A fatal case of ascending spinal and cranial subdural empyema (SDE) caused by Streptococcus intermedius in a socially vulnerable 63-year-old man with repeated emergency department (ED) admissions is reported. Despite persistent lower back pain and neurological symptoms, advanced neuroimaging and additional clinical and laboratory evaluation were delayed, ultimately leading to septic shock and multi-organ failure. Forensic and postmortem investigations identified the pathogen and clarified the cause of death. The case highlights the forensic relevance of missed clinical red flags and the potential interplay between implicit bias - defined as unconscious attitudes and beliefs that may drive intentional discriminatory actions - organizational factors, and diagnostic delays in a life-threatening condition affecting an underserved patient. Increased awareness of vulnerability-associated diagnostic bias, together with systematic organizational interventions, is needed to promote equity in emergency care and reduce healthcare disparities among marginalized populations.

The increasing interest in DNA methylation (DNAm) analysis within the forensic scientific community prompted a collaborative project by Ge.F.I. (Genetisti Forensi Italiani). The study evaluated a standardized bisulfite conversion-based Single Base Extension (SBE) protocol for the analysis of the methylation levels at five age-predictive loci (ELOVL2, FHL2, KLF14, C1orf132/MIR29B2C, and TRIM59). The study encompassed three phases: (1) setting up and validating the protocol to ensure consistency and reproducibility; (2) comparing fresh peripheral blood with blood spots; and (3) evaluating sources of intra- and inter-laboratory variability. Samples from 22 Italian volunteers were analyzed by 6 laboratories in replicates for a total of 528 records. From phase I emerged that the choice of genetic sequencer significantly contributed to inter-laboratory data variation, resulting in separate regression analyses performed for each laboratory. In phase II, blood spots were found to be a reliable source for DNAm analysis, despite exhibiting increased experimental variation compared to fresh peripheral blood. In phase III, a strong correlation between the individual's predicted and true ages was observed across different laboratories. Analysis of variance (ANOVA) of the residuals indicated that one-third of the total variance could be attributed to laboratory-specific factors, whereas two-thirds could be attributed to inter-individual biological differences. The leave-one-out cross-validation (LOO-CV) method yielded an overall mean absolute deviation (MAD) value of 4.41 years, with an average 95% confidence interval of 5.24 years. Stepwise regression analysis proved that a restricted model (ELOVL2, C1orf132/MIR29B2C, and TRIM59) produced results virtually indistinguishable from the five-loci model. Additionally, the analysis of samples in replicates greatly improved the fit of the regression model, balancing the slight effects of intra-laboratory variability. In conclusion, the bisulfite conversion-based SBE protocol, combined with replicate analysis and in-lab calibration of a regression-prediction model, proves to be a reliable and easily implementable method for age prediction in forensic laboratories.

Femicides are grievous, but little is known about risk factors and preventive measures. We present the results of a study conducted across 27 Italian Forensic Institutes. We analyzed 1,238 cases of female homicide and, adopting the definition of femicide as the murder due to the failure to recognize women's right to self-determination, we identified 410 cases as femicides and 395 as non-femicides Current partners were identified as aggressors in a much larger proportion of femicide cases (241 femicides vs. 145 non-femicides; odds ratio (OR) of femicide 2.46, 95 % CI 1.85-3.27), such association being more pronounced for ex-partners (102 vs. 11; OR 11.56, 95 % CI 6.10-21.92). Sharp weapons showed a higher frequency in femicides (168 vs. 140; OR 1.26, 95 % CI 0.95-1.68). Femicides were more often associated with bodies found in vehicles (31 vs. 9; OR 3.50, 95 % CI 1.64-7.45) and outdoor (68 vs. 43; OR 1.62, 95 % CI 1.08-2.45). There was an indication of femicides being more frequently associated with overkilling (87/323 vs. 71/324; OR 1.24, 95 % CI 0.88-1.76) and even more with lesions located in erogenous zones (94/316 vs. 70/325; OR 1.38, 95 % CI: 0.97-1.95). However, the strongest (though statistically imprecise) association emerged for overkilling in erogenous zones (12/398 vs. 6/389; OR 1.95, 95 % CI: 0.72-5.25). The number of lesions showed a nonlinear association with femicide likelihood. These findings offer forensic indicators that could contribute in predicting and potentially prevent femicide occurrence in a Western population such as the Italian one.

