Case report on Cochliomyia hominivorax (Coquerel, 1858) and Co. macellaria (Fabricius, 1775) (Calliphoridae,Diptera) myiasis prior to death: Implications on postmortem interval estimation

The most important contribution of forensic entomology is the estimation of the postmortem interval (PMI) or time since death. The forensic entomologist assumes that the biological clock of necrophagous insects that develop by feeding on cadaver tissues begins when the victim's biological clock is turned off. However, tissues can be invaded while the host is still alive (called myiasis), so the period of activity of necrophagous insects would not be indicative of PMI. The aim of this work was, through the presentation of a case report, to highlight the importance of expertise in the identification of necrophagous species associated with a corpse and their type of relationship in order to avoid an erroneous estimation of PMI. The corpse of a woman, missing for 14 days, was found outdoors in a small river 15 cm deep. Her corpse had numerous lesions infested with dipteran larvae that were collected during autopsy. Entomological evidence included second and third instar larvae of Cochliomyia hominivorax and Co. macellaria. The obligate parasitic nature of Co. hominivorax as a primary producer of myiasis and secondary of Co. macellaria allowed us to establish the time when the victim was still alive and from there to estimate the PMI.     

Validation of temperature methods for the estimation of pre-appearance interval in carrion insects

The pre-appearance interval (PAI) is an interval preceding appearance of an insect taxon on a cadaver. It decreases with an increase in temperature in several forensically-relevant insects. Therefore, forensic entomologists developed temperature methods for the estimation of PAI. In the current study these methods were tested in the case of adult and larval Necrodes littoralis (Coleoptera: Silphidae), adult and larval Creophilus maxillosus (Coleoptera: Staphylinidae), adult Necrobia rufipes (Coleoptera: Cleridae), adult Saprinus semistriatus (Coleoptera: Histeridae) and adult Stearibia nigriceps (Diptera: Piophilidae). Moreover, factors affecting accuracy of estimation and techniques for the approximation and correction of predictor temperature were studied using results of a multi-year pig carcass study. It was demonstrated that temperature methods outperform conventional methods. The accuracy of estimation was strongly related to the quality of the temperature model for PAI and the quality of temperature data used for the estimation. Models for larval stage performed better than models for adult stage. Mean temperature for the average seasonal PAI was a good initial approximation of predictor temperature. Moreover, iterative estimation of PAI was found to effectively correct predictor temperature, although some pitfalls were identified in this respect. Implications for the estimation of PAI are discussed.     

Temperature-dependent appearance of forensically useful flies on carcasses

Flies are frequently used for postmortem interval (PMI) estimations. These estimates are usually based on the age of larval or pupal specimens. However, the age defines only the minimum PMI. In order to move forensic entomology further, a method useful for the estimation of an interval preceding insect appearance on a corpse called the pre-appearance interval (PAI) is needed. Recently, it was demonstrated that the PAI of several carrion beetles is closely related to the temperature prevailing throughout this interval. Hence, it was postulated to estimate PAI from temperature. In order to check premises for using this approach with flies, a test of the relationship between adult or oviposition PAI and temperature was made for nine species of European flies. Data on PAI originated from pig carcasses decomposing under various temperatures. Adult PAI of Hydrotaea dentipes, Hydrotaea ignava, Hydrotaea similis, Phormia regina, and Stearibia nigriceps and oviposition PAI of S. nigriceps were exponentially related to temperature. Only S. nigriceps revealed a close relationship, demonstrating solid premises for PAI estimation from temperature alone. Adult and oviposition PAI of Calliphora vomitoria and adult PAI of Hydrotaea pilipes were not related to temperature. Adult and oviposition PAI of Lucilia sericata and Lucilia caesar responded similarly, with an abrupt and large increase in a narrow range of low temperatures and no response in a broad range of high temperatures. Probably, different mechanisms form the basis for the response of PAI to temperature in flies colonizing carcasses shortly after death and flies colonizing carcasses later in the decomposition process.     

Taphonomic model of decomposition

After death human body is subject to the processes of autolysis and putrefaction. Notably, the changes in cadaver during decomposition complicate its forensic analysis and particularly the estimation of the post-mortem interval (PMI). The process and rate of decomposition is impacted by various intrinsic and extrinsic factors that vary across countries and regions. Studying the decomposition pattern in different regions in the world helps us to understand the process and improve the precision of the PMI estimation of decomposed bodies. With the aim to develop a taphonomic model of decomposition in the province of Barcelona (Catalonia, Spain), this study analyses the influence of several intrinsic and extrinsic factors in the pattern and rate of decomposition in this geographical area. Our statistical model concluded that the most significant factors affecting the decomposition pattern and rate are temperature and PMI. Nevertheless, there are other intrinsic factors such as cause, manner of death and underlying pathological conditions which also have an important role. Moreover, considering the various variables studied in this research, two predictive machine learning algorithms were developed as a probabilistic approach to estimate the PMI. Reliable classification results are obtained for three interval groups (1–2 days, 3–10 days, and > 10 days) and two interval groups (>1 week, < 1 week). Machine learning algorithm is a promising tool to gain objectivity in forensic PMI assessments. The results of this study could potentially assist further research in forensic taphonomy.     

