Antemortem Samples Research Articles

Comparative analysis of the postadmission and antemortem oropharyngeal and rectal swab microbiota of ICU patients

Shotgun metabarcoding was conducted to examine the microbiota in a total of 48 samples from 12 critically ill patients, analyzing samples from both the oropharynx and rectum. We aimed to compare their postadmission microbiota, characterized as moderately dysbiotic, with the severely dysbiotic antemortem microbiota associated with patients’ deaths. We found that, compared with postadmission samples, patient antemortem swab samples presented moderate but not significantly decreased diversity indices. The antemortem oropharyngeal samples presented an increase in biofilm-forming bacteria, including Streptococcus oralis, methicillin-resistant Staphylococcus aureus (MRSA), and Enterococcus faecalis. Although the septic shock rate was 67%, no significant differences were detected in the potential pathogen ratios when the microbiota was analyzed. A notable strain-sharing rate between the oropharynx and intestine was noted. By comparing postadmission and antemortem samples, microbial biomarkers of severe dysbiosis were pinpointed through the analysis of differentially abundant and uniquely emerging species in both oropharyngeal and rectal swabs. Demonstrating strong interconnectivity along the oral-intestinal axis, these biomarkers could serve as indicators of the progression of dysbiosis. Furthermore, the microbial networks of the oropharyngeal microbiota in deceased patients presented the lowest modularity, suggesting a vulnerable community structure. Our data also highlight the critical importance of introducing treatments aimed at enhancing the resilience of the oral cavity microbiome, thereby contributing to better patient outcomes.

Epidemiological Dynamics of Canine Morbillivirus in Resident Dogs of Makurdi Metropolis, Benue State, Nigeria

Canine distemper is an endemic viral disease of dogs in Nigeria. Knowledge of the carrier status of dogs is key to successful control of the disease. In this study, the carrier status of apparently healthy dogs’ resident in parts of Makurdi metropolis was determined using the immunochromatographic rapid antigen assay kit for qualitative detection of canine morbillivirus antigens in ante-mortem samples. Ocular-, nasal-, and rectal- swabs, as well as serum were taken from each of 204 dogs bringing a total of 816 samples. The results showed that 26.96 % of the dogs sampled were positive for Canine morbillivirus antigens. Viral antigens were detected in 9.8%, 11.27%, and 4.41% of nasal, ocular, and rectal swaps respectively, and in 6.37% of the serum samples collected. An age-related susceptibility was observed as viral antigens detection rate was higher in younger dogs compared to older ones. Similarly, 40.43% of vaccinated and 22.93% of unvaccinated dogs were positive for canine morbillivirus antigens. Of significant importance is the prevalence rate in unvaccinated population of dogs. In terms of breed-related detection rate, 23.93% of Nigerian local dogs and 40.63% of exotic breeds tested positive for viral antigens, and it was observed that 25.54% of dogs which had history of contact with other dogs and 40% of dogs which had no such history carried canine morbillivirus antigens. The significance of this study is that it details the current epidemiological dynamics of canine morbillivirus in resident dogs of Makurdi metropolitan area, and the findings are discussed. Subject Areas: Animal infectious disease epidemiology.

Dynamics of antibody response and bacterial shedding of Mycoplasma hyorhinis and M. hyosynoviae in oral fluids from experimentally inoculated pigs

Mycoplasma hyorhinis (Mhr) and M. hyosynoviae (Mhs) are commensal organisms of the upper respiratory tract and tonsils but may also cause arthritis in pigs. In this study, 8-week-old cesarean-derived colostrum-deprived (CDCD) pigs (n = 30; 3 groups, 10 pigs per group, 2 pigs per pen) were inoculated with Mhr, Mhs, or mock-inoculated with culture medium and then pen-based oral fluids were collected at different time points over the 56 days of the experimental study. Oral fluids tested by Mhr and Mhs quantitative real-time PCRs revealed Mhr DNA between day post inoculation (DPI) 5–52 and Mhs DNA between DPI 5–15. Oral fluids were likewise tested for antibody using isotype-specific (IgG, IgA, IgM) indirect ELISAs based on a recombinant chimeric polypeptide of variable lipoproteins (A-G) for Mhr and Tween 20-extracted surface proteins for Mhs. Mhr IgA was detected at DPI 7 and, relative to the control group, significant (p < 0.05) antibody responses were detected in the Mhr group between DPI 12–15 for IgM and DPI 36–56 for both IgA and IgG. In the Mhs group, IgM was detected at DPI 10 and significant (p < 0.05) IgG and IgA responses were detected at DPI 32–56 and DPI 44–56, respectively. This study demonstrated that oral fluid could serve as an effective and convenient antemortem sample for monitoring Mhr and Mhs in swine populations.