Body fluid identification by means of mRNA profiling provides valuable supplementary information in forensic investigations. In particular, the detection of vaginal mucosa mRNA markers is highly relevant in sexual assault cases. Although the vagina undergoes characteristic age-related physiological changes over a lifetime, few studies have evaluated the efficacy of vaginal mRNA markers in women of different ages. In this multicentric study, a 19-plex mRNA profiling assay including vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of 6–20-month-old vaginal swabs obtained from pre- (n = 84) and postmenopausal (n = 55) female volunteer donors. Overall, participating laboratories were able to correctly identify ~85% of samples as vaginal mucosa by mRNA profiling. The assay’s success rate did not differ between the two age groups and was not affected by the time interval between swab collection and RNA analysis. MYOZ1 resulted a less sensitive vaginal marker compared to MUC4 and CYP2B7P1. A significant relative increase in the contribution to the total amplification signal was observed for MUC4, compared to CYP2B7P1 and MYOZ1, in postmenopausal women. Observation of other body fluids and tissues different from vaginal mucosa was also evaluated in connection to information on previous sexual activity and menstrual cycle phase at the time of sampling.

Drowning under drug influence is scarcely reported, especially in relation to lung weight in drug-free or drug-positivity drowning. A novel drowning index is proposed in order to easily compare some factors (drug exposure, fresh/salty water, gender, age, post-mortem interval) acting on drowning deaths. Autopsies and toxicological analyses were performed in 48 drowning cases. Ultra-high-performance liquid chromatography-tandem mass spectrometry for drugs of abuse and headspace-gas chromatography-mass spectrometry GC-MS for volatile substances resulted in positive on 13 cases. The drug-positive group was compared to the drug-free group thanks to a novel drowning index [(lungs plus pleural effusion) / height]. Salty/freshwater, sex, and age influence were studied in the two groups to detect their influence on the novel drowning index. Bromazepam, ethanol, lorazepam, lormetazepam, ethanol plus cocaine were quantified in 13/48 drowning cases. In the univariate analysis, the variables associated with low drowning index were female gender (p = 0.010), freshwater drowned (p = 0.012), and benzodiazepine assumption (p = 0.012). Drowning under ethanol effect compared to drug-free cases did not obtain significant associations. In the multivariate analysis, only the male gender (β = -0.378, p = 0.005) and salty water (β = 0.369, p = 0.006) were independent parameters associated with higher DI (adjusted R2 = 0.241). Comparison between groups of drowning can take advantage of an integrated drowning index that, thanks to its sensibility, can register differences induced by the type of water & intake of exogenous substances & age/sex.

The Ge.F.I. (Italian Forensic Geneticists) board has set up a working group aimed to develop the “Code of conduct of a Forensic Geneticist”. This group includes all practitioners working in the field of forensic genetics. The rules of the code of conduct apply to forensic geneticists belonging to the GeFI group and all of those sharing the GeFI’s mission, vision and values applicable to their professional practice, reciprocal relationship and relationship with third parties.

