Forensic Microbiology: When, Where and How.

Forensic microbiology is a relatively new discipline, born in part thanks to the development of advanced methodologies for the detection, identification and characterization of microorganisms, and also in relation to the growing impact of infectious diseases of iatrogenic origin. Indeed, the increased application of medical practices, such as transplants, which require immunosuppressive treatments, and the growing demand for prosthetic installations, associated with an increasing threat of antimicrobial resistance, have led to a rise in the number of infections of iatrogenic origin, which entails important medico-legal issues. On the other hand, the possibility of detecting minimal amounts of microorganisms, even in the form of residual traces (e.g., their nucleic acids), and of obtaining gene and genomic sequences at contained costs, has made it possible to ask new questions of whether cases of death or illness might have a microbiological origin, with the possibility of also tracing the origin of the microorganisms involved and reconstructing the chain of contagion. In addition to the more obvious applications, such as those mentioned above related to the origin of iatrogenic infections, or to possible cases of infections not properly diagnosed and treated, a less obvious application of forensic microbiology concerns its use in cases of violence or violent death, where the characterization of the microorganisms can contribute to the reconstruction of the case. Finally, paleomicrobiology, e.g., the reconstruction and characterization of microorganisms in historical or even archaeological remnants, can be considered as a sister discipline of forensic microbiology. In this article, we will review these different aspects and applications of forensic microbiology.

The potential of facial nevi in personal identification.

Forensic anthropologists dealing with personal identification (PI) of human remains have recently stressed the need to explore the potential of "secondary identifiers" for identifying victims who died in particular events for whom images often represent the main antemortem data available. Being the face the part most exposed in images, characteristics as pigmented skin lesions (PSLs), can be crucial if combined with other input. Since no data is available on frequencies and distribution of facial PSLs in the general population, this study aims at systematically collecting such data to verify their potential in PI and to open a debate on the aid that "secondary identifiers", regardless of their specific nature, can give to the identification of the deceased in specific forensic contexts. A retrospective analysis on three-dimensional facial models of 1039 Italian subjects (from 4 to 84 years old) was conducted to examine the incidence of PSLs discriminated according to size and position in well-defined facial areas. From the collected data we developed a probabilistic approach providing the likelihood ratio (LR) for two settings: (1) the relative frequencies of nevi in the various facial areas, providing the deriving compound probability of owning a certain facial PSLs pattern; and (2) codes describing the facial nevi pattern of each individual of our population, thus testing their uniqueness and so their potential in PI. The calculated LRs mostly proved high identifying strength, particularly when provided by the compound probability-based approach. Data on incidence and position of facial nevi, their generated codes, and the probabilistic approach here presented, all constitute a starting point for advancing secondary identifiers. Nonetheless, although this preliminary study proved facial PSLs as valuable and potentially useful for identification, their significance and validity should be interpreted with caution as we are still at the first theoretical step clearly based on ideal conditions, and thus further investigations are due on the limitations of their use in practical identifying settings. Therefore, being this systematic study only a preliminary one in its nature, it is recommended not to use this kind of approach until further studies will test its validity in several practical conditions.

Lateralization of the Inferior Alveolar Nerve in the Rehabilitation of Mandibular Atrophy: Case Report.

The lateralization of the inferior alveolar nerve, or LIAN procedure, may be a surgical consideration for implant-prosthetic rehabilitation in the edentulous mandibular posterior region. This technique can be advantageous in that it does not require a donor site and allows for the immediate placement of an implant, potentially leading to reduced morbidity, healing time, and costs. Although such risks as altered sensory nerve function and weakening of the mandibular body are associated with the LIAN procedure, it is a viable alternative to various regenerative techniques to rehabilitate a patient with an implant-supported fixed prosthesis.

Pathological Findings in Hanging: Is the Traditional Knowledge Correct?

BACKGROUND: In forensic pathology, asphyxia presents a problem as frequently as it is thorny and challenging. Some knowledge in forensic pathology is still considered to be traditionally acquired and is not critically assessed with modern statistical or technical tools. In this study, we seek to examine the injuries that are considered to be typical of cases of hanging (neck lesions, haemorrhages, and pleural and epicardial petechiae). METHODS: We evaluated whether there was, indeed, a statistically significant association between these injuries and deaths from hanging as compared to non-hanging deaths. We collected 399 cases (32 deaths by hanging and 367 cases of non-hangings), built contingency tables and performed chi-square tests for each variable (lesion) examined; we also analysed this association in various subgroups of the sample (according to sex, age and weight ranges). RESULTS: Our results did not deviate from the expected outcome based on traditional knowledge, although they do provide a more detailed demonstration and clarification of traditional knowledge regarding this topic. CONCLUSIONS: These findings provide valuable insights for future discussion, examination and deepening of knowledge that is traditionally accepted but often undersupported in the literature.

