A preliminary note on attraction and oviposition preferences of Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae).

Chrysomya rufifacies (Diptera: Calliphoridae) is a blow fly species of forensic importance, documented to have a strong preference for colonisation of substrate already inhabited by heterospecific blow fly larvae, thus exhibiting secondary colonisation behaviour. Larvae exhibit predatory behaviour that may be useful to support development where food substrate is limited or high competition exists, but they may alternately be drawn to pre-colonised substrate to capitalise on the advantages of collective exodigestion by previous/current colonisers. Previous authors have suggested female Ch. rufifacies may use visual orientation to detect substrate currently colonised by heterospecific larvae, rather than chemoreception of volatile organic compounds (VOCs), that signify condition of substrate, which would infer that active colonisation is likely a more important oviposition cue for Ch. rufifacies than substrate condition. This study addressed attraction as well as oviposition, examining whether the condition of substrate (either previously colonised or never colonised) or the presence of heterospecific larvae was more important in the initial choice of food source by female Ch. rufifacies where conspecifics were not present, and whether the condition of substrate and presence of heterospecific larvae affects the number of offspring deposited by a female. Attraction was studied using a Y-olfactometer system, and oviposition using a binary-choice assay, with females responding to pairwise choice between an array of meat conditions (fresh, larval aged or aged) and presence/absence of Lucilia sericata larvae. Females displayed a hierarchy of choice of larval aged substrate > aged substrate > fresh substrate, with the active presence of heterospecific larvae a secondary factor in choice. Females produced higher offspring numbers on meat that was either currently or previously colonised by heterospecific larvae, demonstrating the importance of heterospecific indicators of previous or current colonisation as an oviposition cue. This serves as an important consideration for entomologists working with Ch. rufifacies in any capacity where other blow fly species may be present, and most importantly for forensic entomologists where time of colonisation is utilised to estimate PMI.

Aquatic conditions & bacterial communities as drivers of the decomposition of submerged remains.

Aquatic decomposition, as a forensic discipline, has been largely under-investigated as a consequence of the highly complex and influential variability of the water environment. The limitation to the adaptability of scenario specific results justifies the necessity for experimental research to increase our understanding of the aquatic environment and the development of post-mortem submersion interval (PMSI) methods of estimation. This preliminary research aims to address this contextual gap by assessing the variation in the bacterial composition of aquatic biofilms as explained by water parameter measurements over time, associated with clothed and bare decomposing remains. As part of three field investigations, a total of 9 still-born piglets (n = 3, per trial) were used as human analogues and were submerged bare or clothed in either natural cotton or synthetic nylon. Changes in the bacterial community composition of the water surrounding the submerged remains were assessed at 4 discrete time points post submersion (7, 14, 21 and 28 days) by 16 S rRNA gene Next Generation Sequencing analysis and compared to coinciding water parameter measurements (i.e. conductivity, total dissolved solids (TDS), salinity, pH, and dissolved oxygen (DO)). Bacterial diversity was found to change over time and relative to clothing type, where significant variation was observed between synthetic nylon samples and bare/cotton samples. Seasonality was a major driver of bacterial diversity, where substantial variation was found between samples collected in early winter to those collected in mid - late winter. Water parameter measures of pH, salinity and DO were identified to best explain the global bacterial community composition and their corresponding dynamic trajectory patterns overtime. Further investigation into bacterial community dynamics in accordance with varying environmental conditions could potentially lead to the determination of influential extrinsic factors that may drive bacterial activity in aquatic decomposition. Together with the identification of potential bacterial markers that complement the different stages of decomposition, this may provide a future approach to PMSI estimations.

Prevalence of Coronavirus Disease 2019 Infection and Outcomes Among Symptomatic Healthcare Workers in Seattle, Washington.