The growing use of massively parallel sequencing (MPS) for the whole mitochondrial genome analysis in forensic laboratories, requires the establishment of efficient workflows and interpretation procedures, to support the feasibility of the technology and the reliability of the data. In the case of reference samples, such as blood and buccal swabs, the generation of mtDNA profiles by using MPS is relatively simple. Conversely, many forensic casework samples still pose challenges for the MPS, data interpretation and reporting of mtDNA. This is especially true for the analysis of shed hairs, which are one of the most common evidence types and which are among the most limited in terms of DNA quantity and quality. Due to these limitations, every step involved in the analysis become essentials and should be performed in order to obtain the best performance, optimizing the outcomes and minimizing the errors. In light of this, we present a study focusing on the extraction, quantification and MPS of the whole mtDNA in hair shafts, with the aims of set-up and validate a methodological pipeline to obtain the best sequencing results. The overall performance of the MPS panel, mainly in terms of total coverage, amplicons coverage and different primer pools efficiency, was evaluated also in relation to the different hair fragments, the mtDNA copy number used for libraries preparation and its degradation state.

Cannabis sativa is the most used controlled substance in Europe. With the advent of new and less restrictive European laws on cannabis sale for recreational use (including in Italy), an increase in indoor cannabis crops were observed. This increase was possible due to the availability of cannabis seeds through the internet market. Genetic identification of cannabis can link seizures and if in possession then might aid in an investigation. A 13-locus multiplex STR method was previously developed and validated by Houston et al. A collaborative exercise was organized by the Italian Forensic Geneticists - International Society of Forensic Genetics (Ge.F.I. - ISFG) Working Group with the aim to test the reproducibility, reliability and robustness of this multiplex cannabis STR kit. Twenty-one laboratories from three European countries participated in the collaborative exercise and were asked to perform STR typing of two cannabis samples. Cannabis DNA samples and the multiplex STR kit were provided by the University of Barcelona and Sam Houston State University. Different platforms for PCR amplification, capillary electrophoresis (CE) and genotyping software were selected at the discretion of the participating laboratories. Although the participating laboratories used different PCR equipment, CE platforms and genotyping software, concordant results were obtained from the majority of the samples. The overall genotyping success ratio was 96%. Only minor artifacts were observed. The mean peak height ratio was estimated to be 76.3% and 78.1% for sample 1 and sample 2, respectively. The lowest amount of -1 / + 1 stutter percentage produced, when the height of the parent allele was higher than 8000 RFU, resulted to be less than 10% of the parent allele height. Few common issues were observed such as a minor peak imbalance in some heterozygous loci, some artifact peaks and few instances of allelic drop-out. The results of this collaborative exercise demonstrated the robustness and applicability of the 13-locus system for cannabis DNA profiling for forensic purposes. (c) 2021 Elsevier B.V. All rights reserved.

Methadone overdoses are usually considered "slow" and avoidable deaths. Despite being frequently witnessed, the type and duration of the agonal time are rarely documented and challenging to be reconstructed. Here we report a case in which a young male was found dead in a parked car, shortly after discharge from a compulsory psychiatric treatment. Death scene investigation, clinical records collection, analysis of video recordings from surveillance cameras and private videos, post-mortem examination, GC-MS and LC-MS/MS toxicological analyses were performed and allowed to assess an acute fatal intoxication by methadone and prescription drugs. The case here-in reported is a rare example of a witnessed and recorded prolonged agonal phase due to methadone and drug intoxication, which lasted at least 12 hours. The loss of consciousness and a worsening pattern of respiratory depression, including gasping, were reported by the friends of the victim, but no one called for help, arising the suspect of failure to provide assistance. The possibility of a long-lasting agony with respiratory gasping should be considered in the evaluation of similar intoxication cases.

