P1252428581SEoPN

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Human Identity Testing

• Forensic cases criminal, civil
• Paternity testing -- identifying father, nursery
  mix-ups, immigration, inheritance
• Historical investigations
• Population studies, human diversity, anthropology
• Missing persons investigations
• Military DNA dog tag
• Convicted felon DNA databases
• Patient sample mix-ups
• Mass disasters

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Forensic Science

• Application of science in the investigation of
  legal matters
• Scientific knowledge and technology are used to
  serve as witnesses in both criminal and civil
  matters
• Science may not offer definitive solutions for
  all scenarios it does provide a special
  investigative role
• Goal is attribution i.e., who committed the
  crime

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Forensic Practices

• A crime is recognized
• Identify crime scene
• Crime scene is secured to preserve the quality of
  the evidence
• Properly collect and preserve physical evidence
• Set up a documented chain of custody on all
  evidence collected and record the crime scene
  (scientific and legal integrity)
• Evidence is sent to the laboratory for analysis
• Cadre of analytical procedures may be used to
  identify the material
• A comparison is made of the data obtained from
  the evidence sample(s) with that obtained from
  analysis of a reference sample(s)
• Interpretation of data
• Unique identification of the sample and thus its
  ultimate source or origin is desired
• In many cases that may not be possible

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Mass Disasters
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Embassy Bombings
AUGUST 7, 1998
Nairobi, Kenya 1039 am
Dar es Salaam, Tanzania 1045 am
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World Trade Center (AA 11, UA 175)
9/11/01
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Value of DNA Compared with Other Identity
Tools/Methods

• Tissue sources (blood, semen, muscle, organs,
  bone, tooth hair, saliva, urine)
• Compare different body parts
• Kinship analysis

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Disadvantage of DNA Compared with Other Identity
Tools/Methods

• Cost
• Time
• Labor
• Expectations

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Methods

• RFLP/VNTR typing
• PCR - HLA-DQA1, Polymarker loci by reverse dot
  blot assay (SNP)
• PCR D1S80 locus (VNTR) typing by
  electrophoresis and silver staining
• PCR STR loci typing by capillary
  electrophoresis and fluorescence
• PCR mitochondrial DNA by capillary
  electrophoresis and fluorescence
• PCR Y STR loci by capillary electrophoresis and
  fluorescence
• PCR - SNPs

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Bioterrorism/Biocrime
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Better to be scared by the improbable
possibility, than to be unprepared for the
catastrophic reality.
Congressman Christopher Shays (R-CT), Chairman of
the Subcommittee on National Security, Veterans
Affairs, and International Relations
FMD-infected cattle pyres
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Four Mission AreasBioterrorism or other crime
PREVENT Terrorism by tackling underlying causes
To reduce the THREAT
PURSUE Terrorists and those That sponsor them
To reduce the RISK
PROTECT The public and Nation interests
To reduce VULNERABILITY
PREPARE For the consequences
Forensic Science one approach
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Anthrax Attacks Florida New York
Washington, DC
Demonstrated the need for a coordinated national
response for threat assessments countermeasures
and forensics for biological agents
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Terrorism

• Many may not see the concern and effort

• After all there was only the anthrax incident
  in October 2001.

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Biological Warfare

• Ancient Romans threw carrion into wells to poison
  their adversaries drinking water
• 14th Century the Tartars catapulted bodies of
  bubonic plague victims into the walled city of
  Kaffa
• Conquistadors and British used smallpox and
  measles to help subdue Native Americans (in
  contaminated blankets)
• World War II, the Japanese released
  plague-infested fleas in China
• In 1984 followers of Baghwan Sri Rajneesh
  attempted to incapacitate the population of
  Dalles, Oregon on election day by contaminating
  salad bars with Salmonella typhimurium (more than
  750 people had food poisoning)
• Aum Shinrikyo Cult and sarin nerve gas also
  attempted to spread anthrax (1993)

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Aum ShinrikyoTokyo, Japan
Shoko Asahara
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Terrorism

• Shoko Asahara
• Aum Shinrikyo
• Attempted to require biological materials
• Contacted Russians, Zaire (Ebola)
• 10 separate attempts with aerosolized anthrax and
  botulinum toxin (1990-1995)
• Experimented with botulinum toxin, anthrax,
  cholera, Q fever

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The Aum Shinrikyo Kameido Facility (1993)
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Kameido Isolate Matches the Sterne Strain
pXO2 Minus
Sterne Strain
Kameido Isolate
vrrC1
vrrC2
vrrA
vrrB1
vrrB2
CG3
J. Clinical Micro. 394566-7
pXO1-AAT
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Theories??

