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.

Utility and diagnostic value of postmortem microbiology associated with histology for forensic purposes.

Nowadays, the diagnostic value of postmortem microbiological investigations is still a debated topic, but postmortem microbiology (PMM) remains a discipline with great forensic potential. To evaluate the usefulness and diagnostic-forensic value of postmortem microbiological cultures, it has been conducted a study on cadaveric material sampled during autopsy aiming to identify the correct cause of death. The study analyzed 45 cadavers subjected to judicial autopsy, divided into two groups based on the presence or absence of external or internal macroscopic autopsy signs suggesting infectious pathology. In the same cases, both the microbiological and conventional histological investigations have been simultaneously carried out. From the investigations, mono-bacterial, mono-fungal, mixed and negative cultures were observed. In mono-species microbiological growth, the histological epicrisis confirmed an infectious cause of death due to the presence of signs of acute infection with an aggressive infectious agent. In cases where growth was mixed, it was possible to distinguish between simple postmortal contamination and perimortem colonization. Finally, in some cases where the microbiology was negative, this has been essential in highlighting signs of a vital reaction to viral or parasitic infection. The joint and integrated evaluation of the laboratory results made it possible to correctly understand even those peculiar situations in which the PMM results alone would not have been significant. These methods, when combined, constitute an optimal forensic approach for the identification of the real cause of death and thus reduce the number of unsolved cases.

Histomorphological aspects of cadaveric skin and its possible use in forensic genetics.

The skin is rarely considered as good biological material for successful DNA typing when a corpse is found in a leathery, mummified or partially skeletonised state, as bones and teeth are the gold standard in these cases. This study evaluates the histomorphological aspects of nuclear chromatin (Lillie's staining) in leathery and mummified skin samples as an indicator for possible successful DNA typing. Chromatin was found in samples that underwent mummification or partial skeletonisation but not in samples in a wet type of post-mortem transformation, such as saponification or leathery transformation. As a preliminary result, a positive detection of DNA profiles was only observed in 1-year-old mummified or partially skeletonised samples. These findings suggest that specific areas of skin, even from severely deteriorated cadavers, can show nuclear chromatin and DNA. These preliminary results raise the potential use of skin samples as an alternative source of DNA in highly degraded corpses.

Cutaneous wound metallic residues pattern analysis with SEM-EDX in a forensic case of overkill.

The term overkill identifies the infliction of massive injuries that greatly exceed those needed to kill the victim both in number and intensity. We present the case of a Peruvian transsexual, who was choked and hit by 11 sharp-force wounds all in the facial area. The scanning electron microscopy with energy dispersion X-ray analysis carried out on the skin of the facial wounds proved crucial in proving that two different point-and-edge weapons were used, following the finding of metallic micro-traces of heterogeneous composition. This discovery and the autopsy highlighted the use of two distinct types of injury (mechanical asphyxia and sharp-force trauma) showed that this murder amounted to overkill.

Fungal aneurism of the right posterior inferior cerebellar artery (PICA).

In this case-report, the Authors show the case of a sudden death occurred in a 38-year-old woman submitted to surgical excision of a right acoustic neurinoma. At the autopsy, was detected a cerebral hemorrhage with multifocal localization by a ruptured rare fungal aneurysm of the Posterior Inferior Cerebellar Arthery (PICA). The PCR analysis, carried out on formalin-fixed paraffin-embedded tissue, identified the Aspergillus Penicillioides as the involved pathogen. We discuss the main points of infectious aneurysms, being a potential neurosurgical complication.

The importance of the postmortem interval for the diagnosis of Waterhouse­Friderichsen syndrome by Neisseria meningitidis in a series of forensic cases

The effective value of microbiological post-mortem examinations stands as fundamental in forensic cases involving microbiology. We ran these analyses on five victims, who suddenly died after showing persistent fever. The examinations were conducted between 48 hours and 10 days after death, and adrenal gland apoplexy was detected in all the cases. Microbiological examinations identified Neisseria meningitidis, which was accountable for Waterhouse­Friderichsen syndrome. Diplococci were isolated from three cadavers that underwent forensic dissection between 2 and 3 days after death. The remaining two cadavers showed polymicrobial contamination, and a polymerase chain reaction technique was necessary to identify the pathogen. We assumed that the microbial overlap could lead to diagnostic mistakes and conceal the identification of the lethal pathogen. Therefore, we suggest using molecular techniques for a postmortem interval (PMI) longer than 72 hours. Classical microbiological examination should be performed for PMI within 72 hours.