The efficiency of MPS in forensic mtDNA analysis has been thoroughly proven, although a reliable and well established data evaluation still remains a critical point. Numerous bioinformatics tools have been developed, but most of them require specific operating systems and high costs, while free open-source programs with user-friendly interfaces are few. In this study, 43 full mtGenomes were sequenced using the Ion Personal Genome Machine™ (PGM™) System and analyzed utilizing the plug-in Variant Caller (TVC) of the Ion Torrent Software Suite and the mtDNA-Server (mDS), a free web-based mitochondrial analysis tool for MPS data. The outcomes of these two different analysis tools were compared to variants noted after manual inspection of the aligned reads performed using Integrative Genomics Viewer (IGV). The comparison highlighted the presence of thirty-nine discordant variant calls, which were resolved by Sanger sequencing that confirmed the presence of all variants, except for 7 deletions. The combined adoption of IGV and Sanger type sequencing confirmatory steps, in addition of TVC and mDS analysis, resulted in a more accurate variants assignment with the detection of 32 additional true polymorphisms, which were noted in the final dataset. Regarding the heteroplasmy issue, out of a total of thirty heteroplasmic variants, twenty-eight were detected by the TVC, while the mDS detected twenty-two. Overall, none of the used bioinformatics tools were the perfect choice and a secondary analysis with an expert's opinion in complete mtGenome MPS data evaluation is still required in forensic genetic analysis.

Forensic mRNA profiling assays normally include a set of vaginal-specific markers. Although it is known that vagina undergoes characteristic age-related morphological and physiological changes over a lifetime, few studies have evaluated the efficacy of proposed forensic vaginal mRNA markers in women from different age groups.In this collaborative study involving ten GeFI (Italian working group of ISFG) laboratories, a 19-plex mRNA profiling assay including three vaginal-specific markers (CYP2B7P1, MUC4, MYOZ1) was tested in a collection of vaginal swabs obtained from female volunteer donors in their reproductive years (n = 84) and postmenopausa (n = 55).Differential expression of vaginal markers in the two age categories was assessed by means of: a) overall success rate of mRNA profiling (vaginal mucosa “observed” in the tested sample according to scoring protocol) b) average peak height ratios between vaginal-specific markers within mRNA profiling replicates.Other factors potentially influencing mRNA profiling outcomes, like time interval between vaginal swab collection and analysis, concurrence of menstrual cycle and recent sexual activity at the time of sampling were also investigated.

The Ge.F.I. has by statute the task of elaborating recommendations and their periodic revision. To this aim, in the last years, the board of the Ge.F.I. (Italian Forensic Genetics group) developed recommendations on three topics:1) Personal identification.2) Paternity testing.3) Collection of the biological evidences in rape/maltreatment cases.

To evaluate the pattern of Romanian population from a mitochondrial perspective and to establish an appropriate mtDNA forensic database, we generated a high-quality mtDNA control region dataset from 407 Romanian subjects belonging to four major historical regions: Moldavia, Transylvania, Wallachia and Dobruja. The entire control region (CR) was analyzed by Sanger-type sequencing assays and the resulting 306 different haplotypes were classified into haplogroups according to the most updated mtDNA phylogeny. The Romanian gene pool is mainly composed of West Eurasian lineages H (31.7%), U (12.8%), J (10.8%), R (10.1%), T (9.1%), N (8.1%), HV (5.4%),K (3.7%), HV0 (4.2%), with exceptions of East Asian haplogroup M (3.4%) and African haplogroup L (0.7%).The pattern of mtDNA variation observed in this study indicates that the mitochondrial DNA pool is geographically homogeneous across Romania and that the haplogroup composition reveals signals of admixture of populations of different origin. The PCA scatterplot supported this scenario, with Romania located in southeastern Europe area, close to Bulgaria and Hungary, and as a borderland with respect to east Mediterranean and other eastern European countries.High haplotype diversity (0.993) and nucleotide diversity indices (0.00838±0.00426), together with low random match probability (0.0087) suggest the usefulness of this control region dataset as a forensic database in routine forensic mtDNA analysis and in the investigation of maternal genetic lineages in the Romanian population.

Sudden cardiac death in the young is a very dramatic event that occurs in apparently healthy individuals without an explainable cause of death. The advancement of knowledge in the field of molecular biology has suggested that genetic abnormalities are the leading cause of sudden unexplained death in young people. Three forensic SUD cases were studied. 68 genes most frequently associated with SUD were analyzed by NGS.