Cytologic features of canine melanotroph and corticotroph pituitary adenomas.

The introduction of intraoperative cytology revolutionized neurosurgical procedures in human medicine, providing real-time diagnostic guidance to surgeons and contributing to improved patient outcomes. In the realm of veterinary medicine, the understanding of pituitary tumors in dogs and cats remains limited due to challenges in obtaining antemortem samples of central nervous system lesions. The aim of this study was to describe the cytologic features of pituitary adenomas in 12 dogs that underwent hypophysectomy. The series included nine melanotroph adenomas and three corticotroph adenomas. Definitive diagnosis was based on histopathology and immunohistochemistry. Cytologically, the adenomas had high numbers of bare nuclei and intact cells that were round to polygonal and situated individually or in small clusters. The intact cells had round to oval, eccentric nuclei with finely stippled chromatin and one to three prominent nucleoli and ample to abundant lightly basophilic to amphophilic, grainy cytoplasm with distinct borders, and variable numbers of discrete vacuoles. Mild-to-moderate anisocytosis and anisokaryosis, occasional binucleation, rare and atypical mitotic figures, and nuclear molding were also observed. The results suggest that intraoperative cytology of canine pituitary adenomas holds promise as a valuable diagnostic tool, aiding swift differentiation from other sellar masses before histologic confirmation. Cytologic characterization of pituitary adenomas in dogs is exceptionally rare in the scientific literature, making this study one of the first to offer a comprehensive description of these cytologic features.

Optimal blood tau species for the detection of Alzheimer’s disease neuropathology: an immunoprecipitation mass spectrometry and autopsy study

Plasma-to-autopsy studies are essential for validation of blood biomarkers and understanding their relation to Alzheimer’s disease (AD) pathology. Few such studies have been done on phosphorylated tau (p-tau) and those that exist have made limited or no comparison of the different p-tau variants. This study is the first to use immunoprecipitation mass spectrometry (IP-MS) to compare the accuracy of eight different plasma tau species in predicting autopsy-confirmed AD. The sample included 123 participants (AD = 69, non-AD = 54) from the Boston University Alzheimer’s disease Research Center who had an available ante-mortem plasma sample and donated their brain. Plasma samples proximate to death were analyzed by targeted IP-MS for six different tryptic phosphorylated (p-tau-181, 199, 202, 205, 217, 231), and two non-phosphorylated tau (195–205, 212–221) peptides. NIA-Reagan Institute criteria were used for the neuropathological diagnosis of AD. Binary logistic regressions tested the association between each plasma peptide and autopsy-confirmed AD status. Area under the receiver operating curve (AUC) statistics were generated using predicted probabilities from the logistic regression models. Odds Ratio (OR) was used to study associations between the different plasma tau species and CERAD and Braak classifications. All tau species were increased in AD compared to non-AD, but p-tau217, p-tau205 and p-tau231 showed the highest fold-changes. Plasma p-tau217 (AUC = 89.8), p-tau231 (AUC = 83.4), and p-tau205 (AUC = 81.3) all had excellent accuracy in discriminating AD from non-AD brain donors, even among those with CDR < 1). Furthermore, p-tau217, p-tau205 and p-tau231 showed the highest ORs with both CERAD (ORp-tau217 = 15.29, ORp-tau205 = 5.05 and ORp-tau231 = 3.86) and Braak staging (ORp-tau217 = 14.29, ORp-tau205 = 5.27 and ORp-tau231 = 4.02) but presented increased levels at different amyloid and tau stages determined by neuropathological examination. Our findings support plasma p-tau217 as the most promising p-tau species for detecting AD brain pathology. Plasma p-tau231 and p-tau205 may additionally function as markers for different stages of the disease.