The importance of the postmortem interval for the diagnosis of Waterhouse-Friderichsen syndrome by Neisseria meningitidis in a series of forensic cases.

The effective value of microbiological post-mortem examinations stands as fundamental in forensic cases involving microbiology. We ran these analyses on five victims, who suddenly died after showing persistent fever. The examinations were conducted between 48 hours and 10 days after death, and adrenal gland apoplexy was detected in all the cases. Microbiological examinations identified Neisseria meningitidis, which was accountable for Waterhouse-Friderichsen syndrome. Diplococci were isolated from three cadavers that underwent forensic dissection between 2 and 3 days after death. The remaining two cadavers showed polymicrobial contamination, and a polymerase chain reaction technique was necessary to identify the pathogen. We assumed that the microbial overlap could lead to diagnostic mistakes and conceal the identification of the lethal pathogen. Therefore, we suggest using molecular techniques for a postmortem interval (PMI) longer than 72 hours. Classical microbiological examination should be performed for PMI within 72 hours.

Extensive study on electrocution at the Bureau of Legal Medicine of Milan (1993-2017): Determination of the current mark with scanning electron microscope/energy-dispersive X-ray analysis on paraffin-embedded samples.

Literature on electrocution in Italy is limited. The authors have focused their attention on the use of scanning electron microscope/energy-dispersive X-ray analysis in order to evaluate the current mark even on paraffin-embedded samples. A total of 24,104 autopsies were performed at the Section of Legal Medicine of Milan (1993-2017); all cases of death caused by electrocution (low and high voltage) were selected. We assessed a regular histological examination, a toxicological examination and detected metallisation with energy-dispersive X-ray spectroscopy analysis on paraffin-embedded tissues of typical current marks. We collected 27 high-voltage fatalities and five cases of low-voltage suicide electrocution; the technique revealed itself as sensitive. In all cases, microscopic examination highlighted the typical signs of electric current and thermal damage but the histochemical specific stainings for copper and iron gave negative results. Scanning electron microscope/energy-dispersive X-ray analysis, assessed on these same samples, even when paraffin-embedded, however, enabled us to detect these elements and differentiate between the various metal residues involved in the electrocution process. Based on their experience, authors advise the use of scanning electron microscope/energy-dispersive X-ray analysis on electrocution deaths to provide information for forensic diagnosis.

Pulmonary thromboembolism secondary to pelvic thrombosis related to giant ovarian tumor.

Pulmonary thromboembolism (PTE) is one of the major complications in oncologic patients. The incidence of PTE in these cases is 4 to 7 times higher than in non-oncologic patients. Ovarian tumors, specifically those of large sizes, may impair the blood flow through the pelvic veins as tumor pressure over the pelvic vessels increases the incidence of thrombosis. The authors report the case of the unexpected death of a 74-year-old female due to massive pulmonary thromboembolism, associated with an ovarian tumor almost of 15 kg of weight that filled the abdominal and pelvic cavities. The compressive effect on the walls of the pudendal and periuterine veins somehow facilitated the local thrombosis. According to the histological characterization on post-mortem samples, the mass was identified as an "atypical proliferative (borderline) mucinous tumor." The case emphasizes the important association between pulmonary thromboembolism and ovarian tumors.

Pulmonary thromboembolism secondary to pelvic thrombosis related to giant ovarian tumor

Pulmonary thromboembolism (PTE) is one of the major complications in oncologic patients. The incidence of PTE in these cases is 4 to 7 times higher than in non-oncologic patients. Ovarian tumors, specifically those of large sizes, may impair the blood flow through the pelvic veins as tumor pressure over the pelvic vessels increases the incidence of thrombosis. The authors report the case of the unexpected death of a 74-year-old female due to massive pulmonary thromboembolism, associated with an ovarian tumor almost of 15 kg of weight that filled the abdominal and pelvic cavities. The compressive effect on the walls of the pudendal and periuterine veins somehow facilitated the local thrombosis. According to the histological characterization on post-mortem samples, the mass was identified as an "atypical proliferative (borderline) mucinous tumor." The case emphasizes the important association between pulmonary thromboembolism and ovarian tumors

Can cadaverous pollution from environmental lead misguide to false positive results in the histochemical determination of Gunshot Residues? In-depth study using ultra-sensitive ICP-MS analysis on cadaveric skin samples.