The opportunities to analyse the genetic variations related to the risk of addiction are of interest to forensics, who beside their involvement in drugs-related fatalities may also be required to assess driving and working ability as well as permanent invalidity due to drugs-related conditions. Several genetic variants have been shown to be associated with heroin addiction. The most investigated gene is OPMR1 that encode the μ-opioid receptor. The purpose of this study was to examine the contribution of genetics variants in OPRM1 to the susceptibility to addiction.

Objectives This study investigated the involvement of ADH4 gene polymorphisms in the susceptibility to alcohol use disorders. Methods Thirty-eight single-nucleotide polymorphisms (SNPs) in and around the ADH4 gene were investigated in 136 Italian alcoholics and 276 healthy controls. A new approach based on a bioinformatic method selected 26 SNPs that may affect the splicing sites, destroying or creating binding sites of splicing regulatory proteins. Results Case–control comparisons for allele and genotype frequencies showed that ADH4 SNPs were associated with alcohol dependence but not with alcohol abuse. The association signal was strongest for rs1009145, rs13148577 (both P=0.0008) and rs7689753 (P=0.0007), whose minor alleles were predicted to alter the target protein sequences involved in mRNA splicing. A pairwise linkage disequilibrium analysis showed that all SNPs except five were located in a single haplotype block. Six haplotype tag SNPs were selected to infer haplotypes and to estimate their frequency distributions. A logistic regression analysis confirmed the association between ADH4 variants and alcohol dependence when sex, age, years of education, marital status and the allele genotype, haplotype and diplotype data of the six haplotype tag SNP were considered. Haplotype ATAAAT, which contained the minor allele of rs10009145 and the major allele of rs7689753, increased the risk of alcohol dependence, whereas haplotype GGGGAT, bearing the major allele of rs10009145 and the minor allele of rs7689753, protected against it. Again, there was no evidence of an association with alcohol abuse. Conclusion These data suggest that ADH4 intronic variants play a role in alcohol dependence susceptibility in Italian populations. Functional studies are needed to establish the role of the genetic variations that seem to affect the splicing mechanism.

Aims Alcoholism is a multifactorial, genetically influenced disorder. It is a major health and social issue, a highly frequent disease and a cause of premature death. It is also the most expensive addictive disorder due to morbidity, mortality, societal and legal problems. Besides their involvement in alcohol-related fatalities, forensic scientists are also required to assess driving and working ability as well as permanent invalidity due to alcohol-related conditions. Greater knowledge of the genetic basis of alcoholism could improve prevention by identifying specific risk factors and mechanisms, leading to effective therapeutic strategies and eventually to personalized treatments. Methods This overview of the recent scientific literature on the genetic basis of alcoholism summarizes the analytical strategies currently applied to the identification of candidate genes involved in alcohol-use disorders (AUDs) and discusses some genes and related phenotypes that have been shown to influence the risk of alcoholism. Results Alcoholism is a complex heterogeneous genetic disease. It is a quantitative disorder, in which the combined incidence of multiple genetic factors and environmental factors varies from one subject to another. Family, twin and adoption studies indicate that 50-60% of the risk of alcoholism is due to genetic factors. Risk loci for AUDs include both genes involved in alcohol pharmacokinetics and pharmacodynamics as well as genes moderating neurophysiological responses such as impulsivity, disinhibition, sensation-seeking and externalizing behaviours. Alcoholism also co-exists with other addictions and psychiatric disorders. Such co-morbidity suggests the existence of shared aetiological factors. Conclusions Despite several genes that influence the risk for AUDs having been identified, the genetic bases of alcoholisn remain largely unknown. Particularly the mechanism of action or the understanding of the physiology of some genes, as well as the gene-environment interactions, is still unknown. Technological progress and advances in transcriptomics, epigenomics and proteomics are expected to enhance our knowledge of the genetic susceptibility to alcoholism.