RENAL PATHOLOGY IN CAPTIVE ADULT ALAOTRAN GENTLE LEMURS (HAPALEMUR ALAOTRENSIS).

Full medical histories from captive Alaotran gentle lemurs or Bandro (Hapalemur alaotrensis) > 1 yr old that died between 1990 and 2016 were requested from holding institutions. Eighty-six individuals died during the period analyzed. Full postmortem reports were received from 40 (46.5%) animals from 16 different institutions across Europe (15) and North America (1). Eighteen animals (45%) showed azotemia within three months of death, with accompanying histological renal lesions. Another 17 (42.5%) showed histological renal lesions, but no renal function assessment was carried out antemortem, or results were within normal limits. Only five animals (12.5%) showed no renal lesions. Of the 35 (87.5%) animals with histological renal lesions, 18 were females, and 17 were males, 11 were wild caught, and 24 were captive born. Twenty-seven animals were euthanized, seven were found dead, and in one case, no details were provided. Sixty-four blood samples from 22 animals were available. Azotemia was observed on average 407 d antemortem, with a case observed as early as 2,318 d antemortem. Twenty-nine urinalyses from 12 animals were carried out antemortem. All animals showed hematuria or proteinuria in at least one antemortem sample. A pH decrease from 8.5 to 5.0 was observed in two animals antemortem. Gross renal lesions most frequently reported were irregular surface (n = 14), abnormal shape (n = 12), and/or presence of cysts (n = 9). The most common histological lesions were interstitial nephritis (n = 25), interstitial fibrosis (n = 26), tubule dilation (n = 16), and glomerulosclerosis (n = 12). Development of additional diagnostic tools, standardization of ante- and postmortem diagnostic protocols, and further investigation into potential etiologies, such as diets offered in captivity and genetic factors, should be considered as the next steps for the veterinary management of this species in captivity.

Evaluation of a rapid, chip-based, micro-PCR assay for detection of rabies virus in human and canine specimens.

Rabies, a lethal zoonotic encephalitis, remains a significant global health concern, causing an estimated 60 000 annual fatalities worldwide. Dogs serve as the primary reservoirs and vectors for transmitting this infection to humans. Definitive diagnosis of rabies in both human and animal cases necessitates laboratory testing involving various clinical specimens. However, the complexity of laboratory infrastructure and the need for skilled personnel, along with the challenge of maintaining cold-chain integrity during sample referral, hinder the decentralization of diagnostic facilities. This study aimed to assess the efficacy of the Truenat rabies assay, a rapid, portable, semiautomated, and closed PCR-based system, for the diagnosis of rabies in both humans and animals. The Truenat assay demonstrated a sensitivity of 100% and a specificity of 86.96% when compared with the fluorescent antibody test (FAT), as the reference standard, on 147 canine brain samples tested. Notably, the Truenat assay exhibited a sensitivity and specificity of 100% when tested on 48 human brain specimens. Furthermore, an examination of 148 human antemortem samples (cerebrospinal fluid, saliva, and skin biopsy) using both the Truenat assay and a validated real-time reverse transcriptase PCR assay revealed a κ value of 0.505, indicative of a moderate level of agreement between the two tests. Thus, the Truenat assay offers a robust, reliable, and affordable point-of-care solution to enhance rabies diagnostic capacity in endemic areas.