A case of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae) breeding in a human corpse in Calabria (southern Italy)

Forensic entomology is the study of insects associated with human corpses. The most common and widely accepted application of entomological evidence in criminal investigations is to determine the post-mortem interval (PMI), i.e. the time from death to discovery of the corpse, and other circumstances of the crime scene. The insects that first colonize a human corpse usually belong to the families Calliphoridae, Sarcophagidae and Muscidae, and often are very important elements in forensic investigation. The aim of this study was to highlight the importance of evaluating forensic evidence on a regional and urban scale. To assess the climatic, geographical and environmental influences on carrion-breeding fauna, we present details of a case in Calabria (southern Italy) in February 2007. The entomological evidence consisted only of Calliphora vicina Robineau-Desvoidy, 1830. The presence of this species is related to the climatic and micro-environmental conditions in the urban habitat of Cosenza, southern Italy, and to the location and status of the cadaver.     

Luminol chemiluminescence: contribution to postmortem interval determination of skeletonized remains in Portuguese forensic context

Postmortem interval (PMI) determination is one of the main challenges of forensic anthropology, and there are several referenced methods that consider intrinsic and extrinsic factors to the skeletonized remains. Therefore, there is an important need to develop a precise, economic, easy and reproducible technique, which will operate as a presumptive test. The chemical reaction that occurs in the presence of luminol, chemiluminescence, is used as a work tool to evaluate the PMI. This is given by luminol high affinity for haemoglobin, which is secured in bone tissue by its high mineral content, after the blood supply ceases postmortem. We evaluated a luminol-based technique, in which we measured the chemiluminescence of skeletonized remains as an indicator of the PMI. We assessed the usage of this technique for PMI estimation and for deciding whether remains have forensic interest, particularly in the Portuguese medicolegal context. Additionally, we evaluate the reproducibility of this technique. The test sample consists of 50 skeletonized individuals of both sexes, adults, between 20 and 98 years, with a known time since death and without bone pathologies. The results of Kappa statistic, with values between 0.79 and 0.97, showed high interobserver and intraobserver agreement when applying the luminol test as a presumption test. We concluded that this technique alone is not precise and of limited value to determine forensic relevance in the Portuguese context. However, it can be used as a presumptive test, reducing time and costs in criminal investigation.

     

Age estimation during the blow fly intra-puparial period: a qualitative and quantitative approach using micro-computed tomography

Minimum post-mortem interval (_minPMI) estimates often rely on the use of developmental data from blow flies (Diptera: Calliphoridae), which are generally the first colonisers of cadavers and, therefore, exemplar forensic indicators. Developmental data of the intra-puparial period are of particular importance, as it can account for more than half of the developmental duration of the blow fly life cycle. During this period, the insect undergoes metamorphosis inside the opaque, barrel-shaped puparium, formed by the hardening and darkening of the third instar larval cuticle, which shows virtually no external changes until adult emergence. Regrettably, estimates based on the intra-puparial period are severely limited due to the lack of reliable, non-destructive ageing methods and are frequently based solely on qualitative developmental markers. In this study, we use non-destructive micro-computed tomography (micro-CT) for (i) performing qualitative and quantitative analyses of the morphological changes taking place during the intra-puparial period of two forensically relevant blow fly species, Calliphora vicina and Lucilia sericata, and (ii) developing a novel and reliable method for estimating insect age in forensic practice. We show that micro-CT provides age-diagnostic qualitative characters for most 10% time intervals of the total intra-puparial period, which can be used over a range of temperatures and with a resolution comparable to more invasive and time-consuming traditional imaging techniques. Moreover, micro-CT can be used to yield a quantitative measure of the development of selected organ systems to be used in combination with qualitative markers. Our results confirm micro-CT as an emerging, powerful tool in medico-legal investigations.

