Forensics &Bioterrorism

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FORENSICS

OUTLINE

I. INTRODUCTION

A. BRIEF HISTORY

B. AREAS IN FORENSIC SCIENCE

C. BASICS OF CRIME SCENE INVESTIGATION

II. TECHNIQUES AND METHODS USED

III. EXAMPLES

IV. RECENT PROGRESS AND ISSUES

WHAT IS FORENSIC SCIENCE?

Science is a systematically organized body of knowledgeForensic is derived from the latin word, forum which means “public”

Forensic science - “The science of associating people, places, and

things involved in criminal activities”

Houck and Siegel, 2010

AREAS OF FORENSIC SCIENCE

CriminalisticsCollects and analyzes physical evidences

Forensic PathologyDetermination of cause and manner of death

Forensic AnthropologyIdentification of human remains not possible through soft tissue features

Forensic OdontologyAlso called forensic dentistry

Forensic Engineeringinvestigate and tests materials, products or structures that do not function the way they used to

Forensic ToxicologyDetermination of drugs present in body fluids and tissues

Behavioral Sciences

18TH & 19TH

CENTURY

SCIENCE OF PERSONAL

IDENTIFICATION

- STUDY OF FINGERPRINTS

- FORENSIC BLOOD TYPING

- FORENSIC DNA TECHNOLOGY

EARLY 20TH

CENTURY

COMPARISON

MICROSCOPE

5TH CENTURY

FORENSIC MEDICINE

TOXICOLOGY AS ONE OF THE

NEW EMERGING FORENSIC

SCIENCES

TOXICOLOGY AS ONE OF

THE NEW EMERGING

SCIENCES

BRIEF HISTORY

Houck and Siegel, 2010

FORENSIC SCIENCE begins at the CRIME SCENE

CRIME SCENE INVESTIGATION TEAM

(Jackson & Jackson, 2017)

Securing the scene

Safety and preservation of evidences

Separating the WitnessesGathering

information and Comparison

Scanning the Scene

Determining where photos

should be taken

Seeing the Scene

Photography for present &

future use

Sketching the Scene

Searching for EvidencesGathering information and

Comparison

Securing & Collecting Evidence

Preserving Integrityof Evidences(Placement ofEvidence at X)

12 3 4

5 6 7

CRIME SCENE INVESTIGATION

(Bertino & Bertino, 2012; Jackson & Jackson, 2017)

PHYSICAL EVIDENCESEvidence address to the senses of the court that are capable of being exhibited, examined, or viewed by the court (PNP, 2011)

HAIR BLOOD FINGERPRINT

BODY FLUIDS FIBER GLASS(Bertino & Bertino, 2012; Jackson & Jackson, 2017)

HOW DNA TESTING WORKS- A person’s DNA is unique in every person.

-Only identical twins share the same DNA.

-Scientists identify alleles (small portions of the DNA) & give them a unique number representing a person’s DNA profile.

-At each DNA location a person has 2 alleles (GC-AT)

-Paternity Testing: half of the DNA of a child comes from the mother & half comes from the father

-Potential Sibling: biologically related individuals have more DNA in common than those who don’t (Bertino & Bertino, 2012; Jackson & Jackson, 2017)

ANALYSIS OF EVIDENCES

DNA PROFILING & FORENSIC DNA PHENOTYPING

DNA PROFILING

Comparison of DNA in nucleated cells of person with biological matter found at the

crime scene or with the DNA of another person for the purpose of identification or

exclusion

The Modern DNA fingerprinting procedure or DNA typing

Allows a biological sample at a scene of crime or accident to be linked very strongly to the individual from whom it originated

(Jackson & Jackson, 2017; Jeffreys et al, 1985)

DNA PROFILING PROCEDURE

Extract DNA from

the Sample

DNA Quantitation

PCR Amplification Electrophoresis

Pattern Interpretation &

Matching

-Centrifugation-Agitation-Removal of proteins, lipids & RNA- DNA Purification

- Human DNA Isolation- Quantifiler DNA Quantification Kit-ABI PRISM 7500 Sequence Detection System

Amplification of genetic loci using PCR for forensic analyses

(Bertino & Bertino, 2012; Jackson & Jackson, 2017)

DNA PROFILING TECHNIQUES

Only about 1 ng (10¯⁹ g) of DNA is required for an optimum DNA profiling result. OR LESS.

