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.

Chemical investigation of biological trace evidence; toxicological screening of waste residues obtained from DNA extraction processes.

In a forensic scenario, if biological stains are found in very small quantities, these are usually collected for DNA analyses, considered essential for the forensic investigation and thus excluding possible investigations by other forensic disciplines as forensic toxicology. We developed an experimental study to evaluate the feasibility of analyzing DNA extraction residues obtained from DNA extraction procedures to perform toxicological analysis, with the aim to extract both genetic and toxicological information without affecting or compromising the genetic sample and/or DNA extraction. DNA extraction from four blood samples (fortified with 5 molecules of interest with a final concentrations of 1 µg/mL, 100 ng/mL, 10 ng/mL and 5 ng/mL, respectively) were analyzed with QIAGEN QIAmp® DNA Mini kit. Three waste residues collected from the DNA extraction were analyzed for the toxicological investigation via Solid-Phase Extraction and High-Performance Liquid Chromatography-Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). The analytical investigation revealed that our analytes of interest were detected in two different residues of the DNA extraction procedure, allowing both genetic and toxicological analyses without affecting the DNA identification. At last, the experimental protocol was applied to a hypothetical case, with encouraging results and allowing the identification of our molecules of interest.

The contributing factors to suicide in Italian prisons: An 11-year analysis (2010-2020).

BACKGROUND: Suicide is a leading cause of death globally, with approximately 800,000 deaths annually and accounting for 1.5% of all deaths. Risk factors are multifaceted, encompassing individual factors (such as genetics, family history and mental illnesses) and environmental factors (such as economic conditions, social support and life events). In prisons, suicide rates are markedly higher than in the general population, particularly in Italy, where the prisoner suicide rate is approximately 20 times that of the non-incarcerated population. There is, however, little research on suicide in Italian prisons. AIMS: To analyse the characteristics of all people who died by suicide in Italian prisons between 2010 and 2020. METHODS: We carried out a records-based cohort study analysing official data from the Italian Ministry of Justice on prison suicides between 2010 and 2020. The data were cross-referenced and, when required, supplemented with information from Ristretti Orizzonti, a journal specialising in health and living conditions in prisons, as well as from the website of ISTAT (Italian National Statistical Institute), newspapers, radio broadcasts and news agencies. RESULTS: Factors associated with an increased risk of suicide in prisons were nighttime periods, the months of June, July and October, a relatively brief duration of detention (<6 months), having been convicted of murder, male gender, being about 40 years old, having access to hanging materials and being interned (i.e. subjected to the execution of custodial security measures) or awaiting trial. Prison overcrowding was not a risk factor for suicide. CONCLUSION: Our findings hold substantial implications for suicide prevention in Italian prisons as they suggest both characteristics of individuals and characteristics of the institutions that could be taken as risk indicators. This knowledge can inform the development of targeted interventions to manage both individual and environmental factors better, leading to improved prison conditions and reduced suicide rates. Furthermore, our research establishes a foundation for more systematic and in-depth investigations that could further improve suicide prevention strategies in Italian prisons, ultimately influencing policy changes in both practice and research, including perhaps establishing a national database on every completed suicide in prisons.

Telemedicine in diabetic ulcer management: A pilot study with exploration of medico-legal aspects.

BACKGROUND AND AIMS: This study aimed to evaluate the quality and efficiency of telemedicine in managing patients with chronic diabetic ulcers and provide an overview of the main medico-legal implications associated with telemedicine management of diabetic foot complications. METHODS AND RESULTS: A total of 50 patients with type 2 diabetes mellitus were enrolled from a diabetic foot outpatient clinic in Cyprus between March and May 2022. Participants completed a survey concerning telemedicine services, and the demographic variables and average responses to the PACIC (Patient Assessment of Chronic Illness Care) questions were examined using descriptive analysis. The majority of patients (84%) were male, with a mean age of 60.9 ± 13.05 years. The average PACIC score was 4.42 (min. 2.7 and max. 5.0). Voice calls were the most preferred method of telemedicine delivery, accounting for 53% of all services. CONCLUSION: Telemedicine can effectively manage chronic patients, such as those with diabetic ulcers, by reducing the burden on resources and maintaining service quality. However, healthcare professionals must be well-versed in medico-legal implications to adhere to legal and ethical guidelines, protect patient privacy, and maintain high standards of care while using telemedicine for chronic condition treatment.

Applications of microbiology to different forensic scenarios - A narrative review.