Prevalence of Fasciola species from Cattle Slaughtered at Birnin Kebbi Modern Abattoir

Diagnostic tools to detect and differentiate Fasciola species have improved, but the understanding of the distribution and population structure of this parasite is location dependent. This study was aimed to phenotypically identify Fasciola species from cattle brought for slaughter at Birnin Kebbi Modern Abattoir. Ante-mortem faecal samples were collected from the sampled cattle for worm egg detection and post-mortem inspection of liver samples were performed. Two methods of Parasitological (Flukefinder Kit and formalin-ether sedimentation) methods were performed on the faecal samples. Results have yielded a high prevalence (31.2%) of fluke eggs with the Flukefinder Kit than with formalin-ether sedimentation technique (8.5%). Prevalence was higher (34%) in the males than in female (26.2%) cattle. Younger cattle (34.3%) were also more infected than their older (30.7%) counterpart. Sokoto Gudali had the highest prevalence of 44.0% followed by Red Bororo with 34.0%, and the least prevalence was observed in Bunaji breed with 18.0%. Statistical analyses results had revealed no significant relation between prevalence of fasciolosis and the co-variables (sex, age) as p &gt; 0.05 but there was a significant association between prevalence and breed of cattle (p = 0.018). There was no significant agreement between the methods of faecal analyses used. Prevalence of 15.8% was observed from the post-mortem examination. This have increased the knowledge about the existence of the parasite in the study area. It is recommended that the Flukefinder® Kit should be used in diagnosis of Fasciola species due to its high sensitivity compared to the conventional sedimentation method.

Inflammatory mediators and inflammatory cells as reliable molecular targets for assessment of wound age and vitality in rats

Evaluation of the wound age and vitality is a persistent problem for forensic examinations to determine when injuries occurred and whether during the antemortem or post-mortem period. This study aimed to use the inflammatory cells and mediators as a target marker to determine wound age in antemortem or post-mortem samples in rats. Wound skin samples (n=39) were collected from antemortem groups aged 30, 60, 180, and 360 min and post-mortem groups within the same periods with control samples (unwounded group). Immunohistochemical staining examined all tissue samples for two inflammatory cell markers (CD15, CD68) and 2 inflammatory mediators (IL-6, TNF-α). The results showed that CD15-positive neutrophils were expressed in all time intervals of antemortem, and the expression gradually increased from 30 to 360 min. The CD68-positive macrophages began at 30 minutes and reached the highest level at 360 minutes in the antemortem wound. Because of the negative expression of CD15 and CD68 in all post-mortem groups, they could be a marker for comparing the vital and non-vital wounds. Furthermore, the Immunoreactivity results showed that the IL-6 and TNF-α were negatively expressed in unwounded skin samples. In addition, the periods after the induced wound produced a rise in the expression area of both markers in antemortem wounds. In this study, IL-6 and TNF-α expressions were negative or low in areas far from wound margin, unwounded specimens, and post-mortem wounds. CD15, CD68, IL-6, and TNF-α may be valuable markers for assessing wound age and vitality.

Tonsil biopsy to detect chronic wasting disease in white-tailed deer (Odocoileus virginianus) by immunohistochemistry.

Chronic wasting disease (CWD) continues to spread in wild and farmed cervid populations. Early antemortem CWD testing of farmed cervids is of considerable interest to producers and regulatory agencies as a tool to combat this spread. The tissues accessible for antemortem sampling are limited and include biopsy of the tonsil and recto-anal mucosa-associated lymphoid tissue (RAMALT). The sensitivity to detect CWD by immunohistochemistry (IHC)-the regulatory gold standard-using biopsy samples of RAMALT from naturally infected white-tailed deer (WTD) has been determined by several studies. However, similar information is lacking for tonsil biopsy. In this study, two-bite tonsil biopsies from 79 naturally infected farmed WTD were used to determine the diagnostic sensitivity of tonsil IHC compared to the official CWD status based on results from the medial retropharyngeal lymph nodes and obex. IHC detection of CWD by tonsil biopsy was compared to the result and follicle metrics from the contralateral whole tonsil. The sensitivity of two-bite tonsil biopsy for detecting CWD by IHC was 72% overall. When the stage of infection was considered, the sensitivity was 92% for deer in late preclinical infection but only 55% for early preclinical infection. For deer with early preclinical infection, the sensitivity for deer homozygous for the prion protein gene (PRNP) coding for glycine at codon 96 (GG) was 66% but only 30% when heterozygous for the serine substitution (GS). The results indicate that the sensitivity of two-bite tonsil biopsy in WTD, and consequently its potential utility as an antemortem diagnostic, is limited during early infection, especially in WTD heterozygous for the serine substitution at PRNP codon 96.

