Lecture 3 - Disease and Microbial Forensic Investigations

What is the epidemiological triad?

The interaction of three things a susceptible host, an infectious agent or toxin, and the environment

What are the modes of agent transmission?

• Horizontal

  • Direct

  • Indirect

    • Vehicle or vector

• Vertical

What is an endemic?

The habitual presence of a disease within a given geographical area or as the usual occurrence of a given disease within such an area

What is an epidemic?

The occurrence of a disease in a community or region, clearly in excess of what is normally expected, and generally derived from a common source or from a propagated source

What is a pandemic?

Refers to a worldwide epidemic often involving two or more continents

What are the 3 outbreak investigation questions that determine how and why of an outbreak?

• Who has the disease?

• When did the disease occur?

• Where did the case arise?

What is the phenotypic method?

• Looking at the product of gene expression or the phenotype

• Generally provides little discrimination power

• Ex. Serotyping, Phage Typing, Biotyping, LMEE

What is the genotypic method?

• Analyze genetic structure of organism

• Ex. Restriction enzyme-based, amplification-based, sequence-based analyses

What are the objectives of epidemiology?

• Determine the extent of disease present in community

• Identify the etiology of a disease and the factors that increase a person’s risk for disease

• Study the natural history and prognosis of disease

• Evaluate new preventative and therapeutic measures and new modes of healthcare delivery

• Provide a foundation for developing public policy and regulations

What are the common steps for investigation an infectious outbreak?

1. Verify the diagnosis

2. Establish a case defintion

3. Identify cases

4. Verify you have an epidemic

5. Develop hypotheses

6. Test hypotheses

7. Recommend and implement control/prevention mesures

8. Communicated findings

What are epidemiological clues that may signal biological or chemical attack?

• Single case of disease caused by an uncommon agent without adequate epidemiological explanation

• Unusual, atypical, generally engineered, or antiquated strain or an agent

• Higher morbidity and mortality in association with a common disease or syndrome or failure of such patients to respond to usual therapy

• Unusual disease presentation

• Disease with an unusual geographic or seasonal distribution

• Stable endemic disease with an unexplained increase in incidence

• Atypical disease transmission through aerosols, food, or water in a mode suggesting sabotage

• No illness in persons who are not exposed to common ventilation systems when illness is seen in close-proximity who have a common ventilation system

What are the differences in infectious disease investigations between forensic and epidemiology?

• Direction → police or law authority vs. vet or public health officer

• Main goals → attribution vs. identity cause to prevent reoccurrence

• Secondary goal → deter other perpetrators vs. contribute new scientific or risk management knowledge

• Identification of infectious agents → general possible level of detail vs. to species level at least (subspecies variation)

Ch.8 Epidemiological & Microbiological Investigations for Biosecurity - Natural Outbreaks

• Salmonella linked to contaminated pasta salad and ice cream from raw eggs previously in milk container

• Legionella linked to grocery store misters

• Francisella tularensis from a rabbit hit by a lawn mower

Ch.8 Epidemiological & Microbiological Investigations for Biosecurity - Global travel spreads infectious disease

Ebola caused by Zaire ebolavirus in West Africa

• 18 month old boy from Guinea believed to have been infected by bats

• Weak surveillance systems and poor public health infrastructure attributed to the difficulty of the containment of the outbreak

• EVD spread to 7 more regions

• To prevent cross-border transmission, travellers leaving WA were screened

• The US implemented enhanced entry screening for travellers from those areas to a designated airport

Ch.8 Epidemiological & Microbiological Investigations for Biosecurity - Infected Aedes species mosquitos are great hosts

• Zika virus was originally detected in African rhesus macaques, 2015 outbreak in Brazil

• Yellow fever in 2015 spread to the DRC, Uganda, Kenya, Ethiopia, and China

• Chikungunya virus from 2007-14 spread from Africa and Asia to Italy, France, and the US

• >70% of viruses are detected in human and other mammalian species

Ch.8 Epidemiological & Microbiological Investigations for Biosecurity - Deliberate Salmonella food poisoning in Oregon

• Rare strain of S. typhimurium was used, foul play was not suspected

  • No history, no one claimed responsibility, no motives, no pattern of unusual behaviours, etc.

