Transferability of Human and Environmental Microbiome on Clothes as a Tool for Forensic Investigations.

Considering the growing importance of microbiome analyses in forensics for identifying individuals, this study explores the transfer of the skin microbiome onto clothing, its persistence on fabrics over time, and its transferability from the environment and between different garments. Furthermore, this project compares three specific QIAGEN microbiome extraction kits to test their extraction efficiency on fabric samples. Additionally, this study aims to check if these extracts contain human DNA, providing a chance to obtain more information from the same evidence for personal identification. The results obtained show: (1) variations in the skin microbiome between the volunteers, potentially due to their different sex; (2) differences in microbial composition between worn and unworn clothing; (3) the influence of the environment on the microbial signature of unworn clothing; (4) the potential use of certain phyla as biomarkers to differentiate between worn and unworn garments, even over extended periods; (5) a tendency towards extraction biases in the QIAampMP® DNA microbiome kit among the three tested ones; and (6) none of the extraction kits allow for the typing of human genetic profiles suitable for comparison. In conclusion, our study offers supplementary insights into the potential utility of time-transferred microbiome analysis on garments for forensic applications.

Qualitative Analyses of Textile Damage (Cuts and Tears) Applied to Fabrics Exposed to the Decomposition of Carcasses and Associated Insect Activity in an Austral Summer.

Fatal stabbings are the leading cause of homicide in countries with restricted access to firearms, such as Australia. The analysis of damage on clothing imparted by a sharp object can assist in the characterization of the weapon. However, decomposition and carrion insects can modify the features of the damage, impeding textile damage analysis and crime reconstruction. This study aimed to identify and characterize the modifications of textile damage over 47 days of decomposition during the summer season in Western Australia. Fabric modifications were analyzed on cotton, synthetic, and blended fabrics with standardized cuts and tears, wrapped on 99 stillborn piglets. Six unclothed piglets acted as controls, with three being stabbed. All piglets were placed simultaneously in the field alongside swatches of fabric. Analyses considered taphonomy, insect interactions, and any textile damage using optical microscopy and SEM. The results showed that carrion insects can modify existing cuts and tears and introduce new artifacts on textiles. The 100% cotton fabric was the most affected by mechanical and chemical degradation, especially cuts and areas stained with blood or decomposition fluids. The study highlights the combined effect of multiple factors on textile damage, including the type of fabric, initial damage, bloating, insect activity, and biodegradation.

Corrigendum: Evolution, ecology, and zoonotic transmission of betacoronaviruses: a review.

[This corrects the article DOI: 10.3389/fvets.2021.644414.].

A Summary of Concepts, Procedures and Techniques Used by Forensic Entomologists and Proxies.

Forensic entomology is a branch of forensic science that incorporates insects as a part of solving crime. Insect-based evidence recovered at a crime scene can be used to estimate the minimum postmortem interval, determine if a carcass/corpse has been relocated, and contribute to the cause and manner of death. The current review summarises the stepwise usage of forensic entomology methods at a crime scene and in the laboratory, including specimen collection and rearing, identification, xenobiotic detection, documentation, and referencing previous research and casework. It also provides three standards for the collection of insects when attending a crime scene. The Gold standard attributes to a forensic entomologist (FE) who is likely to be well-trained attending a scene. The subsequent standards (Silver and Bronze) have been added because the authors believe that this information is currently missing in the literature. The purpose is so that an attending crime scene agent/proxy with some basic knowledge and some simple tools can recover almost all the insect information required by an FE to make the best estimation of the minimum postmortem interval.

Corrigendum: Evolution, ecology, and zoonotic transmission of betacoronaviruses: A review.

[This corrects the article DOI: 10.3389/fvets.2021.644414.].

Chasing Flies: The Use of Wingbeat Frequency as a Communication Cue in Calyptrate Flies (Diptera: Calyptratae).