In contrast to other forensic disciplines, forensic microbiology is still too often considered a "side activity" and is not able to make a real and concrete contribution to forensic investigations. Indeed, the various application aspects of this discipline still remain a niche activity and, as a result, microbiological investigations are often omitted or only approximated, in part due to poor report in the literature. However, in certain situations, forensic microbiology can prove to be extremely effective, if not crucial, when all other disciplines fail. Precisely because microorganisms can represent forensic evidence, in this narrative review all the major pathological forensic applications described in the literature have been presented. The goal of our review is to highlight the versatility and transversality of microbiology in forensic science and to provide a comprehensive source of literature to refer to when needed.

Proteomic analysis by mass spectrometry of postmortem muscle protein degradation for PMI estimation: A pilot study.

The determination of the postmortem interval is a topic of great forensic interest. The possibility of using new technologies has allowed the study of postmortem decay of biomolecules in the determination of PMI. Skeletal muscle proteins are promising candidates because skeletal muscle exhibits slower postmortem decay compared to other internal organs and nervous tissues, while its degradation is faster than cartilage and bone. In this pilot study, skeletal muscle tissue from pigs was degraded at two different controlled temperatures, 21 °C and 6 °C, and analysed at predefined times points: 0, 24, 48, 72, 96, and 120 h. The obtained samples were analysed by mass spectrometry proteomics approach for qualitative and quantitative evaluation of proteins and peptides. Immunoblotting validation was performed for the candidate proteins. The results obtained appeared significant and identified several proteins useful for possible postmortem interval estimation. Of these proteins, PDLIM7, TPM1, and ATP2A2 were validated by immunoblotting at a larger number of experimental points and at different temperatures. The results obtained are in agreement with those observed in similar works. In addition, the use of a mass spectrometry approach increased the number of protein species identified, providing a larger panel of proteins for PMI assessment.

Informed Consent in Paediatric Telemedicine: Challenge or Opportunity? A Scoping Review.

The fundamental importance of informed consent as a prerequisite for the lawfulness of the medical act is an indisputable cornerstone of clinical practice. However, the provision of effective information and the collection of informed consent presents important critical issues in the underage patient, even considering that in general terms he or she does not have the power to directly express consent, which must be provided by parents or legal guardians. These critical issues are amplified in the context of telemedicine. The present study aims, through a scoping review of the literature of the past 10 years, to outline the operational practices adopted in the collection of informed consent from children in the context of telemedicine and to identify solutions devised to address the critical issues related to the provision of adequate information to the child in this particular care setting. The results of the research show that the activity of delivering adequate information to the child, itself complex, is made even more complex by the particular setting of telemedicine, which, however, could be effectively exploited to facilitate communication with the child patient.

The role of digital literacy in achieving health equity in the third millennium society: A literature review.

Healthcare in the third millennium is largely delivered through systems involving the use of the technological devices and services, foremost among them telemedicine. For the adequate delivery of digital medicine services, however, it is necessary for users to be digitally literate, that is, able to consciously make use of technology. In order to understand how relevant digital literacy is in determining the effectiveness of e-Health services, we performed a traditional literature review on 3 major databases by combining the terms "Digital Literacy" and "Computer Literacy" with the terms "Telemedicine" and "Telehealth". Starting from an initial library of 1,077 papers, we selected 38 articles. At the outcome of the search, we found that digital literacy is a pivotal element in conditioning the effectiveness of telemedicine and digital medicine services in general, however, with some limitations.

Violence against Women and Stress-Related Disorders: Seeking for Associated Epigenetic Signatures, a Pilot Study.

BACKGROUND: Violence against women is a relevant health and social problem with negative consequences on women's health. The interaction between genome and environmental factors, such as violence, represents one of the major challenges in molecular medicine. The Epigenetics for WomEn (EpiWE) project is a multidisciplinary pilot study that intends to investigate the epigenetic signatures associated with intimate partner and sexual violence-induced stress-related disorders. MATERIALS AND METHODS: In 2020, 62 women exposed to violence (13 women suffering from sexual violence and 49 from Intimate Partner Violence, IPV) and 50 women with no history of violence were recruited at the Service for Sexual and Domestic Violence. All women aged 18-65 were monitored for their physical and psychological conditions. Blood samples were collected, and DNAs were extracted and underwent the epigenetic analysis of 10 stress-related genes. RESULTS: PTSD prevalence in victims was assessed at 8.1%. Quantitative methylation evaluation of the ten selected trauma/stress-related genes revealed the differential iper-methylation of brain-derived neurotrophic factor, dopamine receptor D2 and insulin-like growth factor 2 genes. These genes are among those related to brain plasticity, learning, and memory pathways. CONCLUSIONS: The association of early detection of posttraumatic distress and epigenetic marker identification could represent a new avenue for addressing women survivors toward resilience. This innovative approach in gender-based violence studies could identify new molecular pathways associated with the long-term effects of violence and implement innovative protocols of precision medicine.