Age assessment in unaccompanied minors: assessing uniformity of protocols across Europe.

Age assessment of migrants is crucial, particularly for unaccompanied foreign minors, a population facing legal, social, and humanitarian challenges. Despite existing guidelines, there is no unified protocol in Europe for age assessment.The Forensic Anthropology Society of Europe (FASE) conducted a comprehensive questionnaire to understand age estimation practices in Europe. The questionnaire had sections focusing on the professional background of respondents, annual assessment numbers, requesting parties and reasons, types of examinations conducted (e.g., physical, radiological), followed protocols, age estimation methods, and questions on how age estimates are reported.The questionnaire's findings reveal extensive engagement of the forensic community in age assessment in the living, emphasizing multidisciplinary approaches. However, there seems to be an incomplete appreciation of AGFAD guidelines. Commonalities exist in examination methodologies and imaging tests. However, discrepancies emerged among respondents regarding sexual maturity assessment and reporting assessment results. Given the increasing importance of age assessment, especially for migrant child protection, the study stresses the need for a unified protocol across European countries. This can only be achieved if EU Member States wholeheartedly embrace the fundamental principles outlined in EU Directives and conduct medical age assessments aligned with recognized standards such as the AGFAD guidelines.

Why identification matters: an explorative study on six cases of family reunification.

The escalating phenomenon of migration, accompanied by a disturbing surge in associated tragedies, has persistently violated internationally protected human rights. Absence of physical evidence, namely the presence of adequately identified corpses, may impede the full enjoyment of human rights and-in some cases-the course of justice as it obstructs the initiation of legal proceedings against individuals implicated in causing such catastrophes. It also presents administrative obstacles, as death certificates are indispensable in legitimizing statuses like orphanhood and widowhood. Family reunification, particularly for orphans, plays a significant role for those attempting to reconnect with their relatives all over the world. Likewise, for mothers, the acknowledgment of their marital status or widowhood can be a pathway to regain their marginalized right to social life. To elucidate this issue, we analyzed six representative cases from the tragic October 3, 2013, shipwreck near the Italian island of Lampedusa, where 366 individuals were retrieved dead from the sea. These cases underscore the practical challenges involved, highlighting the compelling need for continued efforts to ensure that this burdensome problem transcends from being a mere ethical, moral, and legal discourse. Although considerable progresses, these cases also reveal that substantial work still lies ahead. There is a pressing need for improved mechanisms to certify kinship ties, which are often the limiting factor in many reunifications, and can hinder the granting of custody to children. The severity and far-reaching implications of this problem necessitate thoughtful attention and action, especially considering the ongoing escalation in migration and related fatalities.

Are autopsies on minors a taboo?: The experience of Milan in a 19-year retrospective study.

Forensic autopsy is an important tool for the proper management of non-natural deaths in minors. However, it seems that autopsy in minors is a practice which may not be performed routinely. In this framework, we conducted a study analyzing autopsies of minors (under 18 years of age in Italy) performed at the Institute of Forensic Medicine in Milan in the period 2001-2019. For the period 2015-2019, we extrapolated all deaths due to non-natural causes in minors to investigate how many and which of these deaths were not subjected to forensic autopsy. Of the total, 344 minors (235 males and 109 females) underwent autopsies, with an overall downward trend of about 80% since 2004. Most autopsies occurred between the ages of 0 and 1 year, and the fewest between the ages of 5 and 9 years. The place of death was home in most cases, and accidental death was most common, followed by natural death, suicide, and homicide, with prevalence varying by age group. Blunt force trauma predominated among accidental death in all age groups, followed by asphyxia. Similar findings were observed for suicides, although there was a more differentiated pattern for suicides between the ages of 15 and 17 years. Among homicides, blunt force trauma, asphyxia, and gunshot wounds were fairly evenly distributed across all age groups. Between 2015 and 2019, a total of 86 minors died of a non-natural cause, and a forensic autopsy was performed in only 33 cases (38%). Our data shows that fewer and fewer autopsies are being performed over the last years, which indicates a dangerous lack of forensic investigation of children and adolescent deaths, with enormous implications for prevention of child abuse.