RFLP(Restriction Fragment Length

Polymorphism)

PCR(Polymerase Chain Reaction)

SNP(Single Nucleotide

Polymorphism)

STR & VNTR(Short Tandem Repeats & Variable

Number Tandem Repeats)Y-chromosomal AnalysisMitochondrial DNA Analysis

(Sharma & Vidya, 2016)

-The in vitro replication of DNA fragments-Used when specimens are very little or the DNA is degraded

PRINCIPLE: Amplification of the biological process of DNA replication but is confined to desired specific DNA sequences.

METHOD:1. Denaturation – 94-96 °C , 5 mins & 30 sec2. Annealing – 62 °C, 30 sec3. Polymerization – 72 °C, 30 sec

Polymerase Chain Reaction (PCR)

(Jackson & Jackson, 2017; Sharma & Vidya, 2016)

Polymerase Chain Reaction (PCR)

DNA Profile of 4 persons using 10 primers (reverse and forward)

Restriction Fragment Length Polymorphism (RFLP)- Variation in the length of a DNA fragment produced by a specific

restriction enzyme acting on DNA from different individuals. Variation usually results from a genetic mutation.

- PRINCIPLE: Restriction enzymes recognize very specific restriction sites in a DNA sequence. Individuals rarely have the same array of restriction sites and distances in every DNA sequence.

Restriction Enzymes

(Losos et al, 2007; Sharma & Vidya, 2016)

Restriction Fragment Length Polymorphism (RFLP) - METHOD:

Restriction Digestion

Restriction enzyme cuts specific restriction

site in the DNA

Gel Electrophoresis-DNA fragments are separated by

length & size-Movement towards (+) Electrode-Smaller fragments move faster

Southern BlottingssDNA fragments migrates

from gel to nylon membrane using electric current

ProbingSLP or MLP

Membrane is incubated with radioactive

fluorescent strand of DNA

HybridizationBinding of DNA fragment with

probeNOTE: Probe will only bind to complementary parts, others

are washed off

Developing Probe Pattern/Autoradiogram

Nylon membrane placed againstX-ray Fillm

Image formation by particle emission of radioactive DNA

1 2 3

4 5 6

(Losos et al, 2007; Sharma & Vidya, 2016)

Restriction Fragment Length Polymorphism (RFLP) -PROBING: Locus serves as the target area of DNA

-DNA SLP or MLP band patterns from stains of blood or sperm can be compared with patterns from persons suspected of leaving the stains.

-Limitation of MLP in statistical interpretation of the multi-banded pattern formed. With this, in 1990, MLP was replaced by SLP.

Multiple Locus Probe (MLP) a probe that hybridizes to a

number of different sites in the genome of an organism

Single Locus Probe (SLP) a probe that is able to hybridize

with DNA from a specific restriction fragment on the

Southern blot.

(Losos et al, 2007; Sharma & Vidya, 2016)

Restriction Fragment Length Polymorphism

(RFLP) - METHOD:

(Giuseppe et al, 2002)

Single-Nucleotide Polymorphism (SNP)-Represents the alterations in the DNA sequence at a single

nucleotide position, either due to base changes, insertion or deletions of one or few bases.

-PRINCIPLE: Alterations create different alleles of that gene.Mutation in SNP is very rare, the sequence tend to be passed unchanged across generations.

-Difference in the sequence itself rather the length of DNASNP occurs every 100-300 bases along entire length of the human genome. - DNA template can be as large as a pair of specific primers ~50bp compared to ~300bp needed in STR. (Sharma & Vidya, 2016)

Short Tandem Repeats (STR) & Variable Number of Tandem Repeats (VNTR)-Are DNA sequences whose length varies from 1,000-20,000 bases and contains

repeating sequences for every 2-9 base pairs (STR) or 10-100 bp (VNTR).

-Number of repeat units vary between individuals.

-Cannot take place without the presence of specific oligonucleotides that serves as primers for the DNA polymerase and deoxynucleotide triphosphates, thebuilding blocks of DNA.