BACKGROUND: Healthcare workers (HCWs) who serve on the front lines of the coronavirus disease 2019 (COVID-19) pandemic have been at increased risk for infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in some settings. Healthcare-acquired infection has been reported in similar epidemics, but there are limited data on the prevalence of COVID-19 among HCWs and their associated clinical outcomes in the United States. METHODS: We established 2 high-throughput employee testing centers in Seattle, Washington, with drive-through and walk-through options for symptomatic employees in the University of Washington Medicine system and its affiliated organizations. Using data from these testing centers, we report the prevalence of SARS-CoV-2 infection among symptomatic employees and describe the clinical characteristics and outcomes among employees with COVID-19. RESULTS: Between 12 March 2020 and 23 April 2020, 3477 symptomatic employees were tested for COVID-19 at 2 employee testing centers; 185 (5.3%) employees tested positive for COVID-19. The prevalence of SARS-CoV-2 was similar when comparing frontline HCWs (5.2%) with nonfrontline staff (5.5%). Among 174 positive employees reached for follow-up at least 14 days after diagnosis, 6 reported COVID-related hospitalization; all recovered. CONCLUSIONS: During the study period, we observed that the prevalence of positive SARS-CoV-2 tests among symptomatic HCWs was comparable to that of symptomatic nonfrontline staff. Reliable and rapid access to testing for employees is essential to preserve the health, safety, and availability of the healthcare workforce during this pandemic and to facilitate the rapid return of SARS-CoV-2-negative employees to work.

The origin of unknown source DNA from touched objects.

The presence of DNA in a criminal investigation often requires scrutiny in relation to how it came to be where it was found. There is a paucity of data with respect to the extent to which one can assume that the last person handling an object, which has previously been touched by others, will contribute to the DNA profile generated from it. There are limited data in detailing the extent to which any foreign DNA is picked-up from a previously touched object and transferred to subsequently touched objects. This study focuses on DNA transfer and persistence on a knife handle after multiple handlings with the knife by different individuals soon after each other, as well as handprints left on flat DNA-free surfaces immediately after touching a knife handle with a known history of prior handling. The profiles of later handlers of a knife are more prominent than earlier handlers; however, the last handler is not always the major contributor to the profile. Proportional contributions to the profiles retrieved from knife handles vary depending on the individuals touching the knife handle. They can also vary when knife handles have been handled in the same manner by the same individuals in the same sequence on different occasions. Hands readily pickup DNA left on objects by others and transfer it to subsequently touched objects. The quantity of foreign DNA picked up by a hand and deposited on subsequently touched objects diminishes as more DNA-free objects are handled soon after each other. Caution is advised when considering how DNA from different individuals may have been transferred to the object from which it was collected.

DNA transfer by examination tools--a risk for forensic casework?

The introduction of profiling systems with increased sensitivity has led to a concurrent increase in the risk of detecting contaminating DNA in forensic casework. To evaluate the contamination risk of tools used during exhibit examination we have assessed the occurrence and level of DNA transferred between mock casework exhibits, comprised of cotton or glass substrates, and high-risk vectors (scissors, forceps, and gloves). The subsequent impact of such transfer in the profiling of a target sample was also investigated. Dried blood or touch DNA, deposited on the primary substrate, was transferred via the vector to the secondary substrate, which was either DNA-free or contained a target sample (dried blood or touch DNA). Pairwise combinations of both heavy and light contact were applied by each vector in order to simulate various levels of contamination. The transfer of dried blood to DNA-free cotton was observed for all vectors and transfer scenarios, with transfer substantially lower when glass was the substrate. Overall touch DNA transferred less efficiently, with significantly lower transfer rates than blood when transferred to DNA-free cotton; the greatest transfer of touch DNA occurred between cotton and glass substrates. In the presence of a target sample, the detectability of transferred DNA decreased due to the presence of background DNA. Transfer had no impact on the detectability of the target profile, however, in casework scenarios where the suspect profiles are not known, profile interpretation becomes complicated by the addition of contaminating alleles and the probative value of the evidence may be affected. The results of this study reiterate the need for examiners to adhere to stringent laboratory cleaning protocols, particularly in the interest of contamination minimisation, and to reduce the handling of items to prevent intra-item transfer.

Internal morphological analysis for age estimation of blow fly pupae (Diptera: Calliphoridae) in postmortem interval estimation.

Larvae and pupae of blow fly species are frequently used in postmortem interval estimation, their age indicating minimum time since death. Most studies have considered age estimation of larvae, neglecting study of pupae. Relative development of external pupal features is useful, but there are also internal changes during metamorphosis that may be indicators of age, utilizing histological techniques. This study aimed to optimize preservation and histological analysis of blow fly pupae, specifically Calliphora vicina Robineau-Desvoidy and Lucilia sericata (Meigen), and to examine internal features with potential for age estimation. Effect of hot-water-killing and different preservatives were examined. It was determined that blow fly pupae should be pierced through the three tagma, hot-water-killed, and preserved in 80% ethanol as the optimal preservation for subsequent analyses. Hematoxylin and eosin stained pupal sections revealed differences in brain and thoracic muscle development throughout the pupal stage with potential for age estimation.

