Forensic Microbiology: When, Where and How.

Forensic microbiology is a relatively new discipline, born in part thanks to the development of advanced methodologies for the detection, identification and characterization of microorganisms, and also in relation to the growing impact of infectious diseases of iatrogenic origin. Indeed, the increased application of medical practices, such as transplants, which require immunosuppressive treatments, and the growing demand for prosthetic installations, associated with an increasing threat of antimicrobial resistance, have led to a rise in the number of infections of iatrogenic origin, which entails important medico-legal issues. On the other hand, the possibility of detecting minimal amounts of microorganisms, even in the form of residual traces (e.g., their nucleic acids), and of obtaining gene and genomic sequences at contained costs, has made it possible to ask new questions of whether cases of death or illness might have a microbiological origin, with the possibility of also tracing the origin of the microorganisms involved and reconstructing the chain of contagion. In addition to the more obvious applications, such as those mentioned above related to the origin of iatrogenic infections, or to possible cases of infections not properly diagnosed and treated, a less obvious application of forensic microbiology concerns its use in cases of violence or violent death, where the characterization of the microorganisms can contribute to the reconstruction of the case. Finally, paleomicrobiology, e.g., the reconstruction and characterization of microorganisms in historical or even archaeological remnants, can be considered as a sister discipline of forensic microbiology. In this article, we will review these different aspects and applications of forensic microbiology.

The Future Is Now: Unraveling the Expanding Potential of Human (Necro)Microbiome in Forensic Investigations.

The relevance of postmortem microbiological examinations has been controversial for decades, but the boom in advanced sequencing techniques over the last decade is increasingly demonstrating their usefulness, namely for the estimation of the postmortem interval. This comprehensive review aims to present the current knowledge about the human postmortem microbiome (the necrobiome), highlighting the main factors influencing this complex process and discussing the principal applications in the field of forensic sciences. Several limitations still hindering the implementation of forensic microbiology, such as small-scale studies, the lack of a universal/harmonized workflow for DNA extraction and sequencing technology, variability in the human microbiome, and limited access to human cadavers, are discussed. Future research in the field should focus on identifying stable biomarkers within the dominant Bacillota and Pseudomonadota phyla, which are prevalent during postmortem periods and for which standardization, method consolidation, and establishment of a forensic microbial bank are crucial for consistency and comparability. Given the complexity of identifying unique postmortem microbial signatures for robust databases, a promising future approach may involve deepening our understanding of specific bacterial species/strains that can serve as reliable postmortem interval indicators during the process of body decomposition. Microorganisms might have the potential to complement routine forensic tests in judicial processes, requiring robust investigations and machine-learning models to bridge knowledge gaps and adhere to Locard's principle of trace evidence.

Positive airway pressure longer than 24 h is associated with histopathological volutrauma in severe COVID-19 pneumonia-an ESGFOR based narrative case-control review.

Background and Objective: A thorough understanding of the pathogenic mechanisms elicited by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still requires further research. Until recently, only a restricted number of autopsies have been performed, therefore limiting the accurate knowledge of the lung injury associated with SARS-CoV-2. A multidisciplinary European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group of Forensic and Post-mortem Microbiology-ESGFOR team conducted a non-systematic narrative literature review among coronavirus 2019 disease (COVID-19) pneumonia cases assessing the histopathological (HP) effects of positive airways pressure. HP lung features were recorded and compared between mechanically ventilated (>24 hours) and control (ventilation <24 hours) patients. A logistic regression analysis was performed to identify associations between mechanical ventilation (MV) and HP findings. Methods: A PubMed and MEDLINE search was conducted in order to identify studies published between March 1st 2020 and June 30th 2021. Key Content and Findings: Seventy patients (median age: 69 years) from 24 studies were analysed, among whom 38 (54.2%) underwent MV longer than 24 hours. Overall, main HP features were: diffuse alveolar damage (DAD) in 53 (75.7%), fibrosis (interstitial/intra-alveolar) in 43 (61.4%), vascular damage-including thrombosis/emboli- in 41 (58.5%), and endotheliitis in only 8 (11.4%) patients. Association of DAD, fibrosis and vascular damage was detected in 30 (42.8%) patients. Multivariate analysis, adjusted by age and gender, identified MV >24 hours as an independent variable associated with DAD (OR =5.40, 95% CI: 1.48-19.62), fibrosis (OR =3.88, 95% CI: 1.25-12.08), vascular damage (OR =5.49, 95% CI: 1.78-16.95) and association of DAD plus fibrosis plus vascular damage (OR =6.99, 95% CI: 2.04-23.97). Conclusions: We identified that patients mechanically ventilated >24 hours had a significantly higher rate of pulmonary injury on histopathology independently of age and gender. Our findings emphasize the importance of maintaining a protective ventilator strategy when subjects with COVID-19 pneumonia undergo intubation.