-STR is a.k.a. microsatellite

(Sharma & Vidya, 2016)

STR are dispersed more evenly than VNTR in the genome

Short Tandem Repeats (STR) & Variable Number of Tandem Repeats (VNTR)

(Jackson & Jackson, 2017;Sharma & Vidya, 2016)

-Analyses based on STR Profiles

1. Y-Chromosomal Analysis- Y-STR typing uses Y-STR markers give specific

information about the presence of male DNA.- Useful for rape cases and male lineage

2. Mitochondrial DNA Analysis-Contains genetic material found only from the

mother-Enables to type ancient samples with only minute

amounts of DNA needed-mtDNA is present in large amounts in the cell and

are highly stable. (ex. Romanov Case)

COMPARISON OF DNA PROFILING TECHNIQUES

(Sharma & Vidya, 2016)

Other Uses of DNA: FORENSIC DNA PHENOTYPING (FDP)

-Makes use of DNA genotype for the prediction of appearance traits (phenotype) of missing individuals or suspects.

-Results of FDP can serve as “biological witness” wherein Externally Visible Characteristics of a missing person or possible perpetrator can be inferred, however remains an issue.

(Kayser, 2015)

CASES OF DNA PROFILING

CASES INVOLVING FORENSIC DNA ANALYSIS

Pitchfork Case (1987) Murder, Narborough, Leicestershire, EnglandFirst case that a man (Colin Pitchfork) was convicted of murder and rape through the aid of DNA profiling of blood & saliva samples and comparing it with the DNA of semen evidence.

Routier Case (1996)Murder, Rowlett, TexasDNA profiled from blood evidences using STR and Y-STR techniques proved that it was the mother (Darlie Routier) who killed her two sons (Devon and Damon) and not an intruder which she initially stated.

(Elvidge, 2016; HubPages Inc., 2017)

CASES INVOLVING FORENSIC DNA ANALYSIS-In the Philippines,

Vallejo (2002) & Yatar (2004) CasesRape-Homicidewere solved after finding DNAs of the perpetrators (Gerrico Vallejo, Joel Yatar) the vaginal swabs of the victims. Note: The 2 are separate cases.

De Villa Case (2001) Rapewas reopened in 2003 for DNA-based paternity testing finding out that the DNA of the child of the said raped victim (niece of the suspect) did not match with the perpetrator

(De Ungria, n.d.)

CASES INVOLVING FORENSIC DNA ANALYSIS-Key to solving ‘Stolen Trees’ Cases,

-Molecular geneticist Keith Woeste at Purdue University was able to prove that the walnut wood came from a tree stolen in Warren County.

-Woeste ground the wood into a fine powder to extract enough DNA evidence to convince investigators the wood matched that of the stolen tree, which had been sold to a sawmill.

(Purdue.edu, 2004)

RECENT PROGRESS AND ISSUES

RECENT PROGRESSOral Microbiota as a tool to estimate Time Since Death (TSD)

RECENT PROGRESSSex determination based on Raman Spectroscopy of saliva traces for forensic purposes

- Raman spectroscopy is a non-destructive technique that can be used at the crime scene to determine the sex of saliva donor

- Can be used in the absence of DNA Profile or in cases where DNA results does not yield a match in a database - RAMAN SPECTRA

RECENT PROGRESSDNA profiling in crimes involving monozygotic twins

Researchers used ultra-deep generation sequencing associated with bioinformatics techniques in differentiating DNAs of monozygotic twins

Results: 5 Single Nucleotide Polymorphisms (SNPs) were present in the twin father and his child but absent in twin uncle

Conclusion: Rare mutations will occur early after the human blastocyst splits into two (the origin of twins) and that such mutations will be carried on into somatic tissue and germline

RECENT PROGRESSForensic Science Institute Act

Establishing Forensic Science Institute in the University of the Philippines System

Objectives:

● Promote R&D● Enhance existing laboratories● To develop a corps of professionals

CURRENT ISSUES

familial searching in CombinedDNA Index System (CODIS)Database could affect innocentpeople whose DNA would partiallymatch with that found in the crimescene (Gloudemans & Shamaprasad,2015).