1. Technical Incompetence
2. Trial Run for Real Event
3. Bad Leadership

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Interior of Mail Sorting Facility
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Air sampling of mail bags
Liquid impinger critical orifice ca. 12 L/min
1) 100 cfu 2) 300 cfu 3) 21,500 cfu
20 bags sampled for 2 min
Spores isolated from 3 bags
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LEAHY LETTER
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Powder Release in Mail Sorter
Courtesy David Bowers, United States Postal
Inspection Service
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Examples of Biological Terrorism

• 1996Minnesota, USA, Four right-wing extremists
• Produced a sizable amount of ricin.
• Targeted Deputy U.S. Marshal.
• Planned to wipe ricin on car door handles and
  steering wheel.
• Were the first to be prosecuted under Title 18,
  Part I (Terrorism).

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Microbial Forensics

• Analysis of evidence from a bioterrorism act,
  biocrime, or inadvertent microorganism/toxin
  release for attribution purposes
• Essentially the same as any other forensic
  discipline

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Intersection of Disciplines
Molecular Biology
Physics
Genomics
Informatics
Epidemiology
Forensic Science
Microbiology, Virology Mycology
Biostatistics Population Genetics
Microbial Forensics
Plant Pathology
Biomedical Sciences Public Health
Ecology
Analytical Chemistry Biochemistry
Veterinary Medicine
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Magnitude of Problem

• Any infectious agent can be used as a biological
  weapon
• Emerging pathogens
• Over 1000 agents known to infect humans
• 217 virus species
• 538 bacterial species
• 307 fungi
• 66 parasitic protozoa
• Additional plant and animal pathogens not counted
• Numerous strain variations
• Potential bio-engineered organisms

Taylor et al, Phil. Trans. R. Soc. Lond. B
(2001) 356, 983-989
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Agents Affecting Human Health or Animal Health
Duck Hepatitis virus Epizootic Lymphangitis
Enzootic Bovine Leukemia Equine
Piroplasmosis Fowl Pox Mareks
Disease Hemorrhagic Septicemia Horse
Mange Infect Bovine Rhinotracheitis
Myxomatosis Ovine Pulmonary Adenovirus
Porcine cystercercosis Porcine resp rep
syndr Rabbit Hemorrhagic Dis Surra
Transmissible Gastroentero
Trichinellosis Trichomonosis
Anthrax Foot Mouth Brucellosis Rift Valley
Fever BSE Swine Fever Nipah
Hendra Botulinum Influenza
Plague VEE/WEE/EEE Rinderpest
Glanders JE NDV Q Fever TBE
Tularemia Hantavirus Salmonella
Microsporidia CCHF Bovine
Tuberculosis African Horse Sickness Meliodosis
Psittacosis Sheep and Goat Pox Rickettsia
rickettsii C perfringens toxin Rabies Ricin
SEB E coli VSV Ebola/Marburg Campylo
bacter Bluetongue Lassa Heartwater Smallpox
Swine Vesicular Disease Typhus Yellow
Fever Akabane Shigatoxin Contagious Caprine
Pleuro. Monkeypox Dengue
S A Hemorrhagic Fevers Shiga-like RIP Lumpy
Skin Disease Shigellosis Hepatitis E Fowl
Cholera T2- mycotoxin Hydatid Disease
Herpes B Abrin Malignant Catarrhal
Fever Peste des petits Avian Infectious
Bronchitis Aujuszkys Disease Bovine
Babesiosis Coccidiodes posadasii Contagious
Equine Metritis Fowl Typhoid Pullorum Conoto
xin
Avian Tuberculosis Bovine cystercercosis Dourine E
nterovirus Encephalitis Equine Infect
Anemia Equine Rhinopneumonia Infectious
Bursal Disease Maedi / Visna Nairobi Sheep
Disease Paratuberculosis Theileriosis Alastr
im (Variola minor) Menangle Avian
Mycoplasmosis Bovine Anaplasmosis Bovine Genital
Campylobacter Contagious Agalactia Dermatophi
losis
Plant pathogens expand list
Derived from DoD, NIAID, OCIE and others
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Pathogen Detection Today

