Immunological Patient Stratification in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex disease characterized by profound fatigue, post-exertional malaise (PEM), and neurocognitive dysfunction. Immune dysregulation and gastrointestinal symptoms are commonly observed in ME/CFS patients. Despite affecting approximately 0.89% of the general population, the underlying pathophysiological mechanisms remain poorly understood. This study aimed to elucidate the relationship between immunological characteristics and intestinal barrier function in ME/CFS patients. ME/CFS patients were stratified into two groups based on their immune competence. After documentation of detailed medical records, serum and plasma samples were collected for the assessment of inflammatory immune mediators and biomarkers for intestinal barrier integrity by ELISA. We found reduced complement protein C4a levels in immunodeficient ME/CFS patients suggesting a subgroup-specific innate immune dysregulation. ME/CFS patients without immunodeficiencies exhibit a mucosal barrier leakage, as indicated by elevated levels of Lipopolysaccharide-binding protein (LBP). Stratifying ME/CFS patients based on immune competence enabled the distinction of two subgroups with different pathophysiological patterns. The study highlights the importance of emphasizing precise patient stratification in ME/CFS, particularly in the context of defining suitable treatment strategies. Given the substantial health and socioeconomic burden associated with ME/CFS, urgent attention and research efforts are needed to define causative treatment approaches.

Decomposition and insect succession of pig cadavers in tents versus outdoors - A preliminary study.

The pattern of insect succession and the species composition on cadavers can be influenced by several parameters, one of which is concealment. It has been previously shown for cadavers inside containers (e.g. suitcases or vehicles) or various indoor situations, that concealment can lead to a delayed arrival, altered species composition and decreased number of taxa present at the cadaver. As no data is yet available for the specific environment of tents on these processes, we placed five pig cadavers inside closed two-person tents during the summer of 2021 in a mixed forest in Germany. Five control cadavers were freely accessible for insects. To minimize disturbances, tents were only opened every fifth day over a period of 25 days for estimating the temperature profiles, insect diversity and quantification of the cadavers' decomposition using the total body score (TBS). The temperature inside the tents was only slightly elevated compared to the ambient temperature during the study. Although the tents prevented direct access of adult flies and beetles, the cadavers were colonized because the flies oviposited on the zippers and fly screens of the inner tents. However, the resulting infestation of the cadavers by fly larvae was reduced and delayed, compared to the exposed cadavers. The dominant fly species on both the tent and exposed cadavers was the blow fly Lucilia caesar. Open cadavers showed the expected decomposition patterns with large larval masses. 25 days after placement, only bones and hair remained from the exposed pigs (TBS = 32), while most of the tissue of the cadavers inside the tents was still present (TBS = 22.5) and post feeding larvae were not able to leave the tents. Regarding the attraction of beetles to both treatments, open cadavers were predominately colonized by the silphid Oiceoptoma thoracicum, while the silphid Necrodes littoralis was the most dominant species in the pitfall traps surrounding the tents. Considering the prolonged pre-appearance interval of fly larvae colonizing the cadavers inside tents, the entomological evidence of forensic cases dealing with concealed bodies should be handled with caution, since the post mortem interval might be significantly underestimated.

Cuticular hydrocarbons for the identification and geographic assignment of empty puparia of forensically important flies.

Research in social insects has shown that hydrocarbons on their cuticle are species-specific. This has also been proven for Diptera and is a promising tool for identifying important fly taxa in Forensic Entomology. Sometimes the empty puparia, in which the metamorphosis to the adult fly has taken place, can be the most useful entomological evidence at the crime scene. However, so far, they are used with little profit in criminal investigations due to the difficulties of reliably discriminate among different species. We analysed the CHC chemical profiles of empty puparia from seven forensically important blow flies Calliphora vicina, Chrysomya albiceps, Lucilia caesar, Lucilia sericata, Lucilia silvarum, Protophormia terraenovae, Phormia regina and the flesh fly Sarcophaga caerulescens. The aim was to use their profiles for identification but also investigate geographical differences by comparing profiles of the same species (here: C. vicina and L. sericata) from different regions. The cuticular hydrocarbons were extracted with hexane and analysed using gas chromatography-mass spectrometry. Our results reveal distinguishing differences within the cuticular hydrocarbon profiles allowing for identification of all analysed species. There were also differences shown in the profiles of C. vicina from Germany, Spain, Norway and England, indicating that geographical locations can be determined from this chemical analysis. Differences in L. sericata, sampled from England and two locations in Germany, were less pronounced, but there was even some indication that it may be possible to distinguish populations within Germany that are about 70 km apart from one another.

It is all about the insects: a retrospective on 20 years of forensic entomology highlights the importance of insects in legal investigations.