The incidental sound produced by the oscillation of insect wings during flight provides an opportunity for species identification. Calyptrate flies include some of the fastest and most agile flying insects, capable of rapid changes in direction and the fast pursuit of conspecifics. This flight pattern makes the continuous and close recording of their wingbeat frequency difficult and limited to confined specimens. Advances in sound editor and analysis software, however, have made it possible to isolate low amplitude sounds using noise reduction and pitch detection algorithms. To explore differences in wingbeat frequency between genera and sex, 40 specimens of three-day old Sarcophaga crassipalpis, Lucilia sericata, Calliphora dubia, and Musca vetustissima were individually recorded in free flight in a temperature-controlled room. Results showed significant differences in wingbeat frequency between the four species and intersexual differences for each species. Discriminant analysis classifying the three carrion flies resulted in 77.5% classified correctly overall, with the correct classification of 82.5% of S. crassipalpis, 60% of C. dubia, and 90% of L. sericata, when both mean wingbeat frequency and sex were included. Intersexual differences were further demonstrated by male flies showing significantly higher variability than females in three of the species. These observed intergeneric and intersexual differences in wingbeat frequency start the discussion on the use of the metric as a communication signal by this taxon. The success of the methodology demonstrated differences at the genus level and encourages the recording of additional species and the use of wingbeat frequency as an identification tool for these flies.

An unsolved case in a culturally diverse community in Italy.

The number of foreign citizens resident in Italy is approximately 5 million, making them the 8.7% of the resident population of the country. However, over the past 20 years, foreign citizens accounted for 24% of the total number of defendants involved in homicides, especially associated with robbery, disputes and assaults, generally connected to prostitution. Investigations of cases in the context of culturally diverse or minority communities can be particularly complex. This is due to the education, the cultural background and the cohesiveness of the community, alongside a negative view of law enforcement. These factors determine a relatively low resolution rate of these violent crimes in these communities. The investigation of the suspicious death of an Indian citizen living in Italy is presented in this paper. The body was discovered in a cattle shed, and despite the initial assumption of an accidental death, the cranial lesions were not consistent with the view that the death was caused by a kick by a cow. The results of a multidisciplinary analysis, including imaging, pathology, toxicology and veterinary forensics, were directed towards a homicide, with the manner of death ultimately filed as undetermined. During the course of the investigation, the collaboration between law enforcement and the community was thwart with difficulty, and as a consequence this case is currently unsolved. In order for law enforcement to continue to play a constructive and supportive role, it is imperative that every country invest in civil education of local population and of any ethnic groups, minorities and culturally diverse people.

Domestic Filth Flies in New Haven, Connecticut: A Case Study on the Effects of Urbanization and Climate Change by Comparing Fly Populations after 78 Years.

Changes in common and widespread insect populations such as the domestic filth fly in urban cities are useful and relevant bioindicators for overall changes in the insect biomass. The current study surveyed necrophagous flies by placing a weekly trap from June-September over a two-year period in the city of New Haven, Connecticut, to compare data on fly abundance and diversity with data collected 78 years earlier. Climate and land cover changes were also assessed in combination with the fly population for each period. The survey results suggest the domestic filth fly population is now less diverse with decreased species richness and changes in the relative abundance of species. In both surveys, 95-96% of the population was composed of only three species. The current survey data indicate the numerical dominance of Lucilia sericata has decreased, the abundance of several species, notably Lucilia coeruleiviridis, has increased, and Lucilia illustris is absent. Species that showed a significant interaction with temperature in the 1940s survey have now increased in abundance, with several of the trapped species continuing to show an interaction with temperature and rainfall. Analysis of the land cover and climate data characterizes the trap site as a region exposed to a prolonged period of industrialization and urbanization, with only 7% of the land cover remaining undeveloped and over 50% impervious, coupled with an increase in temperature and rainfall. This study serves as a model for changes in domestic filth fly populations and other insects in similarly highly urbanized established cities.

Interim recommendations for the management of forensic investigation during the COVID-19 pandemic: An Italian perspective.