• Current culture techniques
• Slow, labor intensive
• Only detect culturable organisms
• Modern diagnostic techniques often look for one
  pathogen at a time (anthrax, plague, smallpox,
  etc.)
• Need too many tests
• May fail to detect newly emergent pathogens
• Could be flawed strategy

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TIGER Biosensor
Triangulation ID for Genetic Evaluation of
Risks
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Electrospray Ionization MS
Triangulation ID for Genetic Evaluation of
Risks By IBIS
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Mass SpectrometryBase composition

• Number of A, G, C, Ts
• Difference in base composition - SNP
• Both strands
• No post-PCR design

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Mass SpectrometryBase composition
Sample 1 --- A-24, G-30, C-18, T-28 Sample 2
--- A-23, G-31, C-18, T-28
A to G transition
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Use of information outside D-Loop
Resolving information of base compositions
signatures assessed over set of sequences that
contain all 6 regions

• Smaller data set 469 total sequences
• Amplicons from 431 full mtDNA genomes with no
  ambiguous base calls
• Amplicons from 38 samples sequenced through all 6
  regions in-house
• Higher overall percentage unique in all regions
  compared to FBI database
• HV1 Base count / sequence resolution somewhat
  inflated in smaller set
• Theoretical limit of base count resolution
  approaches that of sequencing.

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TIGER Process
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TriangulationMeasuring multiple signals from
the threat agent
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TIGER Process
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TIGER Process
Base composition to organism map
Organism Mass Base Count E. coli 40440.652 A3
4G42C29T26 S. milleri 42838.082 A44G33C24T37 M.
leprae 41628.768 A27G43C30T34 Unknown
brucella 40479.625 A30G44C29T29 T.
medium 43515.168 A32G39C27T34
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Multiple Primers Add Resolving Power
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Overt Attack
Courthouse receives letter labeled Anthrax
Anthrax
Covert Attack
Unusual, Disease Clusters, Signs Symptoms
State Local Public Health
CDC/FDA
Law Enforcement
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Clues to Possible Bioterrorism Act?
NY Times, Oct. 16, 1999
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Epidemiologic considerations that may signal a
bioterrorist attack

• Disease caused by an uncommon agent (such as
  smallpox)
• Unusual, atypical, genetically engineered or
  antiquated strain of agent
• High morbidity or mortality associated with a
  common disease or syndrome
• Failure of patients to respond to usual therapy
• Disease with an unusual seasonal or geographic
  distribution
• Increase in normal incidence
• Atypical disease transmission (such as shigella
  in muffins)
• Illness in people who are exposed to same
  ventilation system

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Epidemiologic considerations that may signal a
bioterrorist attack

• More than one unusual or unexplained disease
  existing in a person
• Illness that affects a large disparate population
  
• Illness that is unusual for a population or age
  group
• Unusual death or pattern of illness in animals
  preceding or accompanies death or illness in
  humans and vice versa
• A number of ill persons seeking treatment or
  medicine at the same time
• Same strain or genetic type from spatially or
  temporally disparate sources
• Simultaneous cluster of disease in noncontiguous
  areas
• Large number of unexplained diseases or deaths

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Post-Harvest backgroundHuman Food-borne Illness

• 75 Million Illnesses
• More than 300 Thousand Hospitalizations
• 5 Thousand Deaths
• A Cost of 5-15 Billion per Year

Background Noise!!
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Crime Scene

• Control crime scene
• Document Administrative Log
• Coordinate personnel and make assignments
• Ensure properly equipped
• Survey scene
• Photograph and sketch
• Plan and search
• Collect evidence
• Collect samples
• Package
• Transport
• Etc.

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Authenticity and Integrity

• Sample Collection
• Preservation
• Handling
• Chain of Custody
• Documentation

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Evidence Collection
Sometimes Incompatible with Other Forensic
Techniques
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Evidence - BioSamples

• Preservation
• Varies by microorganism, substrate, device,
• Downstream interoperability

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Challenge Develop better methods for recovery of
BW agents/material bulk, trace or mixed
material
Material recovered from weapons Material
recovered from air filters or ductwork Material
from contaminated surfaces Material recovered
from air collectors Etc.
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Integrated ApproachPoints to Consider

• Will sample aliquot contain sufficient material
  that can be recovered and analyzed?
• Is there an extraction method that can handle the
  bulk/trace and nature of the sample?
• Are there constituents that will interfere with
  extraction or inhibit PCR?