CURRENT ISSUES

In the Philippines,● Need for fingerprints and DNA database (i.e. Automated

Fingerprint Identification System or AFIS)● Cases of malpractice in investigating dead bodies, writing

autopsies and dead reports● Lack of support and participation of the legislative branch

BIOTERRORISM

Outline

I. INTRODUCTION

A. BIOTERRORISM

B. HISTORY

C. BIOLOGICAL AGENTS

II. TECHNIQUES AND METHODS USED

III. EXAMPLES

IV. RECENT PROGRESS AND ISSUES

WHAT IS BIOTERRORISM?Bioterrorism is intentional use of microorganisms or toxins derived from living organisms to cause death or disease in humans or the animals and plants on which we depend.

● Catastrophic terrorism with mass casualties● Microevents using low technology causing

civil unrest, disruption, disease, disabilities and death● Spreading a virulent disease among animal production

facilities● Poisoning water, food crops and supplies

Das and Kataria, 2010

NATIONS DISSIDENT GROUPS

HISTORYThe use of biological agents as weapons is NOT a new concept since it is known to be used as early as the ancient times.

Prakash et al., 2010

650 BC Assyrian politicians dumped fungus (Claviceps purpurea) from rye into their opponents′ wells, giving them fatal ergot poisoning.

400 BC Scythian archers infected their arrows by dipping them in decomposing bodies or in blood mixed with manure.

HISTORYThe use of biological agents as weapons is NOT a new concept since it is known to be used as early as the ancient times.

Prakash et al., 2010

300 BC Persian, Greek, and Roman used dead animals to contaminate water sources such as wells.

190 BC Hannibal, a Carthaginian commander, won a naval victory by firing earthen vessels full of venomous snakes into the enemy ships.

HISTORYThe use of biological agents as weapons is NOT a new concept since it is known to be used as early as the ancient times.

Prakash et al., 2010

1346 Tatar armies spread bubonic plague bycatapulting diseased corpses over the walls of the city of Kaffa

1767 Spreading of smallpox via contaminated blankets by the British to the Native American population loyal to the French.

HISTORYThe use of biological agents as weapons is NOT a new concept since it is known to be used as early as the ancient times.

Prakash et al., 2010

WWI US & Germany developed biological weapons using anthrax & glanders.

WWII Japan operated Unit 731 that carried out human experiments on prisoners exposing them to plague, anthrax, syphilis, and other agents.

CHARACTERISTICS OF BIOLOGICAL AGENTS

EFFECTIVE AT LOW DOSE DIFFICULT TO IDENTIFY

AGENTS OF BIOTERRORISM

CLASSIFICATION, ROUTES OF ENTRY & METHODS OF DELIVERY

CLASSIFICATIONAccording to the Centre for Disease Control and Prevention (CDC),currently known biological weapons can be classified into 3categories.

Das and Kataria, 2010

CATEGORY A● Easily disseminated● Causes high mortality● Causes public panic and social disruption● Requires special attention for public health preparedness

CLASSIFICATIONAccording to the Centre for Disease Control and Prevention (CDC),currently known biological weapons can be classified into 3categories.

Das and Kataria, 2010

CATEGORY A● Examples: Bacillus anthracis (anthrax), Clostridium botulinum

(botulism), Yersinia pestis (plague), Variola major virus (smallpox), Francisella tularensis (tularaemia), Filo viruses and Arena viruses (viral hemorrhagic fevers), Ebola virus (Ebola hemorrhagic fever), Marburg virus (Marburg hemorrhagic fever), Lassa virus (Lassa fever), etc.

CLASSIFICATIONAccording to the Centre for Disease Control and Prevention (CDC),currently known biological weapons can be classified into 3categories.

Das and Kataria, 2010

CATEGORY B● Moderately easy to disseminate● Causes moderate morbidity● Requires enhanced disease surveillance and public health

diagnostic capacity

CLASSIFICATIONAccording to the Centre for Disease Control and Prevention (CDC),currently known biological weapons can be classified into 3categories.

Das and Kataria, 2010

CATEGORY B● Examples: Alpha viruses, Eastern, western, and Venezuelan

equine encephalomyelitis viruses (EEE, WEE, VEE), Brucella species (brucellosis), Burkholderia mallei (glanders), Coxiella burnetti (Q fever), foodborne and waterborne pathogens (Cryptosporidium parvum, Escherichia coli O157: H7, Salmonella species, Shigella dysenteriae, Vibrio cholerae, etc.)