Forensic evaluation of two nucleic acid extraction systems and validation of a RT-qPCR protocol for identification of SARS-CoV-2 in post-mortem nasopharyngeal swabs.

The COVID-19 outbreak has represented a challenge for the international scientific community and particularly for forensic sciences. The lack of Coronavirus post-mortem testing led the National Institute of Toxicology and Forensic Sciences (INTCF) from Spain to verify the performance and utility of a quantitative reverse transcription PCR (RT-qPCR) clinical diagnosis protocol for SARS-CoV-2 detection (TaqPath™ COVID-19 CE-IVD RT-PCR Kit), to shed light on the cause of death (COD) in potentially COVID-19 cases in judicial autopsies. Two different RNA extraction methods were also tested (EZ1® DSP Virus Kit on the EZ1® Advanced XL robot versus MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit) regarding extraction efficiency, precision and contamination. RT-qPCR was evaluated for precision, specificity, limit of detection and concordance. Both the automated and the manual RNA extraction procedures showed good efficiency, but the automated virus extraction by bio-robot produced more reproducible results than the manual extraction. The SARS-CoV-2 RT-qPCR assay showed high sensitivity with a detection limit up to 10 copies/reaction and high specificity, as no cross-reactivity was detected between any of the 12 different RNA viruses tested, including three types of coronaviruses (SARS-CoV, NL63 and 229E). Reproducibility and repeatability of the studied method as well as concordance with other SARS-CoV-2 molecular detection protocols were also demonstrated.

Microbiology in minimally invasive autopsy: best techniques to detect infection. ESGFOR (ESCMID study group of forensic and post-mortem microbiology) guidelines.

This manuscript aims to: 1) provide specific guidelines on PMM techniques in the setting of minimally invasive autopsy (MIA), both for pathologists collecting samples and for microbiologists advising pathologists and interpreting the results and 2) introduce standardization in PMM sampling at MIA. Post-mortem microbiology (PMM) is crucial to identify the causative organism in deaths due to infection. MIA including the use of post-mortem (PM) computed tomography (CT) and PM magnetic resonance imaging (MRI), is increasingly carried out as a complement or replacement for the traditional PM. In this setting, mirroring the traditional autopsy, PMM aims to: detect infectious organisms causing sudden unexpected deaths; confirm clinically suspected but unproven infection; evaluate the efficacy of antimicrobial therapy; identify emergent pathogens; and recognize medical diagnostic errors. Meaningful interpretation of PMM results requires careful evaluation in the context of the clinical history, macroscopic and microscopic findings. These guidelines were developed by a multidisciplinary team with experts in various fields of microbiology and pathology on behalf of the ESGFOR (ESCMID - European Society of Clinical Microbiology and Infectious Diseases - Study Group of Forensic and Post-mortem Microbiology, in collaboration with the ESP -European Society of Pathology-) based on a literature search and the author's expertise. Microbiological sampling methods for MIA are presented for various scenarios: adults, children, developed and developing countries. Concordance between MIA and conventional invasive autopsy is substantial for children and adults and moderate for neonates and maternal deaths. Networking and closer collaboration among microbiologists and pathologists is vital to maximize the yield of PMM in MIA.