Changing trends and characteristics of one punch deaths in Australia (2012–2018)

One-punch assaults also known as ‘coward punches’, are characterised by a single severe blow to the head causing the victim to lose consciousness, resulting in a secondary impact between the head and surrounding environment. Such impacts may result in brain injury leading to fatality or permanent neurological impairment. In a previous publication, there were 90 one punch deaths around Australia between 2000 and 2012, mostly involving young men drinking alcohol at a licensed venue at the weekend. This prompted a surge of public education and awareness campaigns around Australia, in addition to regulatory and legislative changes aimed at curbing social violence. This retrospective descriptive study aimed to examine one punch deaths since 2012 in Australia to determine if there has been a decrease in deaths, and whether the demographics and circumstances of these deaths have changed. A search of the National Coronial Information System was undertaken for all closed coronial cases between 1 January 2012 and 31 December 2018. Additional information was collected from medicolegal reports including toxicology, pathology and coronial findings. There were 80 one punch fatalities in Australia, almost exclusively involving males. The median age was 43.5 (range 18–71) years and there was a decreasing trend in the number of deaths annually. Most fatal assaults occurred in the state of New South Wales (28.8%) followed by Queensland (23.8%), and in metropolitan locations (64.6%) rather than regional areas (35.4%). Alcohol was the most commonly detected drug, found in 47 cases of the 71 cases where toxicology results were available (66%), with a median concentration of 0.14 and 0.19 g/100 mL in antemortem and postmortem samples, respectively (range 0.005–0.32 g/100 mL). Five deaths reported methylamphetamine, with THC detected in 21.1% of cases. Assaults more commonly occurred on a footpath or roadside (41.3%), followed by a home or dwelling (32.5%). 8.8% of assaults occurred inside hotels, bars or other licenced venues. Most transpired on a weekday, which differed from the pre-2012 period when these assaults occurred mainly on the weekend. While some trends are positive, there has been a shift in the victim demographic as well as the typical environment for fatal one punch assaults, highlighting the importance of public health surveillance in providing a current evidence base to inform policy and practice.

Intravitam Diagnosis of Rabies in Patients with Acute Encephalitis: A Study of Two Cases.

Rabies is one of the oldest known zoonotic diseases. Rhabdovirus, an RNA virus belonging to the genus Lyssavirus and family Rhabdoviridae, causes rabies. Rabies diagnosis is challenging as the rabies virus remains confined to neurons after the initial animal bite. It largely remains immune-evasive until the infection reaches the central nervous system. The bottleneck in rabies diagnosis remains the non-availability of technical expertise and failure to collect an appropriate sample. The laboratory confirmation of rabies in both antemortem and postmortem samples is important. The samples were tested for anti-rabies antibodies using quantitative ELISA. In this report, two case studies are presented to demonstrate the suitability of ELISA for the intra vitam diagnosis of rabies using cerebrospinal fluid (CSF) as a diagnostic sample. The interpretation of serology results for both vaccinated and unvaccinated individuals has been discussed in detail, which has helped to confirm the antemortem diagnosis of rabies. In this report, we observed that ELISA can be a viable alternative for anti-rabies antibody detection in CSF and can be used as a viable alternative to more technically challenging tests, such as Rapid Fluorescent Focus Inhibition Test (RFFFIT) and Immunofluorescence Assays (IFA).