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Sample Analyses Flow Chart
Strain ID Genetic Engineering
Bacteria ID Classic bacteriology
Issues Control material Control
environment Maximize recovery
Recover Evidence
Spore density Cfu/ml
Percentage viability Dead vs live spores
Particle sizing
Electron Microscopy (EM)
Analytical chemistry
Transmission EM
Inorganic Silica Silicates Cations and
anions Heavy metals
Organic Carbohydrates Agar (Agarobiose) Peptones H
eadspace
Scanning EM EDX Analysis
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Analytical Methods

• We have methods
• Can always use more

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Direction, Use and Interpretation
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Forensic Questions

• What is the agent?
• Species, strain, or more
• Was the event intentional?
• Obvious in the Anthrax case
• How was it made?
• Where did it come from?
• Who did it?

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Non DNA-based tools for the microbial forensics
toolbox

• Characterization of physical attributes acquired
  during preparation
• Isotope analyses to approximate the age and
  source
• Physiologic methods (e.g., fatty acid
  composition, phage typing, serotyping)
• Analysis of growth media and media components
  adhering to the microorganisms
• Analysis of stabilizers and additives used in the
  preparation of a sample

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Non DNA-based tools for the microbial forensics
toolbox

• Identification of incidental biocontaminants,
  such as environmental pollen and fungi, for
  location and time of year of preparation
• Better understanding of bacterial endemism for
  identifying unique strains that may exist in only
  one location or few locations
• Monitoring changes in the immunological response
  of a host to a pathogen or toxin, such as
  temporal IgG and IgM responses
• Improvements in immunoassays (and antibodies) for
  more effective rapid detection and field
  deployable assays.

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Forensic Genetic Questions

• What might be deduced concerning the nature and
  source of the evidentiary sample?
• Is the pathogen detected of endemic origin or
  introduced?
• Do the genetic markers provide a significant
  amount of probative information?
• Does the choice of markers allow the effective
  comparison of samples from known and questioned
  sources?
• If such a comparison can be made, how
  definitively and confidently can a conclusion be
  reached?

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Forensic Genetic Questions

• Are the genetic differences to few to conclude
  that the samples are not from different sources
  (or lineages)?
• Are these differences sufficiently robust to
  consider that the samples are from different
  sources?
• Is it possible that the two samples have a recent
  common ancestor or how long ago was there a
  common ancestor?
• Can any samples be excluded as contaminants or
  recent sources of the isolate?
• Are there alternative explanations for the
  results that were obtained?

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Clonal Propagation in a Recently Emerged Pathogen
Ancestor
Mutation
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Questions Arise
Was the pathogen engineered? Did it come from a
biowarfare program or is it form nature, a lab,
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Phylogenetic trees
The branching pattern reflects the genetic
relatedness The branch length reflects the
genetic distance, how many mutations/per number
of positions Phylogenetic reconstruction is the
art of assembling these trees based on
evolutionary models In column 1 Did A?T? or did
A?C?T?
A
AATAAGCGGTAG AATAACTGGTAG TTTAAGTGGAAC TTTATGTG
GAAC
B
C
D
A
C
D
B
Evolutionary models consider base frequencies,
substitution rates between bases, and relative
mutation rates in different positions
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MLVA-15 A 15 locus 4-color Genotyping System
B. Anthracis Strain Identification
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Anthrax Worldwide Phylogeny
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AnthraxData and Epidemiology

• Criticism that investigation should look for
  someone who dug up Ames in Texas
• Epidemiology does not support it
• Focuses effort on laboratory
• Assists in interpretation of environmental
  samples that may contain a diverse array of
  samples

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Another Attack?

• San Jose HP Ranch
• -18 Cattle Die of Anthrax

Link to the Anthrax Letters??
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NorthAmericaAnthrax
136
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• 15 VNTR Analysis
• 269 Isolates
• 47 genotypes

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Comparison of Human DNA Forensics and Microbial
Forensics

• Similarities
• QA/QC
• Databases
• Qualitative conclusions of test results
• Quantitative conclusions
• General acceptance, admissibility,

• Differences
• Database size and composition
• Statistical Interpretation methods
• Confidence/uncertainty of outcome of
  interpretation

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Population Genetic Issues

• Origin and maintenance of microbial diversity are
  dependent on
• - mode of reproduction (asexual mostly)
• - inter genic/site variation of mutation rate
• - recent natural vs. laboratory origin
• - experimental/natural stress condition(s)
• - horizontal gene transfer (gene conversion)

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Plausible Statistical Methods

• Comparison of evidence and reference samples will
  generally be based on haplotype/sequence-based
  data (except eukaryotes)
• Statistics should focus on establishing lineage
  instead of individuality
• Extent of mismatch, instead of the counting
  method, may be needed to provide quantitative
  support of the match/non-match evidence
• More like Y STR and mtDNA typing

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Interpretation

• Sensitivity claims of 1copy (Just like LCN)
• Is this reliable?
• What does it mean?
• Should there be an inconclusive category?