This study highlights the importance of insect evidence by evaluating 949 insect-associated cases, including 139 entomological reports, from 2001 to 2019 at the Institute of Legal Medicine Frankfurt/Germany. With a high number of cases in the summer months and a low number in the colder season, 78.5% of the bodies were found indoors, regardless of year or month. In more than 80% of the cases, where PMI information was available (n = 704), the presumed PMI ranged from 1 to 21 days, a period during which entomological evidence can provide a day-specific estimate of PMImin. In cases where insects have been identified to species level (n = 279), most bodies were infested by one or two species with a maximum of 10 different species. Overall, a total of 55 insect species were found. Information on biology, activity and distribution of the most abundant taxa is given and applied for 5 case histories estimating different PMImins of up to over 6 months. Despite proved importance and scientific development of forensic entomology, insects are still rarely considered as a tool in forensic case work. The main reasons are a lack of awareness and (too) late involvement of a forensic entomologist. Our work shows that forensic entomology is an independent discipline that requires specialist expertise.

Evaluation of Immune Dysregulation in an Austrian Patient Cohort Suffering from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a severe multi-systemic disease characterized by debilitating fatigue that is not relieved by rest. The causes of the disease are still largely unexplained, and no causative treatment is currently available. Changes in the immune response are considered as fundamental in the development of ME/CFS. Thus, we aimed to evaluate the immunological profile of ME/CFS patients in a retrospective data analysis. As part of the routine workup for ME/CFS patients, a differential blood count, leukocyte subtyping, and quantification of immunoglobulins and IgG subclasses, as well as a complement analysis, was performed. Out of 262 ME/CFS patients, 64.9% had a reduction or deficiency in at least one of the listed immune parameters. In contrast, 26.3% showed signs of immune activation or inflammation. A total of 17.6% of the ME/CFS patients had an unclassified antibody deficiency, with IgG3 and IgG4 subclass deficiencies as the most common phenotypes. Reduced MBL (mannose-binding lectin) levels were found in 32% of ME/CFS patients, and MBL deficiency in 7%. In summary, the present results confirmed the relevance of immune dysfunction in ME/CFS patients underlining the involvement of a dysfunctional immune response in the disease. Thus, immune parameters are relevant disease biomarkers, which might lead to targeted therapeutic approaches in the future.

To Be There or Not to Be There, That Is the Question-On the Problem of Delayed Sampling of Entomological Evidence.

The aim of the current study was to analyze two major pitfalls in forensic entomological casework: delayed evidence sampling and the effect of low-temperature storage of the body. For this purpose, temperature profiles of heavily infested corpses during cooling and cases in which insect evidence was collected both at the scene and during autopsy were evaluated with regard to species composition and development stages found. The results show that the temperature in the body bags remained at higher average temperatures up to 10 °C relative to the mortuary cooler, therefore, sufficient for larval development, with significant differences in temperature between larval aggregations on one and the same body. In addition, we found large differences both in species number, species composition, and the developmental stages found at the scene and during the autopsy. These data and observations underscore the importance of sampling evidence at the scene and recording temperatures throughout the cooling period of a body.

The applicability of forensic time since death estimation methods for buried bodies in advanced decomposition stages.

Estimation of the postmortem interval in advanced postmortem stages is a challenging task. Although there are several approaches available for addressing postmortem changes of a (human) body or its environment (ecologically and/or biochemically), most are restricted to specific timeframes and/or individual and environmental conditions. It is well known, for instance, that buried bodies decompose in a remarkably different manner than on the ground surface. However, data on how established methods for PMI estimation perform under these conditions are scarce. It is important to understand whether and how postmortem changes are affected under burial conditions, if corrective factors could be conceived, or if methods have to be excluded for respective cases. We present the first multi-methodological assessment of human postmortem decomposition carried out on buried body donors in Europe, at the Amsterdam Research Initiative for Sub-surface Taphonomy and Anthropology (ARISTA) in the Netherlands. We used a multidisciplinary approach to investigate postmortem changes of morphology, skeletal muscle protein decomposition, presence of insects and other necrophilous animals as well as microbial communities (i.e., microbiomes) from August to November 2018 associated with two complete body exhumations and eight partial exhumations. Our results clearly display the current possibilities and limitations of methods for PMI estimation in buried remains and provide a baseline for future research and application.

Stay cool or get hot? An applied primer for using temperature in forensic entomological case work.