Application of a dense SNP PCR multiplex for high throughput kinship determination using Next Generation Sequencing (NGS)

The need to identify large numbers of missing persons from mass disasters, armed conflicts and human rights abuses has become increasingly prevalent. The ForenSeq® Kintelligence Library Prep Kit together with NGS (sometimes called massively parallel sequencing), has been designed to interrogate 10,230 SNPs for the purpose of solving cold cases, including missing persons identification. The kit allows up to 3 samples per sequencing run in order to call sufficient SNPs for detecting relationships up to 5th degree when searching direct to consumer DNA databases, such as GEDmatch PRO™. For missing persons cases with high numbers of post-mortem (P.M.) and ante-mortem (A.M.) samples from the deceased and their family members, respectively, a higher throughput solution is required. In this report, we present the feasibility of increasing sequencing plexity for PM to 12 and AM to 32 samples using the Kintelligence kit and workflow. This generates sufficient SNP calls for kinship determination up to 3rd order. DNA was extracted from bones and dental remains, as well as artificially degraded and low input DNA to simulate P.M. samples. DNA from diverse populations was typed to simulate A.M. samples. We determined the number of called SNPs overlapping between A.M. and P.M samples from these high-plexity sequencing runs and showed the impact on two methods of estimating kinship: kinship coefficients and likelihood ratios (LR). LR and kinship coefficient decrease as fewer SNPs are called (i.e., in more challenging samples) or for more distant relationships. However, filtering on both values achieved high sensitivity and specificity out to 3rd degree. Furthermore, relationship identification out to 3rd degree was robust to loss of heterozygosity in the samples. Overall, increasing plexity can facilitate higher throughput DNA analysis of missing persons cases, without severe loss in kinship estimation.

A multifaceted, multijurisdictional, multiagency, and multidisciplinary approach to investigating unidentified and missing persons cases in Australia

Currently, there are approximately 750 unidentified human remains and 2500 long-term missing persons in Australia. The Australian Federal Police National DNA Program for Unidentified and Missing Persons (Program) is using a multifaceted, multijurisdictional, multiagency, and multidisciplinary approach in a dedicated effort to identify these unknown deceased persons, scientifically link them to known missing persons, and provide answers to their families. The nationally coordinated Program provides its police, forensic, and coronial stakeholders with a suite of contemporary forensic technologies, databases, and experts to forensically examine the skeletonised remains and recover post-mortem data for comparison to the available ante-mortem data for each missing person. Through a number of physical and virtual public outreach activities, families with missing relatives have been encouraged to provide vital ante-mortem forensic information, records, and samples to aid the identification process. To date, this unique Program has assisted to resolve a number of unidentified and missing persons cases from both historical and contemporary contexts, using a combination of genetic and non-genetic techniques, and local and national databases. The centralisation of Program capabilities, expertise, and resources to conduct this type of unique and challenging casework is proving to be the most effective and efficient way to generate investigative leads, identify human remains, and resolve long-term missing persons cases in Australia.

YKL-40 changes are not detected in post-mortem brain of patients with Alzheimer’s disease and frontotemporal lobar degeneration

BackgroundYKL-40 (Chitinase 3-like I) is increased in CSF of Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLD) patients and is therefore considered a potential neuroinflammatory biomarker. Whether changed YKL-40 levels in the CSF reflect dysregulation of YKL-40 in the brain is not completely understood yet. We aimed to extensively analyze YKL-40 levels in the brain of AD and different FTLD pathological subtypes. The direct relationship between YKL-40 levels in post-mortem brain and ante-mortem CSF was examined in a small set of paired brain-CSF samples.MethodYKL-40 was analyzed in post-mortem temporal and frontal cortex of non-demented controls and patients with AD and FTLD (including FTLD-Tau and FTLD-TDP) pathology by immunohistochemistry (temporal cortex: 51 controls and 56 AD and frontal cortex: 7 controls and 24 FTLD patients), western blot (frontal cortex: 14 controls, 5 AD and 67 FTLD patients), or ELISA (temporal cortex: 11 controls and 7 AD and frontal cortex: 14 controls, 5 AD and 67 FTLD patients). YKL-40 levels were also measured in paired post-mortem brain and ante-mortem CSF samples from dementia patients (n = 9, time-interval collection: 1.4 years) by ELISA.ResultsWe observed that YKL-40 post-mortem brain levels were similar between AD, FTLD, and controls as shown by immunohistochemistry, western blot, and ELISA. Interestingly, strong YKL-40 immunoreactivity was observed in AD cases with cerebral amyloid angiopathy (CAA; n = 6). In paired CSF-brain samples, YKL-40 concentration was 8-times higher in CSF compared to brain.ConclusionOur data suggest that CSF YKL-40 changes may not reflect YKL-40 changes within AD and FTLD pathological brain areas. The YKL-40 reactivity associated with classical CAA hallmarks indicates a possible relationship between YKL-40, neuroinflammation, and vascular pathology.

