, diazepam, desmethyldiazepam, fluoxetine and sertraline, that showed a lower concentration on FTA. Additional experiments suggest that the chemicals, adsorbed on FTA, are not the cause of the loss of signal observed for the substances previously mentioned and that methanol should be preferred as extraction solvent. Conclusions: This study proved that FTA cards are a good and a hazard-free alternative sample storage method for analysis of several psychoactive substances in postmortem blood. Supplementary Information: The online version contains supplementary material available at 10.1007/s11419-020-00567-2.

A unique case of traumatic pulmonary food embolism.

We report a unique case of post-traumatic pulmonary food embolism, due to a lethal blunt force trauma occurred in a traffic accident. The subject was a 37-year old man, victim of a road traffic accident while he was riding his motorbike. A forensic autopsy, followed by histological examination, was ordered in order to find out the cause of death and to assess the compatibility of the lesions with the dinamic of the accident. Autopsy revealed a blunt force thoraco-abdominal trauma responsible of the death. The most interesting histological evidences concerned lungs. Here, inside arterious and arteriolar pulmonary vessels, we identified crystal-like corpuscles, of various shape and size, sometimes aggregated in small masses and thin vegetal fibers, refracting at polarized light, both PAS-positive and meat fibers shadows. The presence of alimentary material in the pulmonary vessels was explained by a pulmonary food embolism. The occurring of this kind of embolism implies a communication between the viscera lumen and the venous circulation of his wall (through a small wall rupture) in presence of cardiocirculatory activity, and provides, therefore, a strong proof of vitality.

Identification of cadaveric liver tissues using thanatotranscriptome biomarkers.

Thanatotranscriptome studies involve the examination of mRNA transcript abundance and gene expression patterns in the internal organs of deceased humans. Postmortem gene expression is indicative of the cellular status of a corpse at the time of death, a portion of which may represent a cascade of molecular events occasioned by death. Specific gene biomarkers identify perceptible transcriptional changes induced by stochastic responses to the cessation of biological functions. Transcriptome analyses of postmortem mRNA from a tissue fragment may determine unique molecular identifiers for specific organs and demonstrate unique patterns of gene expression that can provide essential contextual anatomical information. We evaluated the impact of targeted transcriptome analysis using RNA sequencing to reveal global changes in postmortem gene expression in liver tissues from 27 Italian and United States corpses: 3.5-hour-old to 37-day-old. We found that our single blind study using eight liver tissue-specific gene biomarkers (e.g. AMBP and AHSG) is highly specific, with autopsy-derived organ samples correctly identified as tissues originating from postmortem livers. The results demonstrate that 98-100% of sequencing reads were mapped to these liver biomarkers. Our findings indicate that gene expression signatures of mRNA exposed up to 37 days of autolysis, can be used to validate the putative identity of tissue fragments.

Fatal poisoning of four workers in a farm: Distribution of hydrogen sulfide and thiosulfate in 10 different biological matrices.

We evaluate the distribution of sulfide and thiosulfate (TS) in biological samples of four dairy farmers died inside a pit connected to a manure lagoon. Autopsies were performed 4 days later. Toxicological analyses of sulfide and TS were made using an extractive alkylation technique combined with gas chromatography/mass spectrometry (GC/MS). Autopsies revealed: multiorgan congestion; pulmonary edema; manure inside distal airways of three of the four victims. Sulfide concentrations were cardiac blood: 0.5-3.0 µg/mL, femoral blood: 0.5-1.2 µg/mL, bile: <0.1-2.2 µg/mL; liver 2.8-8.3 µg/g, lung: 5.0-9.4 µg/g, brain: 2.7-13.9 µg/g, spleen: 3.3-6.3 µg/g, fat: <0.1-1.5 µg/g, muscle: 2.6-3.5 µg/g. TS concentrations were cardiac blood: 2.1-4.9 µg/mL, femoral blood: 2.1-2.3 µg/mL, bile: 2.5-4.4 µg/mL, urine: <0.5-1.8 µg/mL; liver <0.5-2.6, lung: 2.8-5.4 µg/g, brain: <0.5-1.9 µg/g, spleen: 1.2-2.9 µg/g, muscle: <0.5-5.6 µg/g. The cause of death was assessed to be acute poisoning by hydrogen sulfide (H2S) for all the victims. Manure inhalation contributed to the death of three subjects. The measurement of sulfide and TS concentrations in biological samples contributed to better understand the sequence of the events. Subjects 3 provided the highest concentration of sulfide in brain, thus, supporting the hypothesis of a rapid loss of consciousness and respiratory depression. One by one, the other farmers entered the pit in attempts to rescue the coworkers but collapsed. Despite the rapid death, subject 3 was the only one with TS detectable in urine. This could be due to differences in metabolism of H2S.

Effects of Extended Postmortem Interval on Microbial Communities in Organs of the Human Cadaver.