Transcending time: the forensic anthropological case study of three unidentified transgender women in Italy in the early 1990s.

In forensic anthropology, the estimation of skeletal sex results in a dichotomous outcome: male or female. This poses challenges when dealing with transgender individuals. Italy has the highest rate per capita of transgender murders, making their identification a compelling issue. This study presents three cases of unidentified skeletal remains of transgender women (MtF) that underwent autopsies in the 1990s at the Institute of Legal Medicine of Milan. The transgender status of the individuals was determined from the autopsy records which indicated the co-existence of breast implants and male genitalia. The biological profile was created by estimating sex, age, ancestry, and stature, and the bones were investigated looking for evidence of Facial Feminization Surgery (FFS). Anthropological estimations revealed that the skeletons belong to male, adults, European individuals, between 164 and 184 cm. Fragmented gelatinous masses were found in association with two skeletons, which were interpreted as remnants of breast implants. In two cases, signs of remodeling of the cortical surface were observed on the zygomatic bones, although the bone marks observed here were not specific enough to link them to FFS. Despite some limitations, this study highlights the need for greater awareness among practitioners about this limitedly addressed issue, advocating for a more inclusive forensic anthropology that strives to improve methods and interpretation of evidence for the identification of transgender individuals.

Skin surface debris as an archive of environmental traces: an investigation through the naked eye, episcopic microscope, ED-XRF, and SEM-EDX.

Traces from bodies can be of various nature, for example of biological or inorganic origin. Some of these historically have received more consideration than others in forensic practice. Samplings of gunshot residues or biological fluid traces are commonly standardized, whereas macroscopically invisible environmental traces are usually ignored. This paper simulated the interaction between a cadaver and a crime scene by placing skin samples on the ground of five different workplaces and inside the trunk of a car. Traces on samples were then investigated through different approaches: the naked eye, episcopic microscope, Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray spectroscopy (EDX) and Energy Dispersive X-Ray Fluorescence (ED-XRF). The purpose is to provide the forensic scientist with the awareness of the value of debris on skin and then to highlight implications for forensic investigations. Results demonstrated that even naked eye observation can reveal useful trace materials, for defining the possible surrounding environment. As a next step, the episcopic microscope can increase the number of visible particulates and their analysis. In parallel, the ED-XRF spectroscopy can be useful to add a first chemical composition to the morphological data. Finally, the SEM-EDX analysis on small samples can provide the greatest morphological detail and the most complete chemical analysis, although limited, like the previous technique, to inorganic matrices. The analysis of debris on the skin, even with the difficulties due to the presence of contaminants, can provide information on the environments involved in criminal events that can add to the investigation framework.

Consequences of the lack of clinical forensic medicine in emergency departments.

Most victims of physical violence sooner or later will access a hospital or medical cabinet because of that violence, and in particular emergency departments (EDs). This paper aims to analyze the performance of emergency ward clinicians in the forensic management of such victims by examining the activities carried out and the data reported. A total of 991 medical records were extrapolated from the database of the ED of the Policlinico of Milan in an average pre-pandemic 1-year activity. For each medical record, 16 parameters were analyzed in-depth including epidemiological data, information on the type of violent actions, injuries, and time between the infliction of the lesion and access to the ED. In the vast majority of cases, all the actions with medicolegal implications had been neglected by health professionals causing loss of data not only for the justice system but especially for correctly interpreting what happened and taking appropriate measures to protect the patient/victim. Hence, given that clinicians in EDs are busy with non-forensic clinical tasks (and rightly so), it should be ensured that there be specific forensic clinical personnel. However, it is crucial that when unfortunately there can be no forensic staff, at least the clinicians who work in the ED are properly trained to correctly apply essential medicolegal measures. Overall, timely and informed medical and forensic intervention is possible and necessary for the improvement and maintenance of the mental and physical health of victims of violence.

Stable Isotope Provenance of Unidentified Deceased Migrants-A Pilot Study.