A Two-Year Review of Fentanyl in Driving under the Influence and Postmortem Cases in Orange County, CA, USA.

In recent years, fentanyl is increasingly detected in overdose cases as well as human performance cases due to the current opioid epidemic. The comparison of fentanyl concentrations in deaths to recreational fentanyl use found in driving under the influence of drugs (DUID) cases is important to show how increased availability affects the historical knowledge of fentanyl. A 2-year review of fentanyl cases from Orange County, CA, USA, is presented to highlight fentanyl concentrations in these two case types. Any non-urine sample with fentanyl detected above the limit of detection of 0.5 ng/mL was reviewed in DUID and postmortem (PM) cases. The mean and median fentanyl concentrations in 199 antemortem (AM) blood samples from DUID cases were 14.2 ng/mL and 5.3 ng/mL, respectively, with a range of 0.5-303 ng/mL. In 285 central blood samples from PM cases, the mean was 29.1 ng/mL, the median was 16.9 ng/mL and the range was 0.6-636 ng/mL. In a total of 58 PM peripheral blood samples, the mean, median and range were 14.0 ng/mL, 10.0 ng/mL and 0.9-78.0 ng/mL, respectively. Of the 55 brain samples analyzed, the fentanyl mean, fentanyl median and range of fentanyl concentrations were 50.0ng/g, 31.4ng/g and 1.9-441ng/g, respectively. Fentanyl concentrations in 16 liver samples had a mean of 82.0ng/g, a median of 69.4ng/g and a range of 11.6-226ng/g. The overlap of fentanyl concentrations from the AM and PM samples highlights the importance of evaluating case circumstances when providing an interpretation in PM cases. Additional information such as age, gender, polydrug use and PM redistribution is presented for the cases included in the study.

Challenges in the recovery of the genetic data from human remains found on the Western Balkan migration route.

Traditional DNA-based identification of human remains relies on the system of matching STR profile of the deceased with the family references or antemortem samples. In forensic cases without any available samples for the comparison, the body remains unidentified. The aim of this study was to assess the applicability of massively parallel sequencing (MPS) approach in the forensic cases of five drowned individuals recovered on the Western Balkan migration route. Besides capillary electrophoresis (CE)-based genetic profiling (aSTR, Y STR, and mitochondrial control region sequencing) of postmortem samples, we applied ForenSeq DNA Signature Prep Kit/Primer Mix B on MiSeqFGx platform and concomitant ForenSeq Universal Analysis (UAS) software. The assay showed high reproducibility and complete concordance with CE-based data except in locus DYF387S1. Allele and locus drop was evident in 2.9% of total SNPs that slightly reduced the completeness of the data. We endeavored to predict the phenotype of the tested samples and accurate biogeographical ancestry of European individual. UAS was less informative for the remaining samples assigned to Admixed American cluster. Nevertheless, the application of FROG-kb and Snipper tools along with admixture analysis in STRUCTURE and lineage markers revealed likely Middle Eastern and North African ancestry. We conclude that the combination of the phenotype and biogeographical ancestry predictions, including paternal and maternal genetic ancestry, represent a promising tool for humanitarian identification of dead migrants. Nevertheless, the data interpretation remains a challenging task.

Streptococcus equi subsp. zooepidemicus infection of pigs leads to shedding in faeces and a carrier state.