Human thanatomicrobiota studies have shown that microorganisms inhabit and proliferate externally and internally throughout the body and are the primary mediators of putrefaction after death. Yet little is known about the source and diversity of the thanatomicrobiome or the underlying factors leading to delayed decomposition exhibited by reproductive organs. The use of the V4 hypervariable region of bacterial 16S rRNA gene sequences for taxonomic classification ("barcoding") and phylogenetic analyses of human postmortem microbiota has recently emerged as a possible tool in forensic microbiology. The goal of this study was to apply a 16S rRNA barcoding approach to investigate variation among different organs, as well as the extent to which microbial associations among different body organs in human cadavers can be used to predict forensically important determinations, such as cause and time of death. We assessed microbiota of organ tissues including brain, heart, liver, spleen, prostate, and uterus collected at autopsy from criminal casework of 40 Italian cadavers with times of death ranging from 24 to 432 h. Both the uterus and prostate had a significantly higher alpha diversity compared to other anatomical sites, and exhibited a significantly different microbial community composition from non-reproductive organs, which we found to be dominated by the bacterial orders MLE1-12, Saprospirales, and Burkholderiales. In contrast, reproductive organs were dominated by Clostridiales, Lactobacillales, and showed a marked decrease in relative abundance of MLE1-12. These results provide insight into the observation that the uterus and prostate are the last internal organs to decay during human decomposition. We conclude that distinct community profiles of reproductive versus non-reproductive organs may help guide the application of forensic microbiology tools to investigations of human cadavers.

Distribution of quetiapine and metabolites in biological fluids and tissues.

Quetiapine is an atypical antipsychotic drug, frequently found in post-mortem samples. The quantitative determination of active metabolites may help in the interpretation of the potential toxic effects of the parent drug and its role in death. A fully validated LC-MS/MS method was developed for the identification and quantification of quetiapine and two main metabolites (N-desalkylquetiapine and 7-hydroxyquetiapine) in blood, biological fluids and tissues. Then, the distribution of analytes in different matrices was evaluated. LODs of 0.9, 0.3 and 0.3ng/mL were calculated for quetiapine, N-desalkylquetiapine and 7-hydroxyquetiapine respectively; while a LOQ at the concentration of 10.0ng/mL was defined for the three analytes. 13 post-mortem positive real cases have been included in the experiment. The results revealed that quetiapine and N-desalkylquetiapine might undergo a significant post-mortem redistribution, while 7-hydroxyquetiapine is less affected by this factor. N-desalkylquetiapine could be found in blood in relatively high concentrations in comparison to those of quetiapine; therefore, it should be always advisable to measure both the analytes. The analysis of tissues could provide additional data on potential intoxication with quetiapine.

Determination of fentanyl and 19 derivatives in hair: Application to an Italian population.

Nowadays fentanyl and its analogs represent the most numerous group among synthetic opioid and, due to their higher potency in comparison to traditionl opioids, the main cause of the critical increase of fatal intoxications opioids-intake related in the USA. We developed an LC-MS/MS method for the detection and quantification of fentanyl and its analogs in hair, then applied to 117 real samples, 97 collected from drugs users and 20 from postmortem cases of drugs addicts. The ionization and MRM parameters have been optimized for 27 molecules: 20 reached the acceptance criteria for identification and quantification. LODs and LOQs of 0.2 and 0.5 pg/mg, respectively, were reached for most of the substances, except for five compounds for which were set at 0.5 and 1.0 pg/mg. 2 out of the 97 samples collected from drug users tested positive; one for carfentanil, butyryl fentanyl, THFF and ocfentanil; the other one for 3-methyl norfentanyl. 2 out of the 20 postmortem samples show positive results: one only for fentanyl, the other for furanyl fentanyl, acetyl fentanyl, methoxyacetyl fentanyl, methoxyacetyl norfentanyl, ocfentanil and 4-ANPP. Despite the relatively small number of samples, the results suggest that the method should be included in routine hair analyses for monitoring the new synthetic opioids potential intake by drug users.

Distribution of the Synthetic Cathinone α-Pyrrolidinohexiophenone in Biological Specimens.

We report the analysis of the synthetic cathinones α-pyrrolidinohexiophenone (α-PHP) and α-pyrrolidinopentiophenone (α-PVP), both pyrovalerone derivatives, in blood, urine, gastric contents, main tissues and hair of a deceased person. Qualitative and quantitative analyses were performed by LC-MS-MS. All the biological samples were collected during autopsy and extracted/purified onto a solid phase extraction cartridge before instrumental analysis. The method was validated for blood and urine and proved to be highly sensitive and specific for both the synthetic cathinones (limit of detection: 0.2 ng/mL and limit of quantification: 0.5 ng/mL). Analyses provided negative results for α-PVP in all biological samples except for the 2-cm proximal hair segment, confirming that the young man had consumed in the last 2 months this compound; instead hair analysis proved that the man was a heavy α-PHP user. α-PHP was identified and quantified in biological fluids and tissues. Interestingly, bile and urine concentrations (1.2 and 5.6 ng/mL, respectively) were fairly lower than blood collected into the thoracic cavity (15.3 ng/mL). The highest concentrations were measured for lung (71.1 ng/mL) and spleen (83.8 ng/mL). Concentrations of 3.5, 7.9, 4.7 and 23.6 ng/mL were measured in liver, kidney, brain and heart, respectively. Even if it is not possible to evaluate the presence of this drug in biological samples as a cause of death, to our knowledge, this is the first case of α-PHP finding in postmortem samples, and its potential toxic effects should be elucidated in the future.