Elastic fiber degradation as a possible indicator for PMI estimation on mummified and corified skin: a pilot study.

Mummified and corified bodies are particularly complex scenarios to investigate, starting from identifying the post-mortem interval (PMI), even more so in indoor environments. In these bodies, the skin has the peculiar feature to resist for a long time. Among its components, there are elastic fibers, which are characterized by intrinsic resistance to post-mortem degenerative phenomena. Starting from these considerations, we investigated microscopically the persistence, detectability, and changes of elastic fibers in the skin of mummified and corified bodies with different known PMI. The aim was to evaluate whether they could provide an additional tool to aid in PMI estimation in these cases. Therefore, we collected skin samples from mummified or corified bodies found in a domestic environment with different known PMI, as well as from corified bodies that had been exhumed after 11 years of burial. Histochemical staining specific for elastic fibers, namely, Weigert's resorcin fuchsin, showed their prolonged persistence and a progressive and different degradation between mummified and corified skin as a function of PMI. Moreover, on the whole, we observed greater preservation of elastic fibers in mummified skin than in corified one at the same PMI. Therefore, histological analysis of elastic fibers in mummified and corified skin may help to provide valuable aid in estimating PMI, especially in those particular cases where more reliable alternatives are lacking.

Factors affecting mental health of seafarers on board merchant ships: a systematic review.

OBJECTIVES: Merchant ships represent a peculiar working environment with several challenges and risks. The specific situation on board of ships may affect the mental health of seafarers more remarkably than ashore workers. CONTENT: A systematic review of the literature has been carried out to identify the main causes of mood disorders among seafarers and the impact that these disorders have on their health. This review has analyzed the scientific literature published between January 2006 and December 2021 using the search engines PubMed, Web of Science (WoS) and Cumulative Index to Nursing and Allied Health Literature (CINAHL). SUMMARY: Social isolation, distance from families, fatigue, stress and long work shifts represent the main causes of mood disorders among seafarers. OUTLOOK: Strategies aimed at improving conditions of cohabitation on board, and a greater consideration of these problems are key for improving the mental health of workers at sea.

Verbal Autopsy as a Tool for Defining Causes of Death in Specific Healthcare Contexts: Study of Applicability through a Traditional Literature Review.

Autopsy examination, the gold standard for defining causes of death, is often difficult to apply in certain health care settings, especially in developing countries. The COVID-19 pandemic and its associated difficulties in terms of implementing autopsy examinations have made the need for alternative means of determining causes of death even more evident. One of the most interesting alternatives to the conventional autopsy is the verbal autopsy, a tool that originated in Africa and Asia in the 1950s and consists of a structured interview with the deceased's family members concerning the symptoms manifested by the person and the circumstances of death. In the early 1990s, the first doubts emerged about the validity of verbal autopsies, especially about the real reliability of the cause of death identified through this tool. The objective of the review was to identify studies that had assayed the validity of verbal autopsies through a rigorous comparison of the results that emerged from it with the results of conventional autopsies. When starting from an initial pool of 256 articles, only 2 articles were selected for final review. These are the only two original research articles in which a verbal autopsy validation process was performed by employing the full diagnostic autopsy as the gold standard. The two papers reached opposite conclusions, one suggesting adequate validity of verbal autopsy in defining the cause of death and the other casting serious doubts on the real applicability of this tool. Verbal autopsy undoubtedly has extraordinary potential, especially in the area of health and demographic surveillance, even considering the implementation that could result from the use of artificial intelligence and deep learning. However, at present, there appears to be a lack of solid data to support the robust reliability of this tool in defining causes of death.

Pressure Ulcers from the Medico-Legal Perspective: A Case Report and Literature Review.

INTRODUCTION: The identification of professional liability profiles related to the development of pressure injuries is a very thorny issue from a medico-legal perspective. This is because no matter how strict the applied prevention protocols applied may be, the development of such injuries is largely dependent on endogenous factors. This paper aims to investigate the medico-legal issues related to this topic through the exposition of one case of medico-legal litigation and a traditional review of the literature. METHODS: We performed a literature search using three databases (Pubmed, Scopus, and Web Of Science), restricting the search to the period between 2001 and 2021. We used "pressure ulcers" and "jurisprudence" as the main keywords. From an initial library of 236 articles, our selection resulted in 12 articles, which were included in the review. RESULTS: We identified the ever-increasing expectations of patients and the concept of automatic attribution of responsibility when a pressure ulcer develops as the primary reasons for the increase in litigation over the past 20 years. The related corrective measures are numerous: a strict adherence to guidelines, an adequate documentation of preventive measures, a risk assessment, family involvement, and a successful collaboration between physicians and government institutions. CONCLUSIONS: The biological complexity of the pathogenetic development of pressure ulcers makes the subject very delicate from the medico-legal point of view. In principle, it is possible to state that a very large proportion of such injuries are preventable, but that there remains a percentage of them that cannot be prevented. In such cases, only a proper documentary demonstration of the adequacy of preventive measures can exclude liability profiles.