CLASSIFICATIONAccording to the Centre for Disease Control and Prevention (CDC),currently known biological weapons can be classified into 3categories.

Das and Kataria, 2010

CATEGORY C● Could be engineered for dissemination in the future● Have potential for high morbidity, mortality, and major health

impacts● Examples: Hanta viruses, Multidrug-resistant tuberculosis,

Nipah virus, Tick-borne encephalitis viruses, Tick-borne hemorrhagic fever viruses, Yellow fever

CLASSIFICATIONAccording to the Centre for Disease Control and Prevention (CDC),currently known biological weapons can be classified into 3categories.

Das and Kataria, 2010

CATEGORY C● Could be engineered for dissemination in the future● Have potential for high morbidity, mortality, and major health

impacts● Examples: Hanta viruses, Multidrug-resistant tuberculosis,

Nipah virus, Tick-borne encephalitis viruses, Tick-borne hemorrhagic fever viruses, Yellow fever

ROUTES OF ENTRY

METHODS OF DELIVERY

● Through contaminated food and water

● Through air-handling systems

● Through bomblets delivered by aircrafts

● Through spray tanks released via aircrafts and other

vehicles or via tall buildings

● Through delivery by post

● Through deliberate infiltration of infected animals, vectors,

and pests

SPECIFIC EXAMPLES

ACTS OF BIOTERRORISM IN THE 20TH-21ST CENTURY

GLANDERS● 1915● Dr. Anton Dilger, developed a microbiology facility in

Washington, D.C. that produces large quantities of anthrax and glanders bacteria

● 3,000 horses, mules, sheeps & cattle shipped from Norway, Spain, Romania, and the United States

● To interrupt the flow of supplies to the Allied frontlines● Inoculated using needles and capillary tubes● Effects are unknown since no reports have been made

of disease outbreaks among livestock.

MYCOTOXIN● 1981● Attacks in Vietnam, Laos, Cambodia & Afghanistan

linked to Soviet Union● Mold byproduct of Fusarium spp.● Helicopter or plane flying over releasing a yellow

colored cloud that would fall in a manner that looked, felt and sounded like rain

● Known as “Yellow Rain”● “Naturally occurring phenomenon of a swarm of Asian

honeybees defecating in flight”

SALMONELLA● 1981● Bhagwan Shree Rajneesh cult● Contaminated salad bars with Salmonella bacteria in a

small town in Oregon● Aim: To influence the local county elections.● Largest scale act of bioterrorism in U.S. history● More than 750 cases of salmonellosis

RICIN● 1991● Ricin - a ribosome-binding protein naturally occurring

in the seeds of the castor oil plant.● Patriots Council, an antigovernment extremist group,

members arrested for plotting to kill a U.S. marshal with ricin

● Smearing it on the marshal’s car door handle● Lethal when inhaled or absorbed through damaged

skin.● After 2-5 days, damage to nervous system and internal

organs

SMALLPOX● 1992● Soviet Union had the ability to launch missiles

containing weapon grade smallpox● Smallpox killed at least 300 million people ● At present, smallpox is one of the biggest threat since

most vaccines are destroyed as per recommendations of WHO advisory committee

ANTHRAX● 1994● Aum Shinrikyo, a Japanese religious sect released

anthrax through aerosol from the tops of buildings in Tokyo

● However the attack was a total failure since the group used the vaccine strain of the bacterium.

ANTHRAX● 2001● Letters containing anthrax

spores were mailed to a television news anchor, US senator, and others

● 5 deaths from pulmonary anthrax and 17 other cases of inhalation and cutaneous anthrax.

RECENT PROGRESS AND ISSUES

IMPLEMENTING LAWS ON BIOTERRORISMGENEVA PROTOCOL● Also known as “Protocol for the

Prohibition of the Use in War of Asphyxiating, Poisonous or other Gases, and of Bacteriological Methods of Warfare.”