The Coronavirus disease 2019 (COVID-19) is spreading around the world, representing a global pandemic. In this context, governments from around the world suspended almost all education, industry and business activities, alongside restricting the movement of people. Nevertheless, during this period, the activity of the law enforcement and forensic investigators never stopped. At present, guidelines regarding forensic autopsies of SARS-CoV-2 virus-positive cases and the handling of potentially infected biological samples are available in literature. However, less attention has been given to the development of specific adjustments to the existing crime scene investigation protocols and procedures for this exceptional time. This manuscript aims to share the methods and strategies adopted for the investigation of high priority criminal cases during the pandemic. Furthermore, other pandemic-related processes are critically explored, in order to propose adjustments for any forensic services to be prepared to face similar challenges in the future. The overall goal of this manuscript is to provide a summary of the main measures and the procedures developed to make the operations possible, while safeguarding the technicians in the field and the activity in the forensic laboratory. In order to minimize the risk of infection for personnel, adjustments to the standard practice have been proposed for each of the different phases of crime scene management, i.e. CSI call policy, equipment preparation, working groups, procedure at the scene, chain of custody and analyses of the evidence at the forensic lab. As this is a current study, based on limited cases and limited sources in the literature, changes and updates to the indications provided in this paper may be needed in the near future, according to new virological data epidemiological trends.

Evolution, Ecology, and Zoonotic Transmission of Betacoronaviruses: A Review.

Coronavirus infections have been a part of the animal kingdom for millennia. The difference emerging in the twenty-first century is that a greater number of novel coronaviruses are being discovered primarily due to more advanced technology and that a greater number can be transmitted to humans, either directly or via an intermediate host. This has a range of effects from annual infections that are mild to full-blown pandemics. This review compares the zoonotic potential and relationship between MERS, SARS-CoV, and SARS-CoV-2. The role of bats as possible host species and possible intermediate hosts including pangolins, civets, mink, birds, and other mammals are discussed with reference to mutations of the viral genome affecting zoonosis. Ecological, social, cultural, and environmental factors that may play a role in zoonotic transmission are considered with reference to SARS-CoV, MERS, and SARS-CoV-2 and possible future zoonotic events.

A practical review of adipocere: Key findings, case studies and operational considerations from crime scene to autopsy.

After death, the body begins decomposition, a process that starts with the breakdown of organic matter and typically leads to the complete degradation of a body. Such a process is highly affected by (micro and macro) environmental factors of intrinsic and extrinsic nature. Adipocere is a substance formed from the decomposition of adipose tissue and represents a disruption to the typical decomposition process. Such disruption causes decomposition to slow or arrest completely, placing a body into a state of preservation, and determines complications in the estimation of the time since death (Post-Mortem Interval, PMI). While several studies have been performed on the nature, the formation and the degradation of adipocere, there is still no reliable model to assess the PMI of a body exhibiting it. Case studies are an important source to aid pathologists and investigators during a case. This review presents a summary and an update on the knowledge surrounding the chemistry and the factors affecting adipocere formation and degradation, the timing and the distribution of adipocere throughout a body, and the techniques used to investigate it. Furthermore, a table of the most important case studies involving adipocere since 1950, several images and descriptions of recent cases and operational considerations for the best practice at the crime scene and autopsy are presented to be used as a reference to facilitate forensic professionals in adipocere cases.

The effect of submersion in different types of water on the survival and eclosion of blow-fly intra-puparial forms (Diptera: Calliphoridae).

Blow-fly (Diptera: Calliphoridae) immatures are the main colonizers of decomposing remains, and any information on what influences their growth and development are important to forensic entomologists when they are required to estimate post-mortem intervals during a death investigation. Much of this work has been qualified and quantified in terrestrial environments, but is deplete in aquatic environments. When considering a blow-fly's life history, the longest immature life stage goes from the formation of the puparium to adult emergence, and involves metamorphosis. In an aquatic scenario a corpse may be completely submerged, floating on the surface and or it could be associated with water but neither submerged or floating (e.g. beached on a seashore or washed up after a flood event). The present study concerns two blow-fly species, Lucilia sericata (Meigen) and Calliphora vomitoria (L.), and the effects of the age of the intra-puparial forms ("pupal age") and resultant survival, when submerged in tap, river or salt water for varying times - up to 3 days. The experiment was conducted in two localities, L. sericata in Boston USA and C. vomitoria in Turin, Italy, and full puparia of both species were divided into 4 age cohorts ("white", "young", "medium", and "old') before submergence. L. sericata intra-puparial forms showed a three time greater survival rate compared to C. vomitoria intra-puparial forms when submerged in each of the three water types. Both species had the highest survival rate in tap water. Overall, younger and older intra-puparial forms showed a greater and significant survival rate than medium intra-puparial forms when submerged. The eclosion time following submersion of C. vomitoria and L. sericata was mainly influenced by both the age at which the intra-puparial forms were submerged, and by the type of water, but the duration of the submersion also influenced the eclosion time of L. sericata. These results are discussed considering blow-fly physiology. A deeper understanding of the dynamics of survival and growth rate of blow-fly intra-puparial forms on human remains that have undergone a period of submergence could assist in the estimation of the time of death in criminal cases connected to different aquatic environments.