INTRODUCTION: In a previous work, we wanted to evaluate if the histochemical determination of lead in Gunshot Residues (GSR) on firearm wounds could be misled due to possible environmental contamination produced by heavy metals and, in particular, by lead. The Sodium Rhodizonate test and its confirmation test with 5% HCl Sodium Rhodizonate resulted to be negative and therefore we wanted to verify if these techniques were sensible enough in order to evaluate this element. We have assessed, on these same samples, a more sensitive technique, as inductive coupled plasma mass spectrometry (ICP-MS) is. This technique is able to detect elements in solution at concentrations as low as 10-15gL-1. MATERIALS AND METHODS: Skin samples taken from two groups of victims, whose cause of death was not related to gunshot wounds were analyzed using ICP-MS: group A included 25 corpses found in open spaces after a long time; group B included 16 corpses exhumed after a period of 11 years. As a positive control group we used skin samples from two subjects that had died due to firearm wounds: as a negative control group we used three different types of plain paraffin slides without included biological material. RESULTS: At the analysis by ICP-MS, the evaluation of the samples belonging to groups A, B and for the negative control groups resulted to be negative for traces of lead (Pb), barium (Ba) and antimony (Sb). On the other hand, high concentrations of GSR could be found in the positive control group were victims died for firearm wounds. CONCLUSIONS: On these basis, we can state that environmental Pb does not contaminate cadavers exposed to open air nor those buried in soil, as confirmed using to ICP-MS technique. Sodium Rhodizonate and 5% HCl Sodium Rhodizonate confirmation test have therefore a high sensitivity, highlighting GSRs, for the diagnosis of death caused by firearm wounds.

The detection of metallic residues in skin stab wounds by means of SEM-EDS: A pilot study.

The morphological analysis of stab wounds may often not be accurate enough to link it with the type of wounding weapon, but a further evaluation may be performed with the search for metallic residues left during the contact between the instrument and the skin. In this study, Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM-EDS) was applied to the study of cadaveric stab wounds performed with kitchen knives composed of iron, chromium and nickel, in order to verify the presence of metallic residues on the wound's edge. Two groups of 10 corpses were selected: group A, including victims of stab wounds and a control group B (died of natural causes). Samplings were performed on the lesions and in intact areas of group A, whereas in group B sampling were performed in non-exposed intact skin. Samples were then analysed with optical microscopy and SEM-EDS. In group A, optical microscopic analysis showed the presence of vital haemorrhagic infiltration, while SEM-EDS showed evidence of microscopic metal traces, isolated or clustered, consisting of iron, chromium and nickel. Moreover, in two cases organic residues of calcium and phosphate were detected, as a probable sign of bone lesion. Control samples (group A in intact areas and group B), were negative for the search of exogenous material to optical microscopy and SEM-EDS. The results show the utility and possible application of the SEM-EDS in theidentification of metallic residues from sharp weapons on the skin.

Technical note: A histochemical approach in diagnosing hanging mechanical asphyxia on cadavers undergoing advanced putrefactive phenomena.

The authors used a particular protocol on putrefied corpses to highlight the cutaneous furrow. Two groups of cadavers were selected: in the first group (suicide by hanging mechanical asphyxia), the authors sampled cutaneous lozenges on the furrow still macroscopically recognisable, while in the second group (corpses deceased by different means), we sampled cutaneous lozenges in the hypostatic leaning. All specimens were divided in two: one underwent standard fixation while the other, previously rehydrated in Sandison's solution, was then fixed in formalin. All specimens were stained with hematoxylin and eosin and Resorcin-Fuchsin. Samples processed with formalin and the H&E staining underlined various artifacts; vice versa, the Sandison solution associated with Resorcin-Fuchsin staining demonstrated the compression of the elastic fibres, with focal positivity in hanged corpses, and diffuse positivity in the entire hypostatic leaning. Sandison's rehydrating solution and Resorcin-Fuchsin staining exalt the supposed application of an asphyctic mean even on putrefied corpses in those cases burdened by a diagnostic doubt.

Observations on self-incineration characteristics in 24 years (1993-2016) of autopsies in the city of Milan.