● Signed at Geneva on June 17, 1925 ● Entered into force on February 8,

1928● PH gave its depositary on June 8,

1973● Countries that violated the law:

Spain, France, Japan, US, UK, Germany, Soviet Union, Iraq, Syria

IMPLEMENTING LAWS ON BIOTERRORISMBIOLOGICAL WEAPONS CONVENTION (BWC)● Also known as “Convention on the Prohibition of the Development,

Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on their Destruction.”

● Signed at London, Moscow, and Washington D.C. on April 10, 1972 ● Entered into force on March 26, 1975● PH gave its signed on April 10, 1972 (L, W) and on June 21, 1972 (M);

and gave its depositary on May 21, 1973 (W) ● Occasionally holds review conferences regarding issues and

implementations of the law

IMPLEMENTING LAWS ON BIOTERRORISMREPUBLIC ACT NO. 9271● Also known as “"Implementing Rules

and Regulations of the Quarantine Act of 2004.” or simply “Quarantine Act”

● Creation of the Bureau of Quarantine ● Monitors aircrafts and transport vessels

for any radiological, biological, and chemical (RBC) weapons

● Also responsible for any RBC incidents that may happen inside the country such as bioterrorism

COMMON REACTIONS OF PEOPLE TO BIOTERRORISM● Vicarious Rehearsals - These are emergency protocols or

courses of action done as a safety precaution of people

unlikely to be affected by the bioterrorism attack.

● Denial - This is a reaction wherein people refuse to

acknowledge the existence of a threat or to take necessary

action towards it.

● Stigmatization - This is the fear and isolation of a group

perceived to be contaminated or risky to associate with.

● Fear and Avoidance

● Withdrawal, Hopelessness, and Helplessness

DETECTION AND PREPARATION FOR BIOTERRORISM ATTACKSIntelligence and Information Management

● People’s awareness to biological weapons and other bioterrorism agents

● Scientists’ further research on the matter and help explain in layman’s

terms the bioterrorism agents

● Exchange of information between intelligence, S&T, and public health

communities

BioWatch - “BioWatch air monitoring and analysis, notification procedures, and risk assessment can eliminate or substantially minimize the catastrophic impact of a biological attack. BioWatch also gives public health, law enforcement, emergency management, and laboratory communities a forum to help develop a common understanding of a bioterrorism incident, focus resources, and determine the appropriate response.”

DETECTION AND PREPARATION FOR BIOTERRORISM ATTACKSIdentification of Biological Agents in Environment

● Traditional laboratory detection is slow and limited. However, it is

also the most reliable approach especially in developing countries.

● Develop and evaluate rapid, sensitive, and specific early-detection

technologies.

DETECTION AND PREPARATION FOR BIOTERRORISM ATTACKSSurveillance and Diagnosis of Infection and Disease

● Complications due to lack of relevant medical experience in

handling bioterrorism agents in addition to nonspecific symptoms

of the related disease (fever, flu, etc.)

● Surveillance and diagnosis should focus on three areas: preclinical,

human disease, and agricultural.

ARE WEPREPARED?

BIOTERRORISM IN THE PHILIPPINES

“The Philippines is ill-equipped to deal with a chemical or

biological attack by terrorists because its cash-strapped police

have neither the equipment nor the technology to handle

these "dirty weapons" of mass destruction.”

Singapore Institute of International Affairs (SIIA), 2005

BIOTERRORISM IN THE PHILIPPINESAccording to Maria Auxilia T. Siringan, the head of the

Microbiological Research and Services Laboratory, University

of the Philippines Diliman, Quezon City, the Philippines is:

• Not capable of detecting or stopping the spread of

bioterrorism attacks

• Still in the conceptual stage of creating a biodefense system

prepared to stop the intentional release of biological agents

or toxins such as plague, smallpox, anthrax or the Ebola

virus.BioPrepWatch Reports, 2012

BIOTERRORISM IN THE PHILIPPINES

• Last November 7, 2016 at the 8th Review Conference of the

Biological Weapons Convention in Geneva, Switzerland, the

Philippines, as one of the state parties, highlighted its efforts

in raising awareness in bioterrorism by implementing the

National Chemical Biological Radiological Nuclear (CBRN)

Action Plan and its active engagement in counter-terrorism

measures within the Asia Pacific Region.

DFA, 2016