Illuminating the bacterial microbiome of Australian ticks with 16S and Rickettsia-specific next-generation sequencing.

Next-generation sequencing (NGS) studies show that mosquito and tick microbiomes influence the transmission of pathogens, opening new avenues for vector-borne pathogen control. Recent microbiological studies of Australian ticks highlight fundamental knowledge gaps of tick-borne agents. This investigation explored the composition, diversity and prevalence of bacteria in Australian ticks (n = 655) from companion animals (dogs, cats and horses). Bacterial 16S NGS was used to identify most bacterial taxa and a Rickettsia-specific NGS assay was developed to identify Rickettsia species that were indistinguishable at the V1-2 regions of 16S. Sanger sequencing of near full-length 16S was used to confirm whether species detected by 16S NGS were novel. The haemotropic bacterial pathogens Anaplasma platys, Bartonella clarridgeiae, "Candidatus Mycoplasma haematoparvum" and Coxiella burnetii were identified in Rhipicephalus sanguineus (s.l.) from Queensland (QLD), Western Australia, the Northern Territory (NT), and South Australia, Ixodes holocyclus from QLD, Rh. sanguineus (s.l.) from the NT, and I. holocyclus from QLD, respectively. Analysis of the control data showed that cross-talk compromises the detection of rare species as filtering thresholds for less abundant sequences had to be applied to mitigate false positives. A comparison of the taxonomic assignments made with 16S sequence databases revealed inconsistencies. The Rickettsia-specific citrate synthase gene NGS assay enabled the identification of Rickettsia co-infections with potentially novel species and genotypes most similar (97.9-99.1%) to Rickettsia raoultii and Rickettsia gravesii. "Candidatus Rickettsia jingxinensis" was identified for the first time in Australia. Phylogenetic analysis of near full-length 16S sequences confirmed a novel Coxiellaceae genus and species, two novel Francisella species, and two novel Francisella genotypes. Cross-talk raises concerns for the MiSeq platform as a diagnostic tool for clinical samples. This study provides recommendations for adjustments to Illumina's 16S metagenomic sequencing protocol that help track and reduce cross-talk from cross-contamination during library preparation. The inconsistencies in taxonomic assignment emphasise the need for curated and quality-checked sequence databases.

Evaluation of barnacle (Crustacea: Cirripedia) colonisation on different fabrics to support the estimation of the time spent in water by human remains.

The estimation of the time since death (minimum Post Mortem Interval, minPMI) is an essential aspect of forensic investigations. This is particularly complex when a human body is found submerged, floating or beached in a marine environment. When a cadaver is found in a terrestrial environment the minPMI estimation is generally based on the presence of carrion insects. However, when a cadaver is found in an aquatic environment, a correct crime scene reconstruction is more complex and requires the consideration of the time the remains spent submerged underwater (minimum Post Mortem Submersion Interval, minPMSI) and/or floating (Floating Interval, FI). In marine crime scene scenarios, the use of barnacles (Crustacea: Cirripedia) has recently received some attention, due to their permanent settlement on human remains and their accompanying clothing. Previous research considered barnacle growth on human shoes, but the present research is the first to focus on the colonisation of barnacles on clothing materials (fabrics). Polystyrene floats were covered by either cotton, velvet, satin or neoprene and submerged underwater over a period of six months off the coast of Perth, Western Australia. The aims of this research were 1) the identification of marine species colonising the fabrics, with special attention to barnacles; 2) the identification of which fabric type provides the most desirable environment for colonisation; and 3) the identification of factors that affect the growth rate of the different species. Three species of barnacles, Balanus trigonus Darwin, Amphibalanus reticulatus (Utinomi) and A. variegatus (Darwin), were present in varying numbers and sizes. The colonisation process of the barnacles occurred rapidly, with the first sighting of barnacles observed within the first month on neoprene and control floats. The surface that attracted the largest number of barnacles was neoprene, followed by satin and cotton, while velvet showed an inconsistent colonisation rate. The largest size barnacles were observed on the control floats, while all fabrics showed a similar smaller size. Overall, time spent in water and water temperature had a significant positive relationship with both number and size of the colonising barnacles. This study is the first to provide information that will aid in the investigation of human remains recovered from Western Australian marine waters, using the barnacle colonisation on different fabric types.

