Genetic individual identification from dried urine spots: A complementary tool to drug monitoring and anti-doping testing.

The collection of liquid biological matrices onto paper cards (dried matrix spots [DMS]) is becoming an alternative sampling strategy. The stability over time of molecules of interest for therapeutic, sport drug monitoring, and forensic toxicology on DMS has been recently investigated representing a reliable alternative to conventional analytical techniques. When a tampering of a urine sample in drug monitoring or doping control cases is suspected, it could be relevant to know whether genetic profiles useful for individual identification could be generated from urine samples spotted onto paper (dried urine spot [DUS]). To understand the influence of sex, storage conditions, and time on the quality and quantity of the DNA, five female and ten male urine samples were dispensed onto Whatman 903 paper and sampled after different storage conditions over time, from 1 to 12 weeks. Direct PCR was performed starting from 2-mm punches collected from each spot amplifying a panel of markers useful for individual identification. The female DUS stored in different conditions produced genetic profiles fully matching the reference samples. The same result was obtained for the male DUS but using urine 30X concentrated by centrifugation instead of the original samples. Our data show that this approach is valid for genetic individual identification of urine samples spotted onto paper cards up to 12 weeks after deposition and could be easily incorporated in anti-doping or drug screening protocols to help on the suspicion of evidence tampering or to solve questions on the reliability of samples collection.

Did Italy Really Need Compulsory Vaccination against COVID-19 for Healthcare Workers? Results of a Survey in a Centre for Maternal and Child Health.

Since its early spread, the COVID-19 pandemic has become a health threat globally. Due to their crucial role in the pandemic, Italy declared compulsory vaccination for healthcare workers. Vaccine hesitancy was observed among the healthcare workers and an ethical debate arose about Italian legal statement D.L. n. 44/2021. In this article, we present the results of a survey performed in an Italian center for maternal and infant care and assess the attitudes towards the COVID-19 pandemic and the mandatory COVID-19 vaccination of healthcare workers. Since March 2022, 91.5% of healthcare workers have been vaccinated with an additional dose. Only 2.3% of the respondents refused to take vaccination: the reasons behind this refusal were distrust, doubts over safety, and lack of information. Despite the high rate of response to vaccination, 17.7% of HCWs did not agree with its mandatory nature. In addition, 5.4% stated that they agreed to be vaccinated exclusively because of the sanctions provided for by the legislation. In conclusion, adequate vaccination coverage has been achieved in the hospital under consideration. However, it is still very important to continue to persuade HCWs of vaccine efficacy and safety, considering their social role.

Synthetic Cannabinoids and Cathinones Cardiotoxicity: Facts and Perspectives.

New psychoactive substances (NPS) constitute a group of psychotropic substances, designed to mimic the effects of traditional substances like cannabis, cocaine, MDMA, khat, which was not regulated by the 1961 United Nations Convention on Narcotics or the 1971 United Nations Convention on Psychotropic Substances. Illegal laboratories responsible for their production regularly developed new substances and placed them on the market to replace the ones that have been banned; for this reason, during the last decade this class of substances has represented a great challenge for the public health and forensic toxicologists. The spectrum of side effects caused by the intake of these drugs of abuse is very wide since they act on different systems with various mechanisms of action. To date most studies have focused on the neurotoxic effects, very few works focus on cardiotoxicity. Specifically, both synthetic cannabinoids and synthetic cathinones appear to be involved in different cardiac events, including myocardial infarction and sudden cardiac death due to fatal arrhythmias. Synthetic cannabinoids and cathinones cardiotoxicity are mainly mediated through activation of the CB1 receptor present on cardiomyocyte and involved with reactive oxygen species production, ATP depletion and cell death. Concerns with the adrenergic over-stimulation induced by this class of substances and increasing oxidative stress are mainly reported. In this systematic review we aim to summarize the data from all the works analyzing the possible mechanisms through which synthetic cannabinoids and synthetic cathinones damage the myocardial tissue.