Whole-genome sequencing of Neisseria gonorrhoeae in a forensic transmission case.

Molecular epidemiology and phylogenetic analyses are frequently used in the investigation of viral transmission cases in forensic contexts. Here, we present the methods and results of the analysis of a bacterial transmission episode in an alleged child abuse case using complete genome sequences obtained by high-throughput sequencing (HTS) methods. We obtained genomes of Neisseria gonorrhoeae from the victim, the suspect, and 29 unrelated controls. The analysis of the genomes revealed that the victim and suspect isolates had identical sequences in both the bacterial chromosome and the single plasmid present in them. One of the local controls was very similar (differing in only 2 SNPs) to the case sequences, but the remaining controls were very divergent. Additional cases of identity and very high similarity among controls were observed occasionally, pointing at recent transmission cases. These results were more discriminative than the previous molecular epidemiology analyses performed at the hospital's Microbiology Service, as Multi-Locus Sequence Typing (MLST) could not distinguish between the suspect/victim and the controls isolates, and Pulse Field Gel Electrophoresis (PFGE) was not able to distinguish between the suspect/victim and one of the local controls. These results lead us to conclude that complete bacterial genome sequences obtained with HTS technologies may be a valuable tool for establishing recent transmission cases and, although more studies are needed, they have a great potential for being used in forensic analyses.

Postmortem microbiology sampling following death in hospital: an ESGFOR task force consensus statement.

Postmortem microbiology (PMM) is a valuable tool in the identification of the cause of death and of factors contributory to death where death has been caused by infection. The value of PMM is dependent on careful autopsy planning, appropriate sampling, minimisation of postmortem bacterial translocation and avoidance of sample contamination. Interpretation of PMM results requires careful consideration in light of the clinical history, macroscopic findings and the histological appearances of the tissues. This consensus statement aims to highlight the importance of PMM in the hospital setting and to give microbiological and pathological advice on sampling in deaths occurring in hospital.

[Post-mortem microbiology analysis]. / Análisis microbiológico post mórtem.

Post-mortem microbiology is useful in both clinical and forensic autopsies, and allows a suspected infection to be confirmed. Indeed, it is routinely applied to donor studies in the clinical setting, as well as in sudden and unexpected death in the forensic field. Implementation of specific sampling techniques in autopsy can minimize the possibility of contamination, making interpretation of the results easier. Specific interpretation criteria for post-mortem cultures, the use of molecular diagnosis, and its fusion with molecular biology and histopathology have led to post-mortem microbiology playing a major role in autopsy. Multidisciplinary work involving microbiologists, pathologists, and forensic physicians will help to improve the achievements of post-mortem microbiology, prevent infectious diseases, and contribute to a healthier population.

Clinicopathological features of sudden unexpected infectious death: population-based study in children and young adults.

INTRODUCTION AND OBJECTIVES: Infectious diseases are one of the major causes of sudden death, but data of sudden unexpected death from infectious diseases (SUDID) are sparse in children and young people. The aim of this study is to analyse their epidemiological, clinical, pathological and microbiological characteristics. METHODS: Population observational study of all deaths due to infectious diseases in people aged 0-34 years in Biscay between 1991 and 2010. The data of Mortality Register and Forensic Pathology Service were analysed. The SUDID cases in which a forensic autopsy was performed were identified. RESULTS: There were 56 SUDID cases (16% of the all sudden deaths). Myocarditis (n=20) and acute bacterial bronchopneumonia/pneumonia (n=18) were the most frequent causes, followed by meningococcemia (n=5) and viral pneumonia (n=4). A chronic co-morbid condition (mainly drug abuse in young people and moderate low birth weight in infants) was recorded in one-third and prodromal symptoms in two-thirds. Seventeen young people had consumed illegal drugs and/or methadone. In 17 of the 23 autopsies where microbiological analyses were performed, the organism responsible was identified. The incidence of SUDID was 0.6/100000 inhabitants/year, representing 5% of all deaths from infectious diseases. SUDID rate was 1.87 times higher in males than in females. The highest incidence occurred in infants (3.7). CONCLUSIONS: SUDID is infrequent, but it represents a significant proportion of all sudden deaths in children and young people. The relationship between methadone and bronchopneumonia is noticeable. This article stresses the importance of microbiological analyses in SUDID. Investigation based on forensic pathology provides useful epidemiologic, clinico-pathological and microbiological information for prevention of infectious diseases.