Self-incineration is one of the most dramatic and lethal suicide methods. It is rarely reported in Western countries and is more frequent in developing regions. We illustrate the forensic cases of self-immolation occurring over 24 years in the city of Milan, Italy, highlighting the main issues of such a complex and rare suicide. We selected 33 cases of self-incineration among 23,417 autopsies (4022 suicides) performed at the Department of Legal Medicine of the University of Milan over a period of 24 years (1993-2016). Several parameters were included and analysed: gender and age of the victims, pathological history, previous suicide intentions/attempts, duration of burning, place of death or discovery of the corpse, circumstantial data of fatal events and autopsy findings, with particular attention to thermal injuries. Self-incineration was found in 0.8% of total suicides and 0.14% of total autopsies. One of these cases involved a complex modality (association with plastic-bag suffocation). The typical characteristics of the victim were an Italian man with psychiatric illness, frequently moved by passion, existential discomfort and economic problems. During the 24-year period, the number of cases of self-incineration progressively reduced. This study provides a general overview in one of the biggest metropolitan Italian areas and is one of the few works performed on this topic. It may be helpful in understanding and studying such an unusual manner of suicide.

Characterization With Scanning Electron Microscopy/Energy-Dispersive X-ray Spectrometry of Microtraces From the Ligature Mean in Hanging Mechanical Asphyxia: A Series of Forensic Cases.

The authors applied scanning electron microscopy with energy-dispersive x-ray spectrometry to the furrow derived from hanging means. The study was conducted with the purpose to detect possible extraneous microtraces, deriving from the ligature, that could have had an interaction with the cutaneous biological matrix, thanks to a transfert mechanism, in the proximities of the lesion.Fifteen cutaneous samples of the furrow and an equal number of fragments of graphite tape, directly positioned on the lesion produced by the ligature mean and used as a "conductor" of possible traces, were analyzed using scanning electron microscopy with energy-dispersive x-ray spectrometry.The research of microscopic traces on the furrow using this technique highlights extraneous traces leading to 3 main categories: natural fabrics, and synthetic and metallic materials, excluding possible environmental pollutants. The analysis, run on 7 hanging deaths, made available by the judicial authority, found a morphological and compositional compatibility with the traces found on the cutaneous furrow produced during hanging.The technique used in this study is innovative in the pathological-forensic field, and can be considered useful in clarifying and studying this typology of asphyxia leading to a specific ligature material, when missing, or attributing the cause of death to hanging when the furrow is not macroscopically obvious.

A particularly complex case of matricide by means of plastic bag suffocation: Case report.

A rare case of homicide with plastic bag suffocation is presented in which forensic genetic investigations were carried out on the inner surface of a plastic bag placed over the head of an elderly woman, bedridden after a stroke. The results obtained suggested that she had been murdered and hinted at the perpetrator of the crime. In fact, it emerged that biological traces left by the victim matched those of her principal caregiver, her psychotic daughter, who later confessed to the crime. The old woman also had a son affected by a serious illness, whose genetic profile was found on the same bag. In a later interview, he stated that his sister had tried to kill him too, thus confirming the genetic findings and allowing the investigators to hypothesise that the daughter had used the same means to try to kill him. Based on these results, the usefulness of forensic genetic testing when investigating complex plastic bag suffocation deaths is highlighted. This peculiar case deserves interest, since no murder of this kind has ever been reported in the Italian forensic literature.

Can cadaverous pollution from environmental lead misguide to false positive results in the histochemical determination of gunshot residues? Study on cadaveric skin samples.

Histochemical determination of lead in gunshot residues (GSR) raises the question of possible environmental contamination by heavy metals. Authors assess the specificity of the Sodium Rhodizonate Test in the search of lead derived from environmental pollution. Sodium Rhodizonate Test and 5% HCl Sodium Rhodizonate test were applied to skin samples taken from two groups of victims, whose cause of death was not related to gunshot wounds: group A included 25 corpses found in open spaces after a long time; group B included 16 corpses exhumed after a period of 11 years. The use of these histochemical tests to skin samples from these two groups did not show metal debris histochemically similar to the lead present in GSR. These tests were confirmed to be specific in highlighting the GSR coming from gunshot, without being affected by the potential environmental contamination of the heavy metal pollutants, coming from air or soil.