Evaluating Bioinformatic Pipeline Performance for Forensic Microbiome Analysis*,†,‡

Microbial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories. These differences can potentially affect microbial community composition and downstream analyses. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, varying minimum library size or minimum number of sequences per sample, and sample size. Using the same input sequence data, we found that three open-source bioinformatic pipelines, MG-RAST, mothur, and QIIME2, had significant differences in relative abundance, alpha-diversity, and beta-diversity, despite the same input data. Increasing minimum library size and sample size increased the number of low-abundant and infrequent taxa detected. Our results show that bioinformatic pipeline and parameter choice affect results in important ways. Given the growing potential application of forensic microbiology to the criminal justice system, continued research on standardizing computational methodology will be important for downstream applications.     

Unusual and Fatal Case of an Undiagnosed Intravascular Large B-cell Lymphoma: The Oncologist’s Great Imitator†

Intravascular lymphoma (IVL) is a rare subtype of extranodal lymphomas that is characterized by the selective growth of neoplastic cells within the lumen of small vessels. Authors document the case of an unexpected death caused by an undiagnosed intravascular large B-cell lymphoma with multi-organ involvement, which had initially manifested as an infection and then as an unclarified central nervous system pathology. Histological examination showed a diffuse intravascular large B-cell brain lymphoma with prominent cerebral involvement. The relevance of the case report reveals the importance of an autopsy of an extremely rare and threatening pathology that in most cases is diagnosed only postmortem. As a result, the role of the forensic pathologist becomes particularly important. When specifically performing an in-depth autopsy evaluation with a specific histologic analysis, it is possible to identify the intravascular lymphoma and declare a more accurate cause of death.     

Ethyl Glucuronide Concentration in Hair of Detainees: A Preliminary Study

Through the measurement of ethyl glucuronide in hair (hETG), it is possible to assess chronic alcohol abuse over time. In this paper, we present a study on hETG in Italian prison inmates. Analyses were performed by LC-MS according to a previously published method. Results were evaluated using the cut-offs established by the Society of Hair Testing. Positives samples (ETG > 30 pg/mg) accounted for 6% of all subjects, with concentrations ranging from 42 pg/mg up to 270 pg/mg, abstinent subjects (ETG < 7 pg/mg) accounted for 88%, and moderate alcohol consumption (7 < ETG < 30 pg/mg) for 6% of the subjects. No females displayed ETG values above 30 pg/mg. Among positive samples, only two subjects did not declare heavy alcohol consumption and were found strongly positive at 210 and 270 pg/mg. To the best of our knowledge, this represents the first study on ETG hair concentration on prison inmates.     

A Comparison of Coronary Artery Stenosis Estimates Made by Forensic Pathologists and Medical Students

Previous studies suggest cardiovascular pathologists are less accurate than noncardiovascular pathologists (e.g., clinical pathologists) in estimating the degree of coronary artery stenosis. To further investigate the effect of training on accurate estimation of coronary artery stenosis, we designed a study to compare the accuracy of estimates made by forensic pathologists versus medical students. Six forensic pathologists and twelve medical students each independently examined 24 images of coronary artery cross sections and gave an estimate of the degree of stenosis. When comparing all 24 images, the forensic pathologists had a median difference between the estimated percentage of stenosis and actual percentage of stenosis of −12.380 and the medical students had a median difference of −16.50 (p-value of 0.08542). In estimating the percentage of stenosis, training in forensic pathology does not guarantee significantly improved accuracy compared with medical students. Our study showed no consistent statistically significant difference between estimates given by forensic pathologists and by medical students.     

A Study of the Background Levels of Male DNA on Underpants Worn by Females

This study is the first to examine the background level of male DNA on underpants worn by females in the absence of sexual contact. Here, we examined 103 samples from the inside front of underpants from 85 female volunteers. Samples were examined for the presence of male DNA using NGM SElect and PowerPlex Y23 kits. Only five samples gave a “complete” Y-STR profile, even though 83.5% of our volunteers cohabited with a male. In all cases where a partner reference sample was available, the Y-STR profile matched the cohabiting partner. We have demonstrated that a Y-STR profile is not expected on the inside front of underpants worn by females after social contact alone. The results of this study are informative for evaluating the significance of a Y-STR profile on underpants in cases of alleged sexual assault.     