The reliable reconstruction of the temperature conditions at a crime scene is still a great challenge in forensic-entomological case work. Despite many published standards and guidelines for reconstructing temperature, and studies analysing the influence of various factors on the accuracy on such reconstructions, there are astonishingly many cases in the literature in which the temperature at the place of discovery is not reconstructed at all, i.e. the most common method is using the data of the nearest meteorological weather station without any correlation with on-site data. This study summarizes the state of the art in temperature reconstruction from an entomological point of view and compares the application of generalized additive models (GAMs) and linear regression on the basis of hypothetical death scenarios with various post mortem intervals (PMI) and measurement periods for the correlation between crime scene and weather station. We show that GAMs i.e. analysing the potential delay effect of temperature within a day, are the tools of choice because they give better, i.e. more accurate estimations than linear regression in 95,6% of all analysed cases regardless of the PMI, body discovery site and correlation period. Nevertheless, each case and crime scene is unique and therefore each entomological expertise should discuss the possible strengths and weaknesses of its temperature reconstruction. Even if temperature is not or cannot be reconstructed for various reasons, a comparison of on-site data with those of a meteorological weather station is the minimum forensic experts should do.

A field study to evaluate PMI estimation methods for advanced decomposition stages.

Estimating the postmortem interval (PMI) is one of the major tasks and a continuous challenge in forensic pathology. It is often an exclusion process of available methods, which ultimately can lead to an unsatisfactory outcome due to poor reliability. This problem is most acute in the late PMI, when decomposition proceeds and some methods (such as rigor, livor, and algor mortis) are no longer applicable. Several methods, such as forensic entomology, skeletal muscle protein degradation, and the study of body decomposition by application of a morphological scoring, are expected to provide further information; however, all have certain limitations and weaknesses. Availability of a tool-box of methods allows a case-specific selection of the most appropriate one(s), or eventually provides improvements in the overall accuracy and precision of the PMI estimation by merging and combining methods. To investigate practical (field) application, eventual interferences, and/or synergetic effects, as well as the robustness of these methods towards specific influencing factors, a field study was conducted, using eight pig cadavers of different body weights and physical coverage, left to decompose under natural conditions for 16 days. Morphological changes during decomposition were assessed using the total body score (TBS), muscle samples were collected to analyze protein degradation, and insect colonization was evaluated. The results reveal strengths and current limitations of all tested methods, as well as promising synergistic effects, and thus, provide a baseline for targeted future research.

Estimating the Postmortem Interval of Wild Boar Carcasses.

Knowledge on the postmortem interval (PMI) of wild boar (Sus scrofa) carcasses is crucial in the event of an outbreak of African swine fever in a wild boar population. Therefore, a thorough understanding of the decomposition process of this species in different microhabitats is necessary. We describe the decomposition process of carcasses exposed in cages. Trial 1 compared a wild boar and a domestic pig (Sus scrofa domesticus) under similar conditions; Trial 2 was performed with three wild boar piglets in the sunlight, shade, or in a wallow, and Trial 3 with two adult wild boar in the sun or shade. The wild boar decomposed more slowly than the domestic pig, which shows that standards derived from forensic studies on domestic pigs are not directly applicable to wild boar. The carcasses exposed to the sun decomposed faster than those in the shade did, and the decomposition of the carcass in the wallow took longest. To assess the state of decomposition, we adapted an existing total body scoring system originally developed for humans. Based on our studies, we propose a checklist tailored to wild boar carcasses found in the field that includes the most important information for a reliable PMI estimation.

Precocious egg development in wild Calliphora vicina (Diptera: Calliphoridae) - An issue of relevance in forensic entomology?

The list of factors influencing the development of forensically important insects and the related effects on minimum post mortem (PMImin) estimations is long and well established by various authors in forensic entomology over the last decades. Despite the fact that several authors mention precocious egg development, i.e. first instar larva in the genital tract of some species of Calliphoridae, as a potential error source for PMImin estimations just two studies were performed to examine the occurrence of precocious eggs both in laboratory and field populations of blow flies. In the present study the occurrence of gravid females with precocious eggs in a wild population of Calliphora vicina in Frankfurt am Main was surveyed over 152 days and their seasonal distribution was analyzed as well as the influence of abiotic variables on their abundance. During the sampling occasions, 5.216 females were sampled of which 44 % were gravid. 54 % of all gravid females had a precocious egg/first instar larva in their genital tract. This number varied depending on the season and showed the highest amount in spring with almost 64 % of all gravid females. Hence, precocious eggs in wild populations of blow flies can occur much more frequently than mentioned in forensic literature. Consequences for the evaluation of entomological evidence in forensic casework should not be overrated since focusing alone on the largest larva in a sample is not recommended as the sole reference in an entomological PMImin estimation. The entomological report has to justify its findings, discuss different options and finally state what is considered to be the most likely scenario based on the complete evidence (e.g. including all species and relevant age cohorts as well as succession data) and the case specific history.

An empirical comparison of decomposition and fly colonisation of concealed carcasses in the Old and New World.