In 2019, Streptococcus equi subsp. zooepidemicus was recognized as an emerging pathogen of swine, associated with sudden deaths, increased abortion rates and septicaemia. Limited data are available regarding this disease in pigs. The objectives of this study were to clarify clinical progression, pathogen shedding, transmission, gross and microscopic lesions following infection in pigs. Six weeks old pigs were inoculated with either S. zooepidemicus sequence type 194 (inoculated, n=6) or sham inoculated with sterile culture broth (sentinels, n=4). Animals were housed in the same room, in two pens 2m apart. Pigs were monitored twice daily for clinical signs, and rectal, nasal and oral swabs were collected once daily. A full necropsy was performed if welfare was a concern or at 5 days post-inoculation (dpi). All sentinels remained disease free and their samples tested negative for the pathogen of interest. All inoculated pigs developed fever within 8h of inoculation, and severe disease was observed after 2 dpi. A total of 4/6 inoculated pigs developed clinical signs that compromised animal welfare and were euthanized. Nasal swabs (15/23), followed by rectal swabs (9/23) yield the highest number of positive ante-mortem samples. Clinically healthy, inoculated pigs had detectable levels of S. zooepidemicus in rectal and nasal swabs. Reactive submandibular lymph nodes, kidney petechiae and splenomegaly were found in six of six inoculated pigs. These data suggest that subclinically infected pigs may spread the pathogen through nasal secretions and faeces. Direct contact seems to be required for transmission.

Neuronal-Derived EV Biomarkers Track Cognitive Decline in Alzheimer’s Disease

The hallmarks of Alzheimer’s disease (AD) pathology are senile plaques containing amyloid-beta (Aβ) and neurofibrillary tangles containing hyperphosphorylated tau. Additional pathologies often co-exist, whereas multiple pathogenic mechanisms are involved in AD, especially synaptic degeneration, which necessitate the need for synaptic integrity-related biomarkers alongside Aβ- and tau-related biomarkers. Plasma neuron-derived Extracellular Vesicles EVs (NDEVs) provide biomarkers related to Aβ and tau and synaptic degeneration. Here, to further establish the latter as a “liquid biopsy” for AD, we examined their relationship with ante-mortem cognition in pathologically-confirmed AD cases. We immunoprecipitated NDEVs by targeting neuronal marker L1CAM from ante-mortem plasma samples from 61 autopsy-confirmed cases of pure AD or AD with additional pathologies and measured Aβ42, p181-Tau, total Tau, synaptophysin, synaptopodin and three canonical EV markers, CD63, CD81 and CD9. Higher NDEV Aβ42 levels were consistently associated with better cognitive status, memory, fluency, working memory and executive function. Higher levels of NDEV synaptic integrity-related biomarkers were associated with better performance on executive function tasks. Our findings motivate the hypothesis that releasing Aβ42-laden NDEVs may be an adaptive mechanism in AD.

Novel alphaherpesvirus in a wild South American sea lion (Otaria byronia) with pulmonary tuberculosis.

In 2017, an adult male South American sea lion (Otaria byronia), presenting emaciation and a cervical abscess, stranded alive in Florianópolis, southern Brazil. The animal was directed to a rehabilitation center, dying a few days later. On necropsy, the main gross findings were necrotizing lymphadenitis of the right prescapular lymph node and nodular bronchopneumonia. A novel alphaherpesvirus, tentatively named Otariid alphaherpesvirus 1, was amplified in several tissue samples. No histopathologic findings associated with viral infection were observed. Additionally, pulmonary tuberculosis by Mycobacterium pinnipedii was diagnosed by histopathological, immunohistochemical, and molecular techniques. Several bacteria were cultured from antemortem and postmortem samples, including Proteus mirabilis from the cervical abscess and cardiac blood, and Escherichia coli from the cervical abscess and pericardial effusion. Flavivirus, morbillivirus, and Apicomplexa were not detected by molecular techniques. Herein, we report a novel alphaherpesvirus in a pinniped species of the family Otariidae. Although previously described in Southern Hemisphere pinniped species, including South American sea lions, there is limited information regarding M. pinnipedii impact over this group. Further research is required to determine the associated pathogenesis of this novel herpesvirus, and prevalence of Otariid alphaherpesvirus 1 and M. pinnipedii in the reproductive colonies.