Transferability of Australian diatoms to clothing: Assessment of several extraction methods on different fabric types under laboratory conditions.

Crime scene investigation protocols and procedures are well established when concerning terrestrial environments, but are limited when associated with aquatic environments. In aquatic contexts, the soil, sediments and microscopic algae (e.g. diatoms) may all be used as sources of trace evidence for criminal investigations. Diatoms are one such source that can be used to support the diagnosis of death by drowning and can be used to verify contact between a suspect and a specific water body. For the latter, diatoms can be collected from clothing and shoes for forensic comparisons. Over the years, there have been several methods proposed for the extraction of diatoms from clothing, however, a best practice method is yet to be established and is still open to debate. The present research represents the first investigation that evaluates four different methods for the extraction of native and common Australian diatom species from four different types of fabrics. Diatoms of two different species and shape (pennate and centric) were cultivated in stimulated experimental waters - prepared using either monocultures or mixed cultures of the selected diatoms. Diatom concentrations were set to mimic the natural population (low to bloom events) as commonly found in the Swan River Estuary of Western Australia. Cotton, denim, blend, and acrylic fabric types were placed in experimental waters. Diatom extraction was attempted using methods already proposed in the literature, these include: rinsing with ethanol (RE); rinsing with ethanol together with the application of centrifugal force (RECF); digestion with hydrogen peroxide (H); and a new method of ashing that has never been tested before (dry ash, DA). Results of this research showed that a) the type and the concentration of the mono and mix diatom mixtures significantly affects the transfer and extraction from different types of fabric; b) a noticeable extraction difference is observed between the fabrics, establishing denim, acrylic and blend fabrics as possessing the better extraction rates; c) H treatment had the highest extraction rate of diatoms; d) the application of centrifugal force on the RE method significantly improved the extraction of diatoms; e) DA is a potential method for the extraction of pennate-shaped diatoms from all of the tested fabrics, however, does have a tendency to induce modifications of the morphological structure of the diatoms. Overall, the application of different extraction methods is suggested to maximise the extraction of diatoms that would closely reflect their natural assemblage in the water body under investigation.

Effect of Temperature and Tissue Type on the Development of the Forensic Fly Chrysomya megacephala (Diptera: Calliphoridae).

Chrysomya megacephala Fabricius is one of the most common myiasis-causing and carrion-feeding fly species in the tropics. This species has a worldwide distribution due to globalization and therefore is of critical importance to the public health sector and forensic investigations. Development studies carried worldwide show that Ch. megacephala reared at similar temperatures may require different amounts of time to complete their cycle of growth. The present study determined for the first time the life-history information of this species in Sri Lanka. Chrysomya megacephala colonies were reared on three different food sources (bovine muscle, swine muscle, and swine liver) considering the most common forensic entomology rearing mediums and the capability of Ch. megacephala to cause myiasis in cattle. Furthermore, colonies were reared at four temperature regimes representing several specific development conditions of this blowfly: 1) 20°C representing the estimation of the lower developmental threshold for this species in Sri Lanka; 2) 25 and 27°C representing typical room/environmental temperatures in Sri Lanka; and 3) 38°C as this is the typical living animal temperature, representing myiasis conditions. Results show that temperature significantly affected larval length and width over time; however, tissue type was not a significant factor. The fastest development was recorded at 38°C for immature feeding on bovine muscle (162.15 h), followed by those fed swine liver (184.15 h) and swine muscle (208.00 h). The calculated minimum temperature threshold for Ch. megacephala was 13°C. Data generated will be crucial for future forensic investigations involving living or deceased individuals colonized by this species.