The level of exposure or concealment of a cadaver is known to have an important impact on insect colonisation and decomposition but has been the subject of few investigations. In the present study, 30 pig carcasses were stored in three different types of containers (suitcases, trashcans and drums) with different levels of access for necrophagous insects at two different geographic locations for 100 days. The decomposition proceeded in a similar way in both geographic locations in all three types of container. Both in trashcans and suitcases, the decomposition process was characterised by bones and greasy, brown decomposition fluids left in the containers and an overall moist decomposition. In contrast, decomposition in the drums was characterised by a long bloating phase followed by a slow transition from bloated to deflation. Tissue and the carcasses as a whole were still present till the end of the experiment. Insect occurrence patterns and species composition on suitcases and trashcans were similar for both countries. Mainly flies and some beetles were present in suitcases and trashcans until day 45, with blow flies (Diptera: Calliphoridae) dominating the fauna. There was no insect colonisation in the drums. Our study contributes to the knowledge about insect accessibility of concealed cadavers and the impact of concealment on the speed and type of decomposition. It clearly shows that the degree of exposure of a cadaver is a key factor in decomposition and insect colonisation, which had a larger effect here than the biogeographical location.

Environmental factors influencing flight activity of forensically important female blow flies in Central Europe.

In forensic entomology, evaluation of a possible delay between a person's death and insect colonization is crucial. We monitored the seasonal flight activities of the most abundant blow flies in an urban habitat in Frankfurt/Germany based on 152 sampling days between April and October 2017. Thirty-six thousand female specimens of 12 necrophagous taxa were sampled as a possible groundwork for establishing a prediction tool for the activity of certain forensically relevant taxa. The most abundant taxon was Lucilia sericata (n = 19,544), followed by Lucilia caesar (n = 8025), Calliphora vicina (n = 5224), and Lucilia ampullacea (n = 1834). Up to six environmental parameters were statistically significant predictors of fly presence, leading to unique patterns of seasonal and daily activity for all four species. In detail, our analysis proved that L. sericata is a sun-loving, high-summer species that dominates the warmer months and is mostly influenced by mean day temperature. In contrast, L. caesar seems to be a shade-loving species that dominates in autumn resp. late-season and is mainly influenced by mean day temperature and wind speed. The activity of L. ampullacea was highly related to mean day temperature and relative humidity. In contrast to all other species, C. vicina behaved differently, particularly due to its occurrence throughout the entire sampling interval and the higher tolerance limits for the measured abiotic parameters, especially temperature. The present study is groundwork for establishing a prediction tool for the flight and oviposition activity of forensically relevant taxa.

Carcass concealment alters assemblages and reproduction of forensically important beetles.

Most forensic studies have examined decomposition and insect colonization for estimating the minimum postmortem interval (PMImin) using carcasses that are readily accessible to insects but in homicides, cadavers are often concealed to a certain extent. Here, we tested the hypothesis that the type of concealment of a decomposing resource and the permeability of the material used affect to different extents the animals attracted to the resource. To this end, beetle assemblages were documented for 100days on 15 domestic pig carcasses, placed individually in soft shell suitcases, trashcans with a hinged lid, and sealable drums in an untended open field in New Brunswick, Canada. Five pigs were allocated to each treatment. During the study, almost 33,000 beetle occurrences from 50 recognizable taxonomic units were documented around containers that concealed carcasses. Results indicated that trashcans, drums, and suitcases had different effects on the patterns of beetle arrival and departure from the vicinity of concealed carcasses, on beetle assembly, on their breeding strategies, as well as on the interspecific relationships between beetles of forensic importance. Of the 50 recognizable taxonomic units, only six species exhibited a somewhat predictable occurrence and yielded information about the time of placement or the type of container. Results also suggested that some of the abundant Silphidae species opted to breed or feed in suboptimal conditions or at a later period to avoid competing with the dominant silphid Necrodes surinamensis (Fabr.). This suggests the occurrence of preferential colonization and/or asymmetrical competition between beetle species, which would affect the potential of these species for PMImin estimations on concealed carcasses.

Species identification of adult African blowflies (Diptera: Calliphoridae) of forensic importance.

Necrophagous blowflies can provide an excellent source of evidence for forensic entomologists and are also relevant to problems in public health, medicine, and animal health. However, access to useful information about these blowflies is constrained by the need to correctly identify the flies, and the poor availability of reliable, accessible identification tools is a serious obstacle to the development of forensic entomology in the majority of African countries. In response to this need, a high-quality key to the adults of all species of forensically relevant blowflies of Africa has been prepared, drawing on high-quality entomological materials and modern focus-stacking photomicroscopy. This new key can be easily applied by investigators inexperienced in the taxonomy of blowflies and is made available through a highly accessible online platform. Problematic diagnostic characters used in previous keys are discussed.