Entomological evidence: lessons to be learnt from a cold case review.

Insects are known to be useful in estimating time since death, but this is only possible if samples are collected and preserved correctly according to best practices. This report describes a case where an 18-year old female was found dead and during the first medico-legal investigation which determined it was a homicide, entomological samples were collected but not considered. The case was then closed with no suspect. However, 9 years after the first investigation the courts decided that the case needed to be re-examined. In doing so the new review team decided that although the remaining entomological evidence was poorly preserved some extra information may be gained from its analyses. On inspection of the remaining samples of larvae no normal morphological analyses could be conducted. Molecular analyses were combined with an unorthodox morphological analysis to provide an estimate of the post-mortem interval based on insect evidence, indicating the value of multidisciplinary approaches to both cold and contemporary cases.

An alternative for the extraction and storage of DNA from insects in forensic entomology.

An important area of recent research in forensic entomology has been the use of insect DNA to provide identification of insects for fast and accurate estimation of time since death. This requires DNA to be extracted efficiently and in a state suitable for use in molecular procedures, and then stored on a long-term basis. In this study, Whatman FTA cards were tested for use with the Calliphoridae (Diptera). In particular, testing examined their ability to effectively extract DNA from specimens, and store and provide DNA template in a suitable condition for amplification using the polymerase chain reaction (PCR). The cards provided DNA that was able to be amplified from a variety of life stages, and thus appears to be of sufficient quality and quantity for use in subsequent procedures. FTA cards therefore appear suitable for use with calliphorids, and provide a new method of extraction that is simple and efficient and allows for storage and transportation without refrigeration, consequently simplifying the handling of DNA in forensic entomological cases.

Isolation and detection of ingested DNA from the immature stages of Calliphora dubia (diptera: Calliphoridae) : A forensically important blowfly.

The forensic entomologist frequently bases time since death (TSD) estimation on fly larvae. In some cases, the food source on which these larvae have completed their development may be questionable, and requires verification to ensure the accuracy of the TSD estimation. Ingested DNA may be isolated from the alimentary canal of immature insects. Previous studies have confirmed the ability to extract ingested DNA from the alimentary tract of third instar blowfly larvae. This study considers the potential to detect ingested DNA from immature stages of the blue-bodied blowfly Calliphora dubia (Macquart) that had fed on sheep liver. Individuals from early first instar larvae through day 3 pupae were surface decontaminated, followed by DNA isolation and detection by amplifying the sheep satellite I region. Fragments of 197 basepairs (bp) and 87 bp were successfully isolated and detected in all stages of immatures until 2-day-old pupae, with detection at this stage being unsuccessful on 3-day-old pupae. This study presents a suitable protocol for the isolation and detection of ingested DNA from immature stages of C. dubia.

Mitochondrial DNA cytochrome oxidase I gene: potential for distinction between immature stages of some forensically important fly species (Diptera) in western Australia.

Forensic entomology requires the fast and accurate identification of insects collected from a corpse for estimation of the postmortem interval (PMI). Identification of specimens is traditionally performed using morphological features of the insect. Morphological identification may be complicated however by the numerical diversity of species and physical similarity between different species, particularly in immature stages. In this study, sequencing was performed to study the mitochondrial DNA (mtDNA) as the prospective basis of a diagnostic technique. The sequencing focused on a section of the cytochrome oxidase I encoding region of mtDNA. Three species of calliphorid (blow flies) commonly associated with corpses in western Australia, Calliphora dubia, Chrysomya rufifacies and Lucilia sericata, in addition to specimens of Calliphora augur and Chrysomya megacephala were studied. Phylogenetic analysis of data revealed grouping of species according to genus. The DNA region sequenced allowed identification of all species, providing high support for separation on congeneric species. Low levels of variation between some species of the same genus however indicate that further sequencing is required to locate a region for development of a molecular-based technique for identification.