     

Decomposition variability between the scene and autopsy examination and implications for post-mortem interval estimations

Forensic experts rely on scene and/or autopsy photographs to estimate the post-mortem interval (PMI) when an in-situ assessment of decomposition is unfeasible. The degree of decomposition may vary between the scene and autopsy, which importantly could affect estimations of the unknown PMI in forensic casework. This study aimed to investigate decomposition variability between the scene and autopsy and assess the subsequent effect on the accuracy of PMI estimations. Scene and autopsy photographs from 94 cases with known PMI were used from the Allegheny County Office of the Medical Examiner in Pittsburgh, United States. The total decomposition scoring (TDS) method measured the overall decomposition level, and 28 markers of decomposition were recorded as a percentage of the total body surface area (TBSA). In 60% of cases the TDS had increased at autopsy causing significant overestimations of the autopsy PMI and 86% of decomposition markers varied between the scene and autopsy. Decomposition progressed during mortuary time lags (MTL) of 3–44 h, where bodies were stored in a pre-autopsy refrigerator at 4 °C, suggesting that refrigeration may not always delay decomposition. This research also assisted in validating photographs as a proxy for real-time decomposition assessments. While the autopsy photographs conferred higher quality than the scene photographs, the scene photographs produced more accurate PMI estimations. Forensic experts should exhibit caution when estimating the PMI from autopsy photographs alone, as they may not accurately reflect scene decomposition. To prevent misinterpretation of the PMI estimation, both scene and autopsy photographs should always be requested.     

A novel method for determining postmortem interval based on the metabolomics of multiple organs combined with ensemble learning techniques

International Journal of Legal Medicine - Determining postmortem interval (PMI) is one of the most challenging and essential endeavors in forensic science. Developments in PMI estimation can take...     

A standard protocol for the analysis of postmortem muscle protein degradation: process optimization and considerations for the application in forensic PMI estimation

The analysis of postmortem protein degradation has become of large interest for the estimation of the postmortem interval (PMI). Although several techniques have been published in recent years, protein degradation-based techniques still largely did not exceed basic research stages. Reasons include impractical and complex sampling procedures, as well as highly variable protocols in the literature, making it difficult to compare results. Following a three-step procedure, this study aimed to establish an easily replicable standardized procedure for sampling and processing, and further investigated the reliability and limitations for routine application. Initially, sampling and processing were optimized using a rat animal model. In a second step, the possible influences of sample handling and storage on postmortem protein degradation dynamics were assessed on a specifically developed human extracorporeal degradation model. Finally, the practical application was simulated by the collection of tissue in three European forensic institutes and an international transfer to our forensic laboratory, where the samples were processed and analyzed according to the established protocol.

     

Reliability of weathering in the estimation of the post-mortem interval of human remains buried in coffins

Although the post-mortem interval (PMI) is essential in forensic sciences, dating skeletonized remains is a complex task for forensic scientists, not only because of the lack of reliable methods that currently exist for its estimation but also due to the variability of the cadaveric decomposition process, which involves a series of factors. Weathering and breakdown of remains progresses with time, from cracking to loss of shape, and is accepted to occur in observable stages. When dealing with buried remains, these processes will take longer because many of the most destructive agents will be shut out. Burial represents a unique environment which has not been examined often. This study analysed bone weathering (weight loss, grease, degradation, fractures and flaking) of the atlas, axis, humerus, femur and first metatarsal of 86 individuals recovered from a Portuguese cemetery plot. Sex, age-at-death, burial period and storage period were taken into account, but several problems may have affected the outcome of the study. However, PMI estimation based on weathering is not reliable, confirming that burial contexts create distinct micro-taphonomic environments.     

Intra- and intermuscular variations of postmortem protein degradation for PMI estimation

International Journal of Legal Medicine - In recent years, protein decomposition has become of increasing interest for the use in forensic estimation of the postmortem interval (PMI). Especially...     

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.

     

Metabarcoding to investigate changes in soil microbial communities within forensic burial contexts

The estimation of the time elapsed since death (post-mortem interval, or PMI) is one of the key themes that forensic scientists have to address frequently. However, the estimation of PMI still suffers from poor accuracy and biases especially when decomposition stages are prolonged, so further improvements in methods for PMI estimation are desirable. Soil microbial communities associated with decomposing bodies have been shown to be good candidates for the estimation of the PMI of exposed bodies. Nevertheless, further research is required to better understand the bacterial succession associated with decomposition of buried carcasses in order to test its reliability and applicability for the estimation of PMI and to better understand the dynamics involved with decomposition within this particular scenario. Therefore we explored the succession of soil microbial communities associated with four decomposing pig carcasses (from one to six months PMI) using a metabarcoding approach. The sequencing of the bacterial 16S rRNA variable region 4 (V4) revealed trends linking particular microbial taxa with specific PMIs, and notably an increase in Proteobacteria, Firmicutes and Bacteroidetes at specific PMIs as well as a decrease in Acidobacteria. Our results, in accordance with previous studies conducted on exposed bodies of different mammalian species (including humans), also showed a general reduction of the taxonomic richness from two months PMI onwards, as well as an incomplete re-establishment of the starting soil microbial conditions after six months PMI. We also found specific mammal-derived taxa, such as Bacteroides spp., being still present in the soil after six months PMI. As such, this study serves as a baseline for additional research to allow the characterisation of biomarkers associated with specific PMIs. Due to the similarity between the results presented here and those reported in other types of decomposition studies we believe that the metabarcoding approach has considerable potential in the estimation of the PMI, particularly to clarify cases involving heavily skeletonised bodies or for the investigation of clandestine graves in which the carcass has been moved from its original place of deposition.     