Outbreak of invasive group A streptococcal disease among children attending a day-care center.

BACKGROUND: Most cases of invasive group A streptococcal (GAS) disease arise sporadically in the community, but outbreaks of severe invasive GAS infections have been reported in closed environments, such as military populations, family communities and hospitals. An outbreak of invasive GAS disease involving 3 cases of streptococcal toxic shock syndrome (TSS), one with a fatal course, occurred among children attending a day-care center located in Cantabria, Northern Spain. OBJECTIVE: To determine the characteristics of GAS isolates obtained from the outbreak environment. METHODS: GAS isolates obtained from children attending the same day-care facility, staff members, and family contacts were assayed for emm typing, pulse-field gel electrophoresis (PFGE), and toxin-gene content. One isolate obtained from the fatal case was also characterized by multilocus sequence typing. Antimicrobial susceptibility testing was done. Strains from patients unrelated to the outbreak were included for comparison. RESULTS: All GAS isolates from children attending the day-care center, including those from streptococcal TSS cases, shared the same emm type 4, genomic pattern by PFGE (A) and toxin-gene profile. Neither the emm type nor the PFGE pattern or toxin gene profile of the outbreak-associated strains were encountered among GAS isolated from household or staff contacts. CONCLUSIONS: A clone of GAS belonging to emm type 4 and characterized by a specific PFGE pattern and toxin-gene profile was responsible for a community outbreak of streptococcal TSS disease in a child day-care center in Spain. This is the first day-care outbreak reported in our country.

Detection of human herpesvirus-6, Epstein-Barr virus and cytomegalovirus in formalin-fixed tissues from sudden infant death: a study with quantitative real-time PCR.

BACKGROUND: The role of viruses in the context of sudden infant death in early childhood is still unclear, although there are many findings pointing to a viral infection possibly leading to death. OBJECTIVES: To analyse the prevalence and viral loads of human herpesvirus-6 (HHV-6), Epstein-Barr virus (EBV), and cytomegalovirus (CMV), three viruses that have been previously detected in some cases of sudden death in infants, in tissues from sudden infant death syndrome (SIDS) patients and controls. MATERIALS AND METHODS: Thirty-nine formalin-fixed paraffin-embedded tissue sections of eleven consecutive cases of SIDS, and thirty-nine formalin-fixed paraffin-embedded tissue sections of nine control cases were analysed by a specific quantitative real-time PCR for the detection of HHV-6, EBV, and CMV. RESULTS: The comparison of the whole viral DNA prevalence in cases and tissue sections between SIDS and controls showed a statistical significance (72.7% vs. 22.2%, p=0.025; 41.1% vs. 10.3%, p=0.001, respectively); in particular, we found a statistical significant difference for the EBV DNA prevalence among cases (p=0.042) and tissues (p=0.048), and a statistical significant difference for the HHV-6 DNA prevalence among cases (p=0.017). CONCLUSIONS: This is one of the first studies using quantitative real-time PCR for virus detection in cases of SIDS, and the results suggest that some herpesvirus infections, and particularly those caused by EBV and HHV-6 could be related with some cases of SIDS. Further studies will be necessary to understand the real significance of these findings in the context of SIDS.

[Outbreak of streptococcal toxic shock syndrome in a day care center in Cantabria, Spain, 2006]. / Brote de síndrome de shock tóxico estreptocócico en una guardería de Cantabria en 2006.

