Forensic and Autopsy Microbiology

The role of microbiology in forensic science and post-mortem examinations: a special focus on molecular diagnosis

Amparo Fernández©-Rodríguez by author Microbiology Laboratory, Biology Service InstitutoESCMID Nacional Online de Toxicología Lecture Library y Ciencias Forenses (INTCF), Madrid, Spain Glamorous forensic medicine is in the media

For more than 100 years, physicians have relied on isolation and characterization of bacteria to understand disease and develop both patient- specific treatment plans Koch’s postulates link specific microorganisms to specific diseases that remain today the “gold standard” in medical microbiology

Isolation of a pathogen microorganism in pure culture from a patient: association with infectious disease © by author However, clinical microbiology laboratories are increasingly moving away from primary isolation of pathogensESCMID to culture independent Online diagnostic testingLecture (CIDT)… Library …CIDT provides multipathogen diagnostic panels with remarkably brief turn-around times. Clinical laboratories are increasingly using CIDT methods and are less likely to conduct primary isolation Autopsy microbiology Determining the cause of death Or predisposing factors to death Applications in forensic / clinical pathology

Main goals of forensic microbiology

©Is there by aauthor crime? Attribution: who committed a crime? ComparisonESCMID of strains isolated Online in a victim Lecture to those associated Library with the assailant “Autopsies tell communities why people are dying. Take for example West Nile Virus. When eight New Yorkers died of brain infections in 1999, most everyone assumed they had a common virus. But New York City's medical examiners insisted on autopsies. And, what do you know, they discovered the first U.S. cases of West Nile Virus.” © by author ESCMID Online Lecture Library Something is moving…

2014: Huge number of articles regarding forensic & autopsy microbiology

© by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Mycological examination of the respiratory and gastrointestinal tracts in newborn from the forensic medicine‘s point of view. DOSA A. Kiserl Orvostud. 1958 Feb;10(1):1-10. Hungarian. No abstract available. Role of the forensic microbiologist: STI Medico-legal issues On people Person to person STI in Other transmission of children biocrimes Environment infection Bioterrorism Other infections Infants Natural deaths: sudden-unexpected Transplantation Children

Paleomicrobiology Forensic Malpractice Adults Microbiologist Judicial complaint Outbreaks © by author investigations

Crime scene Violent death ESCMIDTiming the investigation Online Lecture LibraryDrowning death Identification Tracing the purposes perpetrator Forensic microbiology today

Water and food Clinical Molecular Environmental microbiology analyses microbiology Soil Plants

Contamination© by author issues Preventive measures at the crime scene and autopsy ESCMID Online Lecture Library Chain of custody In developing countries: Causes of death unknown

The Problem of Mortality Statistics in Resource Poor Settings: The Upsurge of the Verbal Autopsy © by author ESCMID Online Lecture Library In developed countries

Feb. 22, 2010 – “Every year, 48,000 Americans die of infections they caught while in the hospital -- and that's a conservative estimate, a new study finds”. We know what happen in hospitals… Or we don’t? The Vanishing Nonforensic Autopsy Kaveh G. Shojania, M.D., and Elizabeth C. Burton, M.D. N Engl J Med 358;9 february 28, 2008 “Autopsies are performed after less than 10% of all U.S. deaths. At the majority of nonacademic institutions, few or no autopsies are ever performed”.

Proposed © by author alternatives minimally invasive postmortem ESCMID Online Lectureless accurate? Library investigations, such as needle specialized resources or personnel biopsy or endoscopy, and radiology Statistics talk about less clinical autopsies What about deaths out of the hospital?