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Interpretation

• Biowatch (semi-fictitious)
• F. tularensis
• Positive reaction on mall
• Endemicity (of strains) may be helpful

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Genomics and Pathogenesis
Ignorant of diversity and distribution in food,
water, and environment
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The challenge for Microbial Forensics is
to advance from sample matching to INFERENCES
Comparison among evidence samples or between
evidence and pre-existing reference samples
Simple sample matching
Match criteria - identical to within
analytical precision
Information about processes that can be deduced
even if there is no exact match between
evidence and pre-existing reference samples -
False positive rates - False negative rates
Spoofing
Showing that an end-to-end process results in a
reference sample that matches an evidence sample
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Molecular Evidence of HIV-1 Transmission in a
Criminal Cases
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Could HIV be used as a biowarfare agent?

• HIV is not readily transmitted without intimate
  contact or through blood-blood transfer
• HIV takes, on average, 10 years to kill the host

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Could HIV be used in a biocrime?

• Rape
• Not informing partner(s) of being HIV positive
• Health care transmissions positive providers,
  unsterile conditions -- responsibility for
  damages?
• Criminal threats with blood or body fluids -
  robbery at needle point, spitting on policemen or
  prison guards with intent to harm, deliberately
  injecting someone with HIV

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Louisiana v SchmidtCase

• Allegations
• Gastroenterologist made a mixture of blood (or
  blood products) from two patients one infected
  with HIV-1 and one infected with hepatitis C
• Infected girlfriend (affair) by intramuscular
  injection (Vitamin Shot August 4, 1994)

Metzker, et al. PNAS 99(22)14292-14297, 2002.
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Louisiana v Schmidt Risk factors

• 1984-1995, victim reported sexual contact with
  seven men, including the doctor
• All tested HIV-1 negative
• Victim was a nurse, but no documented needle
  sticks
• Victim did report splashing of saliva onto her
  skin by HIV-1 infected patient in the mid 1980s
• Victim had tested negative as blood donor up
  through April 1994

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Louisiana v Schmidt

• In January 1995, Victim tested positive for
  HIV-1
• Investigators identify HIV-1 infected patient
  that blood was drawn on August 4, 1994 (one of
  last recorded in log book)
• Patients blood draw recorded differently
• Hypothesis the patient is the source of the
  HIV-1 observed in Victim

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RT region
patient
victim
controls
Victim sequences embedded in patient sequences
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Dr. Schmidt was found guilty of second degree
attempted murder and is serving a 50 year
sentence

• The admissibility of the conclusion that the HIV
  samples were closely related was challenged on
  appeal
• Use of DNA evidence is well-established in
  Louisiana, but its use to establish similarities
  between viral infections was without precedent
  (Note no statistical strength)
• The appeal was rejected by the Louisiana State
  Supreme Court in 2000 
• The case was then appealed to the United States
  Supreme Court, and the appeal was rejected
  March, 2002

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Is there a law against it? In many countries,
criminal justice systems are constrained by
inadequate legal frameworks governing the
detection and repression of bio-weapons.
Frequently, no law is violated until the disease
or biological agent is actually deployed. Law
enforcement officers are therefore unable to
begin preliminary investigations into the
development of such weapons. Without laws which
criminalize activity relating to bio-weapons,
there is no basis for legal assistance or
co-operation to prevent their production and
transport.
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There is therefore an urgent need to ensure that
countries are adequately prepared for, protected
from, and able to respond to bioterrorism
attacks. Law enforcement agencies have a crucial
role to play, with significant support from, and
in collaboration with a range of other national
and international bodies.
Arthur P. Sloan Foundation
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The Challenge for Any Forensic Analysis of a
Bioweapon Source Exclusion, Association and
Attribution
Five minutes before a party is NOT the time to
learn how to dance
Snoopy to Woodstock
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