Barnacle colonization of shoes: Evaluation of a novel approach to estimate the time spent in water of human remains.

Estimating the time since death (minimum Post Mortem Interval, minPMI) is a necessary part of a forensic investigation. Besides considering the typical signs of death, minPMI can be estimated using the insects and other arthropods that colonize the remains (forensic entomology). In an aquatic environment, both insects and crustaceans may provide information regarding the time spent in water of the remains (minimum Floating Interval, minFI and minimum Post Mortem Submersion Interval, minPMSI), and this can also assist in determining the minPMI. Barnacles (Crustacea: Cirripedia) are common crustaceans that colonize solid substrates in marine environments and they can be found in association with organic and inorganic remains recovered from the sea. Barnacles colonize both floating and submerged remains and their growth rate is temperature dependent. Despite their potential to be indicative of the minFI and/or minPMSI, only a few case studies have considered it for this purpose, and scant research has been conducted in this field. Assuming that the vast majority of the bodies found in the sea are clothed, this research is focused on the barnacle colonization of two different types of shoes placed in the sea, in order to (1) identify the colonizing species in the chosen environment; (2) identify the settlement preferences of the barnacles associated with the shoes; and (3) determine the factors affecting the growth rate of the barnacles associated with the shoes. In April 2016 64 sport shoes (SS) and 64 patent leather shoes (PLS) were placed in the Boston Harbor (MA-USA) at 8/10m below sea level. Four of each shoe type were collected every two weeks for seven months. Individual barnacles from each shoe were sampled and measured to determine species and age. The overall colonization density and settlement preference was statistically analyzed. Results show that (a) Amphibalanus improvisus (Darwin) (Crustacea: Cirripedia: Sessilia) colonized the vast majority of shoes; (b) colonization occurred in less than 30 days and continued throughout the research period; (c) a significant difference in colonization densities was found between the SS and PLS, with PLS seeing higher densities; (d) barnacles showed preferential colonization of specific sections on both shoe types; (e) barnacle growth was found to be significantly affected by water temperature and shoe type but not by the time spent in water; (f) time spent in water and shoe type had a highly significant effect on the total number of barnacles per shoe, whereas water temperature did not.

Development and validation of an HPLC-MS/MS method for the detection of ketamine in Calliphora vomitoria (L.) (Diptera: Calliphoridae).

Entomotoxicology is a branch of forensic entomology that studies the detection of drugs or other toxic substances from insects developing on the decomposing tissues of a human corpse or animal carcass. Entomotoxicology also investigates the effects of these substances on insect development, survival and morphology to provide an estimation of the minimum time since death. Ketamine is a medication mainly used for starting and maintaining anesthesia. In recent years ketamine has also been used as a recreational drug, and occasionally as a sedating drug to facilitate sexual assault. In both activities, it has resulted in several deaths. Furthermore, ketamine has been also implicated in suspicious deaths of animals. The present research describes for the first time the development and validation of an analytical method suited to detect ketamine in larvae, pupae, empty puparia, and adults of Calliphora vomitoria L. (Diptera: Calliphoridae), using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This research also considers the effects of ketamine on the survival, developmental rate and morphology (length and width of larvae and pupae) of C. vomitoria. The larvae were reared on liver substrates homogeneously spiked with ketamine concentrations consistent with those found in humans after recreational use (300 ng/mg) or allegedly indicated as capable of causing death in either humans or animals (600 ng/mg). The results demonstrated that (a) HPLC-MS/MS method is applicable to ketamine detection in C. vomitoria immatures, not adults; (b) the presence of ketamine at either concentration in the food substrate significantly delays the developmental time to pupal and adult instar; (d) the survival of C. vomitoria is negatively affected by the presence of ketamine in the substrate; (e) the length and width of larvae and pupae exposed to either ketamine concentration were significantly larger than the control samples.