16% unexpected deaths in young people: infectious origin “Most deaths occurred out of hospital. Approximately one- third of the subjects © by author were found dead in bed.” ESCMID Online Lecture Library Sudden unexpected deaths Need of molecular analyses

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No. 3, March 2016 11 (42%) of 26 fatal cases had been misdiagnosed or undiagnosed and were reclassified as IMD after rPCR showed meningococcal DNA in all available specimens  PCR: taken postmortem. © by authorrespiratory tract  central nervous system

ESCMID Online ValidationLecture to use it in fixed Library tissues from postmortem samples Outbreaks forensic studies: survivors and deceased First outbreak of a GAS disease in a child day-care centre in Spain

PFGE FoodESCMID-borne outbreaks Online Lecture Library NGS: complete genome 100 Salmonella Bareilly

• 2011/12: Outbreak in US • Isolates from natural sources, and from unrelated historically and geographically disparate foods. • Pathogen genomes were© by linked author to geography • Phylogenetic analysis of WGS data revealed a common originESCMID for outbreak Online strains: Lecture Library • Patients in Maryland and New York were infected from sources originating at a facility in India. Food-borne Outbreaks

PulseNet

• CDD: Plans are underway to transition to routine Whole Genome Sequencing for Shiga toxin– producing E. coli, Salmonella, and Campylobacter

FDA Foods Program: © by authorGenomeTrakr WGS network and database >12000 food-borne pathogens publically ESCMID Online Lecture Libraryavailable Using Genomics to Identify the Source of an Outbreak • WGS is a precise forensic tool

• coupled with epidemiological analysis, enables new opportunities to investigate outbreaks

• Tracing global food© -byborne author contamination events back to their source. ESCMID Online Lecture Library Violent death

Is that one a violent death or a natural one? Clinical-forensic microbiology

Assessing bacterial markers of water the cause of faecal pollution: © by author faecal coliforms death and faecal Timing the streptococci Drowning death Also AeromonasESCMID Online Lecture Library Quantification of 3 intestinal bacterial genera by quantitative PCR (qPCR) group- specific primers targeting 16S rRNA genes

• Bacteroides and Lactobacillus relative abundances declined exponentially with increasing PMI • Bifidobacterium did© not by change author significantly: BacteroidesESCMID and Online Lactobacillus Lecture abundances Library could be used as quantitative indicators of PMI. Crime attribution: linking the assailant with the victim &/or the crime scene

Sexual Assault: STI Bioterror: Identifying strains

Identifying the type of Tracing the fluid: microbioma perpetrator © by author

Environmental Fungi matching Soils Microbial ESCMID OnlineComparison Lecture Library Forensic genetics

Crime scene investigation  Identification of biological fluids: semen, blood stains, saliva, vaginal fluid

 Human Identification:  Aproximately 10 pg nuclear DNA  Mixtures female/male 40/1 © by author

ESCMIDMicrobial Online Forensics Lecture applications Library? Identifying the type of fluid

In the context of forensic casework, inferring the type(s) of cell material present in an evidentiary sample is only of value when accompanied by information regarding the possible donor of the cells

Pattern of bacteria characteristic of vaginal fluid

© by author ESCMID Online Lecture Library Real-time PCR panel for Species of Lactobacillus Microarray analysis of a sample: to detect multiple species from various genera Probes for species: (1) only detected in vaginal samples (2) detected in all or the majority of vaginal samples (3) less common in vaginal samples© but by frequently author found on other body sites.

It will render a microbial flora pattern that is probably best analysed in a probabilisticESCMID approach Online resulting Lecture in support Library for hypothesis A or B (body site A versus B). The salivary microbiome for differentiating individuals: proof of principle • Potential for bacteria found in the salivary microbiome to be used to differentiate individuals.

• Combination of 16S rRNA + rpoB: to maximise coverage of the salivary microbiome increasing the power of differentiation (identification).

• Streptococcus, (Firmicutes), one of the most abundant aerobic genera found in saliva and targeting Streptococcus rpoB

• Samples from the same person© group by together author regardless of time of sampling. It is ESCMIDpossible to distinguishOnline Lecture two people Library using the bacterial microbiota present in their saliva • Microbial communities show unique structure and composition based on surface type, the identity of the person interacting with the surface, and geographic location.

• It is possible to infer individual identities based on the microbial community associated with their smart phone surface © by author • it is less likely that this assemblage could be used to track where that person has been recently located in space due to theESCMID rapid turnover Online of the surface Lecture-associated Library microbial community.