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.

Is Hospital Autopsy Auditing Suitable for Clinical Risk Manage ment? Actualities and Perspectives of Auditing in the Autopsy Room Following Italian Law 24/2017 on Patient Safety.

BACKGROUND: Autopsy is a valuable tool for understanding the physiopathology of any disease, and it is the gold standard to assess the cause of death. The clinical autopsy is the ultimate medical service for a patient and plays a crucial role in the context of quality control, education of physicians and other medical personnel, as well as mitigation of risk of malpractice claims. OBJECTIVE: This study aims to demonstrate the importance of improving an autopsy service and the relevance of this investigation procedure in daily clinical practice by evaluating the rate of major discrepancies between the assumed cause of death and the ascertained cause of death after a complete post mortem investigation. A further aim is to classify these discrepancies as class I or class II discrepancies according to the Goldman's criteria in order to assess performance quality. METHODS: A retrospective study of the hospital autopsies performed from June 2018 to March 2020 was conducted by considering a diversified dataset, including age and sex of the deceased as well as the clinical and pathological causes of death. RESULTS: 362 cases were taken into consideration. Major discrepancies were found in 71.3% of cases, with a class I error of 22.7% and a class II error of 48.6%. The most frequent misdiagnosis were cardiovascular disorders, embolism, and aneurism rupture. DISCUSSION: The rate of major discrepancies and the rate of class I and class II errors are way above the rate found in the literature. Despite the high rate of a major discrepancy, evidence collected from hospital autopsies (i.e., certainty of the cause of death, unknown comorbidities) has strengthened the legal defense in cases of medical malpractice litigation. In our experience, by accurately determining the cause of death, revealing new or unexpected findings, and any possible diagnostic or technical errors, post-mortem examinations can significantly contribute to the improvement of team performance and quality of care. CONCLUSION: The presence of clinicians during an autopsy and the early sharing of results can be considered a new auditing strategy for hard clinical cases. Finally, by providing a clearer understanding of the nature and cause of the illness, the autopsy results assist in the grieving process by reassuring family members that action or inaction on their part had not contributed to the death.

Assessment of the Precision ID Identity Panel kit on challenging forensic samples.

The performance of the Precision ID Identity Panel (Thermo Fisher Scientific) was assessed on a set of 87 forensic samples with different levels of degradation for which a reference sample from the "same donor" or from a "first degree relative" was available. PCR-MPS analysis was performed with DNA input ranging from 1 ng to 12 pg and through 21-26 PCR cycles, in replicate tests, and a total number of 255 libraries were sequenced on the Ion Personal Genome Machine™ (PGM™) System. The evaluation of the molecular data allowed to set a fix threshold for locus call at 50 x which suitably worked even when low amounts of degraded DNA (12 pg) were investigated. In these analytical conditions, in fact, 25 PCR cycles allowed the genotyping of about 50 % and 35 % of the autosomal and the Y-specific markers on average, respectively, for each single amplification with a negligible frequency of drop ins (0.01 %). On the other hand, drop out artefacts reached 18-23 % when low copy number and degraded DNA samples were studied, with surviving alleles showing more than 600 reads in 2.9 % of the cases. Our data pointed out that the Precision ID Identity Panel allowed accurate typing of almost any amount of good quality/moderately degraded DNA samples, in duplicate tests. The analysis of low copy number DNAs evidenced that the same allele of a heterozygous genotype could be lost twice, thus suggesting that a third amplification could be useful for a correct genotype assignment in these peculiar cases. Using the consensus approach, a limited number of genotyping errors were computed and about 37 % of the autosomal markers was finally typed with a corresponding combined random match probability of at least 1.6 × 10-13, which can be considered an excellent result for this kind of challenging samples. In the end, the results presented in this study emphasize the crucial role of the expert opinion in the correct evaluation of artefacts arising from PCR-MPS technology that could potentially lead to genetic mistyping.