Sample Mining and Data Mining: Combined Real-Time and Retrospective Approaches for the Identification of Emerging Novel Psychoactive Substances

Novel psychoactive substances (NPS) are synthetic drugs that pose serious public health and safety concerns. A multitude of NPS have been identified in the United States, often implicated in forensic investigations. The most common and effective manner for identifying NPS is by use of mass spectrometry and the true utility lies within nontargeted acquisition techniques. During this study, a liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) assay was developed, validated, and implemented for forensic toxicology testing. A SCIEX TripleTOF™ 5600 + with SWATH^® acquisition was used. Resulting data were compared against an extensive library database containing more than 800 compounds. The LC-QTOF-MS assay was applied to the reanalysis of biological sample extracts to discover emergent NPS. More than 3,000 sample extracts were analyzed, and more than 20 emerging NPS were detected for the first time. Among these were isopropyl-U-47700, 3,4-methylenedioxy-U-47700, fluorofuranylfentanyl, N-methyl norfentanyl, 2F-deschloroketamine, 3,4-methylenedioxy-alpha-PHP, eutylone, and N-ethyl hexedrone.     

Empirical Evaluation of the Reliability of Photogrammetry Software in the Recovery of Three-Dimensional Footwear Impressions

This paper examines the reliability of Structure from Motion (SfM) photogrammetry as a tool in the capture of forensic footwear marks. This is applicable to photogrammetry freeware DigTrace but is equally relevant to other SfM solutions. SfM simply requires a digital camera, a scale bar, and a selection of oblique photographs of the trace in question taken at the scene. The output is a digital three-dimensional point cloud of the surface and any plastic trace thereon. The first section of this paper examines the reliability of photogrammetry to capture the same data when repeatedly used on one impression, while the second part assesses the impact of varying cameras. Using cloud to cloud comparisons that measure the distance between two-point clouds, we assess the variability between models. The results highlight how little variability is evident and therefore speak to the accuracy and consistency of such techniques in the capture of three-dimensional traces. Using this method, 3D footwear impressions can, in many substrates, be collected with a repeatability of 97% with any variation between models less than ~0.5 mm.     

Detection of Mitragynine in Mitragyna Speciosa (Kratom) Using Surface-Enhanced Raman Spectroscopy with Handheld Devices

A simple, quick, selective, sensitive, and effective field-friendly method capable of being used by nonexperts has been developed for detecting mitragynine in Mitragyna speciosa (kratom) using surface-enhanced Raman spectroscopy (SERS). Over 100 samples and blanks (known to be either positive or negative for the presence of mitragynine) were examined in duplicate using five identical handheld Raman spectrometers, which provided a data set of over 1,000 examinations. Based on the results of these analyses, the method yielded a true-positive rate of 99.3%, a true-negative rate of 97.9%, a false-positive rate of 2.1%, and a false-negative rate of 0.7%. The average minimum detectable concentration (C_m) of mitragynine that reproducibly yielded a match for one of the library spectra on all five instruments was determined to be 342 ng/mL (ppb). This C_m value is a conservative estimate considering that the extraction process was not fully optimized by this study, which was not necessary since the C_m value achieved was well below typical mitragynine concentrations in kratom (1.3–2.3%). The method is ideal (i) for prioritizing samples for additional testing using other more time-consuming laboratory-based techniques needed to detect and quantify mitragynine and (ii) for field use at international mail facility (IMF) satellite laboratories to help interdict kratom and prevent this dangerous product from reaching the U.S. supply chain.     

Postmortem Changes of Female External Genitalia and Their Importance in Suspected Sexual Abuse

Examination of the female external genitalia to assess for sexual abuse is performed in living individuals, and the interpretation of the findings is based on evidence-based studies. However, in the deceased, no such studies are available, and postmortem changes could present as suspicious findings that can be mistaken for trauma. Patches of discoloration in the hymen were reported previously in one case as hypostasis (i.e., livor and lividity), and based on this finding, it was listed as a finding that is not associated with trauma. This was a retrospective study that was conducted in the Center of Forensic and Legal Medicine in Dammam, Saudi Arabia over a 4-year period. The study included 30 deceased women in whom photographic documentation of their external genitalia was assessed for postmortem changes. The postmortem interval ranged from less than 24 h to more than 100 days, and the ages of these deceased women were in the 20–40 year-old age group. In cases where the hymen, vagina, and/or fossa navicularis were clearly visible, none of these areas showed any hypostatic discoloration. A comparison between antemortem and postmortem appearance of the hymen in one case clearly showed the absence of hypostatic changes in the hymen. In conclusion, any discoloration of the external genitalia that is detected in a female decedent requires serious consideration.