Microbial Forensics Scientific discipline dedicated to the analysis of forensic evidences to identify and criminally prosecute those responsible for a biological attack, and it involves a detailed analysis of the weapon--that is, the pathogen

Capability to respond to biocrime and bioterrorism incidents to aid in the complex task of distinguishing between natural outbreaks and deliberate acts. Goal

individualization to track unambiguosly its origin© by author Prosecution aims Prevention Identify, quickly and in an unbiased manner, previously undetectable genomeESCMID differences betweenOnline closely Lecture related isolates. Library “AMERITHRAX” INVESTIGATION Whole-genome sequence analysis Strains comparisons: Identification, characterization and attribution Some of the most deadly bacterial diseases are caused by bacterial lineages with extremely low levels of genetic diversity. Availability of high-quality reference© by databases author of bacterial whole-genome sequences.

Need to beESCMID curated and accurate Online in terms Lecture of sequences, Library metadata, and genetic diversity coverage. Sexual assault / abuse To take into account…

In children the isolation of a sexually transmitted organism may be the first indication that abuse has occurred.

Give sexual assault patients priority as emergency cases © by author ESCMID Online Lecture Library General guidelines…some of them from the Judicial System

Most of them lack of specific recommendations about microbiological© by author techniques!! ESCMID Online Lecture Library Creation of: a) Customized protocols b) Sexual Assault Response and Resource Teams

Laboratory investigations Microbiologist

Victim Forensic advocate examiner Physical /prosecutor trained in STI examination

Integrated team © by author Psychosocial Psychologist Pediatrician evaluation Sexual ESCMID Onlineassault Lecture Library nurse Main goals: health and forensic: attribution

Sexual Assault Evidence Collection Kit: including microbiology sampling!!

Preserve evidence and maintain the chain of custody.

Encourage prophylaxis /treatment for STI as well as follow-up STI exams

Lesion exudate Marker HSV-1 ESCMID Online Lecture Library(FAM) From Standard microbiological methods…

…To Nucleid Acid Amplification Tests (NAAT) with a higher sensitivity compared with conventional culture method

Sometimes, this is the only forensic evidence

Neisseria gonorrhoeae: the most common organism. • N. gonorrhoeae strain typing can also be used to identify the abuser:

Assessing whether a person suspected of involvement could have been on the crime site

The marker of choice: 16S rRNA gene small ribosomal subunit

0,25 mg Already used as a forensic evidence

Results of the analyses on the soil samples compared with two specimens © by authorof soil (CarR , CarL), found respectively on the right and left carpets of the suspect's car floor. ESCMID Online Lecture Library

Concheri G, Bertoldi D, Polone E, Otto S, et al. (2011) Chemical Elemental Distribution and Soil DNA Fingerprints Provide the Critical Evidence in Murder Case Investigation. PLoS ONE 6(6): e20222. doi:10.1371/journal.pone.0020222 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020222 High Throughput Sequencing

Fungi, Arthropoda, Nematoda, Rotifera or Porifera © by author ESCMID Online Lecture Library Microorganisms as markers of pollution: soil, water, sediments

(Vibrio fischeri NRRL B-11177) Environmental luminiscence crimes

ESCMID Guidelines: post©- mortemby author and forensic microbiology Sampling Methodology:ESCMID including molecular Online analyses Lecture Library Analysis of archival specimens

Fixed brain White purulent meningeal exudate

Real-time PCR © by author + Streptococcus pneumoniae ESCMID Online Lecture Library At the sampling…Some tips for molecular methods “flocked swab with liquid Amies media”

Allow: Molecular analyses, bacteriology and immunochromatography Blood collection tube for whole blood specimens to extend the stability of nucleic acids for molecular analyses © by author

For viral molecular analyses: ESCMIDcontainer/tube Online with sterile Lecture saline or an RNALibrary stabilization solution Forensic microbiology at the 21th century

Thanks to Veroniek Saegeman, Juan Alberola & Catherine Moore for their useful comments And my colleagues B Aguilera, C Albarrán, JC Cordero, B Morentin, R Mosquera, MP Suárez, M Sánchez de León & I Téllez for sharing their cases