• A terrorist does not always take credit from their actions

• Two documented cases of bioterrorism in the US - this one and anthrax letters

Ch.8 Epidemiological & Microbiological Investigations for Biosecurity - Pulsenet, the national molecular subtyping network for food-borne disease surveillance

• Began subtyping a single pathogen and expanded to include other pathogens

• Salmonella infections linked to Kellogg’s Honey Smack cereal across 33 states in 2018

• Increases the ability to link apparently unrelated outbreaks and identify a common source

Ch.8 Epidemiological & Microbiological Investigations for Biosecurity - Accidental escape of pathogens with letal or pandemic potential from research laboratories

• Unintentional release of viable anthrax spores and potential exposure of non-inactivated Ebola virus

• 86 facilities in the US and 7 foreign received “inactivated” anthrax spores that were viable

• Accidental release of variola virus, SARS coronavirus and the 1977 Influenza A/H1N1 virus

Ch.8 Epidemiological & Microbiological Investigations for Biosecurity - How to distinguish between natural & deliberate exposure?

• Community outbreak of individuals with smallpox-like lesions

  • Disease eventually identified as monkeypox

  • Individuals were infected by prairie dogs purchased as pets, acquired monkeypox while co-house with giant Gambian rats from Ghana

• Hemorrhagic fever in demilitarized zone in the Republic of South Korea

  • Linked to rodent hosts at training sites

What is microbial forensics?

An extension of epidemiological principles to enable investigation of unlawful acts involving biological agents

What are 3 microbial forensic concepts?

• Attribution: Assignment of a sample of questioned origin to a source of known orgin to a high degree of scientific certainty while, at the same time, excluding that is originated from other sources

• Conventional crimes: fingerprints, blood, semen, saliva, fibers, documents, powders, tool marks, etc.

• Bicrimes: All listed + microbial evidence

What are key investigative questions?

• Did a crime occur?

• What happened?

• How did it occur?

• When, where, & why did it occur?

• Who was involved?

• What evidence exists?

• What does the evidence tell us?

• How string are the links?

• How reliable and credible is the evidence?

• What alternative explanations are there for evidence?

• What extra safety precautions must be taken?

• What biological agents are suspected?

• How should the microbial evidence be handled?

What is included in microbial evidence?

• Chain of evidence/custody

• Collection, transport, and storage of microbial evidence differs compared to other physical evidence

• This collection creates new challenges in the investigation

  • Must protect the people collecting, transporting, storing, working with agents

  • Must preserve and protect the microbial evidence

What are the sampling activites?

1. Sampling strategy development

• Sample size, locations to collect samples from, method of collection

1. Sample collection

2. Sample transportation

3. Sample extraction

4. Sample analysis

• Preliminary and confirmatory tests

What are the points to consider when establishing a sampling plan?

• Appropriate sampling strategy depends upon the underlying questions to be answered or mission to be accomplished

• Targeted collection is an appropriate approach for law enforcement when use of a biological agent is suspected or when information on the source of a possible biological agent is available

• A Bayesian acceptance sampling model combines information derived from targeted and randomly collected samples is designed sampling after decontamination or demonstrate the cleanliness of an areas that is presumed not to be contaminated during the original event

How do you establish a sampling strategy?

• Assess the scene/situation

• Create a sampling plan of action

  • Safety of personnel

  • Compliance with all regulations and legal requirements

  • Prioritization of sampling

  • Determining the appropriate personnel and equipment

  • Timetable

• Documentation of:

  • Location, area, building, animal, subject

  • Sample provenance and chain of possession

• Applying validated collection techniques and equipment

• Preservation and storage of samples

How does collection work?

• Samples must be appropriately packages, labelled, surface decontaminated, and maintained in a secure, temporary manner until final packaging and submission to secured storage or an analytical lab

• Bacterial viability or viral integrity in specimens must be maintained using appropriate media and recommended temperatures

How do you sample microbes?

• 3 approaches

  • Bulk collection of an entire item

  • Collection of a portion of an item by vacuuming or collection of liquids

  • Swabbing or wiping the surfaces

• Pre-moistened swabs are better than dry and contact plates or tapes may be better than swabs on flat nonporous, non-absorbent, and porous surfaces

• Collection and preservation methods must permit genetic analysis of DNA, RNA, and rRNA, as well as ligand, visual and mass spectrometry analyses, along with the potential future analysis withe emerging methods

What are the goals for collecting microbial evidence?

• To obtain sufficient bioloigcal agent to support both species/strain or toxin identification for critical public health decisions and complete signature characterization for valuable lead information

• Traditional forensic evidence must also be collected at the scene

• The techniques developed for microbial forensics will be applicable in investigations for intelligence, non-proliferation, and verification purposes

How is shipping done?