Technical note: A comparison between rehydrating solutions in the pretreatment of mummified and corified skin for forensic microscopic examination.

Microscopic examination of mummified or corified skin may be of extreme importance for forensic purposes. However, standard histological samples in these cases are low-end, and preparation is burdened by several problems and so are diagnostic results: an improvement of these types of specimens is therefore advantageous. This study aims to identify the best performing rehydration solution among a fabric softener, a body lotion, and Sandison's rehydrating solution. Samples of skin undergoing mummification or corification were collected from 25 corpses and each sample was divided into 4 fragments: one of these fragments was directly fixated in 4% formalin, one was previously treated with a tissue softener, another one was previously treated with a body lotion, and the last one was treated with Sandison's solution. After 72 h, the pretreated samples were post-fixated in 4% formalin and then prepared for standard histological examination staining the histological slides with hematoxylin-eosin and Masson's trichrome. At the microscopic examination, samples directly fixated in formalin were characterized by usual marked structural alterations and altered stainability, typical of such dry tissues. Vice versa, those previously treated appeared to be better-preserved even though with different improvement levels: body lotion made a medium-low-grade restoration of the tissues, and fabric softener a high-grade restoration, while Sandison's rehydrating solution produced an optimal grade restoration. Sandison's rehydrating solution was confirmed to be the best rehydrating substance for mummified and corified skin. Fabric softener could be, however, considered a valid substitute, being productive of high-grade microscopic yield.

Use of cadaveric vitreous humor as an innovative substrate for diatoms research and forensic diagnosis of drowning.

The search for diatoms is the test that most of all may contribute to the forensic diagnosis of drowning. Diatoms can be extracted through different methods, which are all complex and long. In this preliminary study, we assessed human vitreous humor as an innovative substrate on which to research diatoms. Sampling and analyses were performed on 3 groups of 10 corpses each. The first one was composed of drowned victims. The second one of victims of traffic accidents with subsequent projection in water; based on the severe traumatic injuries reported at autopsy, we considered such victims as ideally assimilable to bodies immersed in water postmortem. The third group was composed of subjects who died for natural causes and without any relationship with water. The vitreous humor was centrifugated and cytocentrifuged and spotted on two different histological slides: one was left blank and the other one was assessed with hematoxylin and eosin staining. Microscopic observation successfully revealed diatoms in all cases of the first group; in the second group diatoms were not detected in 6 cases; in the third group, no diatoms at all were observed. Diatoms were always qualitatively and quantitatively consistent with those found in the corresponding viscera and waters, which had been investigated through classical acid digestion. Our analyses have demonstrated that the vitreous humor behaves in a completely similar way to the other viscera in cases of drowning. Although further investigations are necessary, vitreous humor has proved to be an innovative, suitable, and reliable substrate for the forensic research of diatoms.

Analysis of the corrosive effects of hydrochloric acid (HCl) on human bone: Preliminary microscopic study and observations for forensic purposes.

The destruction of a corpse in caustic acid can cause complex forensic scenarios to deal with. Furthermore, the literature on the subject is poor, having been the few studies carried out only on animal bones. We carried out an experimental analysis on human cranial, ribs, vertebrae, and femur bone fragments. These samples were sent for dissolution by hydrochloric acid (HCl) at two different concentrations in the lab: 10% and 37%. We have performed macroscopic and microscopic histological and cytological observations at set time intervals: 3, 4, 19, 24, 48, and 72 h of immersion in acids. The purpose of the study was threefold: to investigate the temporal evolution of bone dissolution, evaluate the destructive effect of the two hydrochloric acids, and establish whether or not the human pattern of histological structure could be recognized. A more significant destructive action of HCl at 37% has been observed. In the 10% acid, the bone nature of the samples was demonstrable up to 24 h of immersion, but the human pattern of histological structure was already compromised at 19 h, being lost at 24 h. Instead, in the 37% acid, the bone nature of the sample was demonstrable only within 4 h of immersion, and the human pattern of histological structure was markedly compromised within 3 h. At 19 h of immersion, neither the recognition of the bone nor its human nature was feasible. These preliminary findings and observations may be of practical use in forensic investigations of bodies found in acidic substances, for which there is no scientific evidence to refer.