In the global migration crisis, one of the challenges in the effort to identify deceased migrants is establishing their region of origin, which facilitates the search for ante mortem data to be compared with the post mortem information. This pilot study explores the potential of using stable isotope analysis to distinguish between individuals coming from West Africa and the Horn of Africa. Six individuals (four of known origin and two of unknown origin) were sampled. δ13CVPDB(keratin), δ15NVPDB(keratin) and δ18OVSMOW(keratin) of hair were analysed using Elemental Analyzers coupled with Isotope Ratio Mass Spectrometry (IRMS). δ18OVSMOW(carbonate) and δ13CVPDB(carbonate) of bone were analysed using GasBench II with IRMS, while 87Sr/86Sr composition was determined in bone and dental enamel using laser ablation multi-collector inductively coupled plasma mass spectrometry. The stable isotope compositions of the individual from the Horn of Africa differed from the other individuals. The differences found between 87Sr/86Sr of enamel and bone and between δ18O and δ13C in bone and hair reflect changes in sources of food and water in accordance with regionally typical migration journeys. The analysis of multiple stable isotopes delivered promising results, allowing us to narrow down the region of origin of deceased migrants and corroborate the information about the migration journey.

Trends in mortality from non-natural causes in children and adolescents (0-19 years) in Europe from 2000 to 2018.

BACKGROUND: Non-natural mortality in children and adolescents is a global public health problem that varies widely from country to country. Data on child and adolescent maltreatment are not readily available, and mortality due to violent causes is also underestimated. METHODS: Injury-related mortality rates (overall and by specific causes) from 2000 to 2018 in selected European countries were analysed to observe mortality patterns in children and adolescents using data from the Eurostat database. Age-standardized mortality rates per 100,000 person-years were calculated for each country. Joinpoint regression analysis with a significance level of 0.05 and 95% confidence intervals was performed for mortality trends. RESULTS: Children and adolescent mortality from non-natural causes decreased significantly in Europe from 10.48 around 2005 to 5.91 around 2015. The Eastern countries (Romania, Bulgaria, Poland, Slovakia, Czech Republic) had higher rates; while Spain, Denmark, Italy, and the United Kingdom had the lowest. Rates for European Country declined by 5.10% per year over the entire period. Larger downward trends were observed in Ireland, Spain and Portugal; smaller downward trends were observed for Eastern countries (Bulgaria, Czech Republic, Poland, Slovakia) and Finland. Among specific causes of death, the largest decreases were observed for accidental causes (-5.9%) and traffic accidents (-6.8%). CONCLUSIONS: Mortality among children and adolescents due to non-natural causes has decreased significantly over the past two decades. Accidental events and transport accidents recorded the greatest decline in mortality rates, although there are still some European countries where the number of deaths among children and adolescents from non-natural causes is high. Social, cultural, and health-related reasons may explain the observed differences between countries.

Toxicological analysis in victims of sexual and domestic violence: A retrospective study of a 3-year period (2018-2020).

This manuscript presents an epidemiological investigation carried out on abuse victims who accessed the Sexual and Domestic Violence Service (SVS&D) of IRCCS Ca' Granda in Milan, Italy. The focal point of this research was the detection of alcohol, prescription medications, and illicit substances in victims who solicited help from the SVS&D center between 2018 and 2020. Over this three-year span, biological samples of blood and urine were procured from 207 victims, out of a patient pool of 2470. All collected samples were analyzed via High Performance Liquid Chromatography - Tandem Mass Spectrometry (HPLC-MS/MS) and Gas Chromatography - Mass Spectrometry (GC-MS). Toxicological examination results demonstrated that 43% of the cases tested positive for substances in 2018, 39% in 2019 and 60% of the cases in 2020. Overall, 45% of the victims tested resulted positive to some substance over a 3-year period, equivalent to 3.6% of the overall cases (2470 victims). Substances of toxicological interest were detected in 104 samples (out of 377, corresponding to 27.6%) belonging to 94 patients. The most detected classes of drugs were stimulants, antidepressants, benzodiazepines and antipsychotics. Moreover, BAC (Blood Alcohol Concentration) indicated positivity in 25 cases (out of 184 cases analyzed - 14% of positive cases). Based on this study's findings, we recommend broadening the range of substances evaluated in drug-facilitated sexual assaults and establishing standardized protocols for both national and international implementation. Implementing procedures would significantly enhance forensic support provided to victims of abuse seeking healthcare services post-incident.

Rumor has it: A narrative review on the use of skeletal non-metric traits and variants for personal identification.