The micro-CT evaluation of enamel-cement thickness,abrasion, and mineral density in teeth in the postmortem interval (PMI): new parameters for the determination of PMI

International Journal of Legal Medicine - The estimation of time of death or the determination of the postmortem interval (PMI) is one of the most important issues in forensic medicine and...     

Insects' colonization of human corpses in warm and cold season

The succession of insects is commonly used for estimation of the postmortem interval (PMI). In the warm season there are many insect species, which enable us to estimate the time of death, but in the cold season there are less species. For the estimation of a seasonal pattern, we examined 117 human corpses found in domestic environment during the year 2001. We found three typical 'summer species', active from May until October, three further species active from April until November. One blowfly species (Calliphora vicina) and the family of scuttle flies were active all over the year. These insects may be useful for PMI estimation in winter; however, further research concerning the developmental rate of scuttle flies is required. The distinct seasonal pattern of the early colonizing blowfly species allows to allocate the time of death to a particular time interval even in cases of long PMIs if empty puparia of these insects are found.     

Thick quilt may severely impact the estimation of postmortem interval using forensic entomology-based methods -two case reports

This paper introduces two indoor cases in China, characterized by bodies lying on a mattress, covered with thick quilts and wearing clothes. There were obvious deviation in the estimated minimum postmortem interval (PMI_min) of the corpses using entomological methods. Based on the forensic entomology evidence from the scene, the PMI_min estimated using temperature data from the nearest weather station was longer than the actual postmortem interval (PMI) based on the police investigation and the security camera footage. The most probable cause of the errors in PMI_min estimation was the hindrance in heat dissipation since the corpses were covered with thick quilts while lying on the mattress. Therefore, the heat generated by the decomposition process and larval activity was hard to lose, resulting in the rapid development of insects. These case reports emphasize the importance of temperature collection in forensic entomological investigations. Our findings call for standardized temperature acquisition procedures, including which temperature measurements (body, microenvironment or ambient temperature) should be used in forensic entomological investigations when handling similar cases.     

The changes of attenuated total reflection-Fourier transform infrared after rat’s death under different humidity conditions

Estimation of the time since death (post-mortem interval, PMI) is one of the most difficult problems in forensic investigations and many methods currently are utilized to estimate the PMI. The goal of this study was to investigate the changes of attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra of rat tissues from post-mortem time zero to 6 days in 40% and 80% humidity conditions. The spectra of rat tissues displayed similar change tendencies with increasing post-mortem interval in 40% and 80% humidity conditions, and the changes were more obvious in 80% humidity. Our study further confirmed that humidity was a factor affecting the estimation of PMI.     

A preliminary study on early postmortem submersion interval (PMSI) estimation and cause-of-death discrimination based on nontargeted metabolomics and machine learning algorithms

Postmortem submersion interval (PMSI) estimation and cause-of-death discrimination of corpses in water have long been challenges in forensic practice. Recently, many studies have linked postmortem metabolic changes with PMI extension, providing a potential strategy for estimating PMSI using the metabolome. Additionally, there is a lack of potential indicators with high sensitivity and specificity for drowning identification. In the present study, we profiled the untargeted metabolome of blood samples from drowning and postmortem submersion rats at different PMSIs within 24 h by liquid chromatography–tandem mass spectrometry (LC–MS/MS). A total of 601 metabolites were detected. Four different machine learning algorithms, including random forest (RF), partial least squares (PLS), support vector machine (SVM), and neural network (NN), were used to compare the efficiency of the machine learning methods. Nineteen metabolites with obvious temporal regularity were selected as candidate biomarkers according to “IncNodePurity.” Robust models were built with these biomarkers, which yielded a mean absolute error of 1.067 h. Additionally, 36 other metabolites were identified to build the classifier model for discriminating drowning and postmortem submersion (AUC = 1, accuracy = 95%). Our results demonstrated the potential application of metabolomics combined with machine learning in PMSI estimation and cause-of-death discrimination.