Iatrogenic femoral nerve injuries: Analysis of medico-legal issues through a scoping review approach.

PURPOSE: Accidental femoral nerve injury is a well-known iatrogenic complication of orthopaedic, abdominal, and pelvic surgery. Because of the largely transitory nature of the symptoms associated with nerve damage, its true incidence is in all likelihood underestimated. This work aims to illustrate the surgical contexts within which this nerve injury is reported, based on the evidence obtained from a Scoping Review of the literature of the last 20 years, with specific reference to the underlying etiopathogenetic mechanisms and prognostic outcomes, to highlight the evaluation issues of medico-legal interest related to this pathology. METHODS: We conducted a Scoping Review of iatrogenic femoral nerve injuries reported between 2000 and 2021 by searching the electronic databases Pubmed, Scopus, Ovid Medline, Ovid Emcare, and Web of Science.We conducted the review according to the five-step methodology outlined by Arksey and O'Malley. RESULTS: The literature search identified 104 papers, including case reports, case series, and retrospective studies. Surgical contexts within which iatrogenic femoral nerve injuries were reported include orthopaedic, abdominal, gynaecological, urological, vascular, and plastic surgery, as well as locoregional anaesthesiological procedures. The long-term prognosis was generally favourable. CONCLUSIONS: Because of its frequent iatrogenic genesis, femoral nerve injury is a topic of intense medico-legal interest. From the perspective of estimating the patient's disability, the mostly favourable nature of the prognosis makes the medico-legal assessment, in some respects, complex, thus requiring a precise evaluation methodology.

Fatal hemorrhage from peripheral varicose vein rupture.

Varix of the lower extremities is a common entity that eventually presents fatal outcome. Fatal massive bleeding due to rupture of a peripheral varicose vein is rare. The estimated incidence of these cases is 1/1000 autopsies. The case we present is unique among 26,054 autopsies performed in Milan from 1993 to 2020. It describes the investigations carried out in the suspicion of a non-natural event in an elderly woman. She was found dead at home with a large volume of blood near her feet that drained from the right leg. Pathological examination disclosed that the hemorrhage occurred by the rupture of a venous varix of the lower limb. Cases of fatal hemorrhage from peripheral variceal rupture are insidious and require proper characterization. The bloodstain pattern analysis, careful autopsy dissection by layers to demonstrate the rupture, and histologic examination of the lesion are the essential elements to find out the actual cause of death.

Uncommon suicide methods in the detention regime in Milan (1993-2019): Forensic contribution on autopsy cases.

We analysed the recorded cases of suicides committed other than by hanging in prison in Milan. A retrospective analysis was conducted on 25,512 autopsies performed from 1993 to 2019, selecting all the suicides in prison but our attention was focused solely on cases where an alternative mode to hanging was used. From a total of 97 suicide events in prison, 15 were consistent with the established search criteria: 6 victims died from plastic bag suffocation, 4 by direct inhalation of butane gas, 2 associated plastic bag suffocation to inhalation of butane gas, one committed suicide with an edged weapon, one by self-burning and, finally, one by voluntary ingestion of a food to which he was allergic, with the intent of inducing an anaphylactic shock. Our analysis has shown that the landscape of prison suicides is diverse, not limited solely to hanging. Therefore, it is necessary for the forensic scientific community to raise awareness of potentially unusual suicide methods in prisons and, in the same way, for the Penitentiary Administration to put adequate preventive measures and strategies in place.

Mortality in Prisons: The Experience of the Bureau of Legal Medicine of Milan (Italy) (1993-2017) : Suicides and natural deaths in prison.

We undertook a retrospective analysis of deaths that took place in prisons in Milan between 1993 and 2017, by identifying cases from a total of 24,101 autopsies that were performed at the Section of Forensic Medicine of the University of Milan. From the archives of this institution, we found 227 autopsy reports relating to deaths that had taken place in one of Milan's three detention facilities. These deaths were divided into two types: natural deaths (n=135; 59.5%) and violent deaths (n=92; 40.5%). The groups have different characteristics: while natural deaths mostly resulted from cardiovascular diseases, suicides were mainly the result of hanging. Further, people who died by suicide often had a history of psychiatric disease and/or drug abuse, and over a quarter of them had previous suicide attempts and/or had declared suicidal intentions. This study confirms the need for good quality healthcare services for prisoners, given that they remain a population at high risk of early death.