The human skeleton displays an immense array of traits and variant features that are elements of inter-individual variability. The general assumption is that they may represent individualizing markers for the personal identification of unidentified decedents, but very few works consider them as such. This review provides an overview on the possible use of non-metric traits and skeletal variants for personal identification. The paper discusses the issues related to unquantified comparisons, then it presents a statistical approach based on frequencies of these features for identifying unknown remains. Narrowing down an initial number of 1000 papers, the core of the review is represented by 10 papers that considered non-metric traits and skeletal variants as individualizing features, according to both qualitative and quantitative assessments. Despite visual examination remains the gold-standard, more sound methods are requested to quantify the strength of a match or a mismatch. This especially applies in the wake of juridical demands, hence also satisfying the desire of prosecutors and judges to rely on a "quantified" risk. To this purpose, non-metric traits and skeletal variants seem to be a suitable tool to provide quantified evidence, when related frequencies are known.

Assessment of Anatomical Uniqueness of Maxillary Sinuses through 3D-3D Superimposition: An Additional Help to Personal Identification.

Paranasal sinuses represent one of the most individualizing structures of the human body and some of them have been already analyzed for possible applications to personal identification, such as the frontal and sphenoid sinuses. This study explores the application of 3D-3D superimposition to maxillary sinuses in personal identification. One hundred head CT-scans of adult subjects (equally divided among males and females) were extracted from a hospital database. Maxillary sinuses were segmented twice from each subject through ITK-SNAP software and the correspondent 3D models were automatically superimposed to obtain 100 matches (when they belonged to the same person) and 100 mismatches (when they were extracted from different individuals), both from the right and left side. Average RMS (root mean square) point-to-point distance was then calculated for all the superimpositions; differences according to sex, side, and group (matches and mismatches) were assessed through three-way ANOVA test (p < 0.017). On average, RMS values were lower in matches (0.26 ± 0.19 mm in males, 0.24 ± 0.18 mm in females) than in mismatches (2.44 ± 0.87 mm in males, 2.20 ± 0.73 mm in females) with a significant difference (p < 0.001). No significant differences were found according to sex or side (p > 0.017). The study verified the potential of maxillary sinuses as reliable anatomical structures for personal identification in the forensic context.

Comparing Genetic and Physical Anthropological Analyses for the Biological Profile of Unidentified and Identified Bodies in Milan.

When studying unknown human remains, the estimation of skeletal sex and ancestry is paramount to create the victim's biological profile and attempt identification. In this paper, a multidisciplinary approach to infer the sex and biogeographical ancestry of different skeletons, using physical methods and routine forensic markers, is explored. Forensic investigators, thus, encounter two main issues: (1) the use of markers such as STRs that are not the best choice in terms of inferring biogeographical ancestry but are routine forensic markers to identify a person, and (2) the concordance of the physical and molecular results. In addition, a comparison of physical/molecular and then antemortem data (of a subset of individuals that are identified during our research) was evaluated. Antemortem data was particularly beneficial to evaluate the accuracy rates of the biological profiles produced by anthropologists and classification rates obtained by molecular experts using autosomal genetic profiles and multivariate statistical approaches. Our results highlight that physical and molecular analyses are in perfect agreement for sex estimation, but some discrepancies in ancestry estimation were observed in 5 out of 24 cases.

Mass spectrometry-based proteomic strategy for ecchymotic skin examination in forensic pathology.

Mass spectrometry (MS)-based proteomics has recently attracted the attention from forensic pathologists. This work is the first report of the development of a shotgun bottom-up proteomic approach based on rapid protein extraction and nano-liquid chromatography/high-resolution mass spectrometry applied to full-thickness human skin for the differential analysis of normal and ecchymotic tissues to identify new biomarkers for bruise characterization and dating. We identified around 2000 proteins from each pooled extract. The method showed excellent precision on independent replicates, with Pearson correlation coefficients always higher than 95%. Glycophorin A, a known biomarker of vital wounds from immunochemical studies, was identified only in ecchymotic tissues, as confirmed by Western blotting analysis. This finding suggests that this protein can be used as a MS-detectable biomarker of wound vitality. By focusing on skin samples from individuals with known wound dating, besides Glycophorin A, other proteins differentially expressed in ecchymotic samples and dependant on wound age were identified, although further analysis on larger datasets are needed to validate these findings. This study paves the way for an in-depth investigation of the potential of MS-based techniques for wound examination in forensic pathology, overcoming the limitations of immunochemical assays.