• Transport of infectious substances/dangerous goods

• State/Provincial and Federal authorities for guidance, instruction, permission

• Receiver must have permission and approval to receive the goods

What is the preservation and storage?

• Microbial samples must be properly packaged/stored

  • Store in appropriate media at proper temperature

• Liquid samples are preserved differently than dry samples

How does the chain of evidence continue at the lab?

• Evidence is categorized as individual or class

  • Individual: evidence associated with a reference or source sample to a high degree (fingerprints, human DNA markers)

  • Class: evidence that can only be included within a group (blood groups, mtDNA, hair)

What are microbial forensic limitations?

• Unique identification difficult because of clonal reproduction

• Most evidence is expected to fall into the class-characteristic category whether limited or informative

What are the difference in forensics vs. microbial forensics?

• Species

  • Conventional - only 1

  • Microbial - large #

• Analysis

  • Conventional - rigorous standardized DNA methods

  • Microbial - no specific method used

• Safety

  • Conventional - samples are mostly ‘safe’

  • Microbial - safety precautions required during all steps

In what way is microbial forensics a young discipline?

• largely built on science and technology developed and used for other fields and purposes - transposed to forensic applications

• Some strategy, review, gap identification, policy, papers, and validation guidelines have been published

• Limited independent, external, peer reviewed studies, and limited validated methods of analysis but decreasing

• Few court cases to date, few case law precedents

• No legal challenges or rulings to date: admissibility of novel scientific evidence or methods, evidence, results or conclusions at trial

What are microbial forensic tools?

• DINA methodologies are predominant, and biomarkers are already available

  • Problem - for organisms without a history of investigation we do not know where the variable regions of all genomes are located or how to rapidly detect change

  • Some techniques have been used (PFGE, MLVA, WGS)

• Not just DNA

  • Physical attributions, isotope analysis, growth media remnants, preparation additives

What is the purpose and regions of DNA for discrimination between individual isolates/clonal lineages?

• “Fingerprinting” - tracking transmission of genotypes/identifying sources of infection and risk factors

• Highly variable genetic markers that are not under selection by the host

What are the components of microbial forensiscs?

• Detection and identification

  • DNA-based, analytical chemistry and physical analysis, culture, immunoassays, bioassays

  • Information and databases

    • Genomic sequence data, controlled access

    • Relational databases, encyclopedic databases

• A strain repository

  • To provide reference samples for comparison

• Newly validated analytical methods

• Quality assurance guidelines

What are challenges in microbial forensics?

• Integrating and sharing data across multiple databases

• Molecular technologies cannot assess whether a live organism was released, if people were exposed, or a deliberate release occurred

• Analytic methods must be refined to improve pattern recognition and integration of multiple streams of epidemiologic and lab data to enable more rapid detection of outbreaks

• Environmental monitoring must be further refined to enable rapid identification of biological agents present in the environment so that public health and medical response efforts can be initiated

• Considering information from public health and epidemiological investigations when interpreting information from environmental monitors

What are the technological advanced and knowledge gaps?

• Gene editing may result in a loss of function through knock-out, a change of function through knock-in technologies

• Influenza A/H5N1 virus has been modified so that it was transmissible by the airborne route in ferrets

• It has been demonstrated that mail-order DNA can be used to create horse pox virus de novo

• The direct identification of resistance genes by PCR or similar methods of limited used because only a fae resistance genes are strongly associated with phenotypic resistance

What is SWGMGF

• Scientific Working Group on Microbial Genetics and Forensics

• Initiated in 2002 to contribute to the microbial forensic infrastructure and development of a research agenda

• Baseline requirements for a compre program:

  • Detection and identification

  • Information databases

  • A strain repository

  • Validated analytical methods

  • Quality assurance guidelines

Ch 21. Collection and Preservation of Microbial Forensic Samples - The US Government Accountability Office (GAO)

• Report of the sample collection and analysis by the US Postal Service, the CDC, and the EPA following 2001 anthrax letter investigations

  • Assessment of sampling strategy for anthrax after cleanup of postal facilities

  • Concluded results unreliable due to the use of targeted sampling approach, and unvalidated collection/analytical procedures

• Argued probability sampling would have better allowed agencies to determine, with some defined level of confidence whether a building was contaminated