BACKGROUND: Beta hemolytic group A streptococcus only exceptionally produces aggressive disease with high lethality. Even more uncommon is the occurrence of an outbreak. In Spain, no outbreak in child care center has been previously described. METHODS: Descriptive study of an outbreak of streptococcal toxic shock syndrome (3 cases, one lethal) in a child care center, which motivated the health care intervention with chemoprophylaxis, the closure of the child care center and the study of contacts. We analyzed the determinants of infection in the invasive and non-invasive cases, and the results of the pharyngeal culture of contacts. RESULTS: We identified 3 invasive and 14 non-invasive cases between 40 children attending the child care center (attack rate 42.5%). We studied 19 possible determinants of the infection, finding only an association with being over the age of 24 months and the assistance to the handouts classroom (that of the oldest children). It was not associated with chickenpox. All children attending the child care center, its staff (4 women) and 258 contacts were microbiologically investigated. In 12 children the emm 4 strain was isolated, including 2 of 3 cases with invasive disease. In 13 of 258 contacts other strains of beta hemolytic group A streptococcus were isolated, but in none of them the strain responsible of the outbreak was found. Azytromicin chemoprophylaxis was implemented for all children and contacts, and in those with a positive isolation, the culture was repeated until negative. CONCLUSIONS: The invasive strain circulated only in the child care center. Azytromicin chemoprophylaxis eradicated effectively the infection.

Real-time polymerase chain reaction detection of Neisseria meningitidis in formalin-fixed tissues from sudden deaths.

Accurate identification of meningococcal sudden deaths is needed to avoid underestimation of the true incidence of the disease. This study analyzed the usefulness of a real-time polymerase chain reaction (PCR) protocol using MGB (3'-minor groove binder) probes to detect Neisseria meningitidis in formalin-fixed paraffin-embedded tissues from sudden deaths where a meningococcal fulminating infection was suspected. The protocol included detection of meningococcal DNA (ctrA gene), multiplex B/C PCR serogrouping (siaD gene), and rapid confirmation of PCR products by microcapillary electrophoresis. Sixty-nine tissues from 15 culture-confirmed meningococcal sudden deaths were analyzed (positive cases). Validation studies were performed. In each positive case, both the ctrA and the B/C siaD genes were detected. The ctrA was detected in 81.2% of the samples, whereas the serogroup (B or C) was identified in 44.9% of them. Therefore, this protocol may improve nonculture diagnosis and case ascertainment in meningococcal disease deaths, particularly when formalin-fixed tissues are the only available specimen.

Challenges of DNA profiling in mass disaster investigations.

In cases of mass disaster, there is often a need for managing, analyzing, and comparing large numbers of biological samples and DNA profiles. This requires the use of laboratory information management systems for large-scale sample logging and tracking, coupled with bioinformatic tools for DNA database searching according to different matching algorithms, and for the evaluation of the significance of each match by likelihood ratio calculations. There are many different interrelated factors and circumstances involved in each specific mass disaster scenario that may challenge the final DNA identification goal, such as: the number of victims, the mechanisms of body destruction, the extent of body fragmentation, the rate of DNA degradation, the body accessibility for sample collection, or the type of DNA reference samples availability. In this paper, we examine the different steps of the DNA identification analysis (DNA sampling, DNA analysis and technology, DNA database searching, and concordance and kinship analysis) reviewing the "lessons learned" and the scientific progress made in some mass disaster cases described in the scientific literature. We will put special emphasis on the valuable scientific feedback that genetic forensic community has received from the collaborative efforts of several public and private USA forensic laboratories in assisting with the more critical areas of the World Trade Center (WTC) mass fatality of September 11, 2001. The main challenges in identifying the victims of the recent South Asian Tsunami disaster, which has produced the steepest death count rise in history, will also be considered. We also present data from two recent mass fatality cases that involved Spanish victims: the Madrid terrorist attack of March 11, 2004, and the Yakolev-42 aircraft accident in Trabzon, Turkey, of May 26, 2003.