A multi-analyte LC-MS/MS method for screening and quantification of 16 synthetic cathinones in hair: Application to postmortem cases.

A multi-analyte method for detection and quantification of 16 synthetic cathinones (known also as "bath salts") in human hair has been developed and fully validated using liquid chromatography-tandem mass spectrometry system. About 20 mg of hair samples, previously washed and homogenized, were ultrasonicated with 1 mL HCl 0.1 M solution. Samples were then extracted using a solid phase extraction procedure (SPE), taken to dryness and reconstituted in 100 µL mobile phase. Finally, they were directly injected into a liquid chromatographic system, coupled with tandem mass spectrometer detector. The validation criteria parameters were satisfactory according with the international guidelines. A LOQ of 5 pg/mg was obtained for 4-fluoromethcathinone (4-FMC), buphedrone, ethcathinone, methcathinone, mephedrone and naphyrone, while the method proved to be more sensitive for 4-methylethcathinone (4-MEC), methedrone, alpha-pyrrolidinopentiophenone (α-PVP), alpha-pyrrolidinohexiophenone (α-PHP), methylenedioxypyrovalerone (MDPV), butylone, ethylone, 3,4-dimethylmethcathinone (3,4-DMMC), pentedrone and pentylone, reaching a LOQ of 1 pg/mg. Potential use of bath salts was investigated in postmortem cases of young subjects previously tested positive at least to one traditional drug of abuse. Two samples out of 17 cases analyzed provided positive results for synthetic cathinones. One sample has been divided in two segments of 2.5 cm length each. Both segments were positive for 8 different cathinone derivatives, namely: 3,4-DMMC, 4-FMC, 4-MEC, α-PHP, α-PVP, methcathinone, methedrone and pentedrone. The second case provided positive results for ethcathinone.

Drug Abuse-Related Neuroinflammation in Human Postmortem Brains: An Immunohistochemical Approach.

The aim of the study was to investigate blood-brain barrier alterations, neuroinflammation, and glial responses in drug abusers. Five immunohistochemical markers (CD3, zonula occludens-1 [ZO-1], intracellular adhesion molecule 1 [ICAM-1], vascular cell adhesion molecule [VCAM-1], and glial fibrillary acidic protein [GFAP]) were assessed on postmortem brain samples collected from drug abusers who died from acute intoxication of cocaine, heroin, or a combination of both, compared with controls. CD3 and ICAM-1 immunopositivity were significantly stronger in drug abusers than in controls. VCAM-1 immunopositivity was similar across drug abuser and control groups. In heroin abusers, significantly lower ZO-1 immunopositivity was observed relative to controls. GFAP positivity did not show significant differences between groups, but its distribution within the brain did differ. Both cocaine and heroin abuse promoted neuroinflammation, increasing expression of ICAM-1 and recruiting CD3+ lymphocytes. Heroin affected the molecular integrity of tight junctions, as reflected by reduced ZO-1 expression. The outcomes of the present study are, overall, consistent with prior available evidence, which is almost exclusively from studies conducted in vitro or in animal models. These findings provide important information about the downstream consequences of neuroinflammation in drug abusers and may help to inform the development of potential therapeutic targets.

Determination of benzodiazepines in blood and in dried blood spots collected from post-mortem samples and evaluation of the stability over a three-month period.

We successfully developed and validated an LC-MS/MS method for the identification of 27 and quantification of 9 benzodiazepines and metabolites in whole blood and DBSs. The results provided a good qualitative and quantitative correlation between DBSs stored at room temperature and whole blood stored at -20°C. A good stability for a three-month period was observed for most of the compounds detected in real post-mortem samples.

Vaccination policy in Italy: An update.

The effective control of vaccine-preventable diseases generally requires indefinite maintenance of extremely high rates of timely vaccination. Therefore, vaccine hesitancy is of paramount importance and needs to be addressed. In Italy, regulations about vaccinations are controversial and, to some extent, inconsistent. Even though the childhood vaccinations are mandatory by law (Italian Law n. 891/1939, n. 292/1963, n.51/1966 and n. 165/1991), the limited deterrent effectiveness of the sanctioning system, and the changes introduced by the Italian Constitutional Law n. 3/2001 (devolution of almost all the competences and responsibilities in health matters to the Regions and the Autonomous Provinces), were the fertile ground in which new vaccine policies were generated and developed, radically different from the existing ones: many Regions, based on what was decided in 2005 - on an experimental basis - by the State-Regions Conference, decided to abolish the vaccination obligation and/or to stop the imposition of administrative sanctions on non-compliant parents. In addition, since then, there is a worrying tendency to decline vaccinations due to the parents' mistrust in pharmaceutical companies and health policies. Therefore, recently, the Italian government decided to deploy an emergency ordinance (Italian Decree Law n. 73/2017). In this article, the authors are going to illustrate the current situation in Italy concerning vaccination policy, from a legislative and social point of view.