Deliverable 4.3

Transcription

Deliverable 4.3

ClassificAtion Level: Unclassified
4.3
TASK
FORENSIC RESPONSE
PLAN AND IMPROVED
FORENSIC SAMPLING
AND DNA ANALYSIS
With the financial support from the Prevention of and
Fight against Crime Programme of the European Union
European Commission – Directorate – General Home Affairs
WP4
TASK 4.3
FORENSIC RESPONSE PLAN
AND IMPROVED FORENSIC SAMPLING
AND DNA ANALYSIS
Task leader
Swedish National Laboratory of Forensic Science (SKL)
Task partners
National Veterinary Institute (SVA)
University of Lund (ULUND)
main Authors of this report
Birgitta Rasmusson, SKL/ULUND, Cecilia Vahlberg, SKL (4.3.1),
Johannes Hedman, SKL/ULUND (4.3.2), Therese Ottinger, SVA (4.3.3),
Anders Lindström, SVA (4.3.4), Mats Forsman, FOI (4.3.5)
and Gunnar Andersson, SVA (4.3.5)
How to refer to this document
Rasmusson, B., Vahlberg, C., Hedman, J., Ottinger, T.,
Lindström, A., Forsman, M. Andersson, M. G. (2013),
Forensic Response Plan and Improved Forensic Sampling and DNA Analysis,
CWA 15793, ISBN number 978-91-87147-13-5
ISBN number 978-91-87147-13-5
Layout To Be Frank
Printed by Davidsons Tryckeri 2013
WP4: EUROPEAN LABORATORY RESPONSE NETWORK (LRN) FOR ANIMAL BIOTERRORISM THREATS
TASK 4.3: FORENSIC RESPONSE PLAN AND IMPROVED FORENSIC SAMPLING AND DNA ANALYSIS
The AniBioThreat project was in 2010 awarded a
grant by Directorate General Home Affairs under
the programme “Prevention of and Fight Against
Crime”. One issue stated in the call text in 2009
under this programme was animal bioterrorism
threats. The focus of AniBioThreat is therefore
based on threats to living animals, animal feed
and food of animal origin. As part of this, it is
foreseen that the project will enhance inter
national cooperation and promote networking for
bridging security with animal and public health.
The objectives are furthermore based upon
some of the identified actions in the EU Chemical,
Biological, Radiological and Nuclear (CBRN)
Action Plan (2009), the recommendations of the
CBRN Task Force Report (2009) and especially
the work that took place in the Biosubgroup
threats to animal, and food and feed for animals
(2008), and the Biosubgroup detection and
diagnosis (2008, June).
The project is divided into the following six
work packages (WPs); WP1 the establishment
of a network between law enforcement, forensic
institutes, first responders, intelligence, veterinary
institutes, public health agencies and universities,
WP2 threat assessment, WP3 early warning/
detection, WP4 European Laboratory Response
Network for animal bio-terrorism threats, WP5
detection and diagnostics and WP6 dissemination.
Specific objectives of the WPs
are as follows:
• To facilitate effective international cooperation,
improve training and establish a network
between law enforcement, forensic institutes,
first responders, intelligence agencies,
veterinary institutes, public health agencies
and universities (WP1).
• To improve monitoring and threat assessments
(WP2).
• To investigate early warning and rapid alert
for animal disease outbreaks caused by
criminal acts (WP3).
• To establish a European Laboratory Response
Network approach to counter animal bioterrorism threats (WP4).
• To enhance research and development of
detection methods of animal diseases, such as
anthrax, botulism and viral diseases caused
by criminal acts (WP5).
• To disseminate the outcome of the project
to relevant stakeholders through exercises,
workshops, publications, and academic
courses and to strengthen research through
existing EU projects (WP6).
The overall objective of AniBioThreat is to improve
the EU’s capacity to counter biological animal bioterrorism threats
in terms of awareness, prevention and contingency.
3
Capacity and Capability
The overall goal of the EU CBRN Action Plan is
an all-hazards approach to reduce the threat of
damage from CBRN incidents of accidental,
natural or intentional origin, including acts
of terrorism.
This deliverable has improved EU’s capacity
and capability to counter biological animal
bioterrorism threats in terms of awareness,
prevention and contingency in following areas:
Education and training capacity and capability
Research capability
Risk assessment capability
Cooperation/interoperability capability
Surveillance and rapid alert capability
Diagnostic and laboratory response
network capacity and capability
Forensic awareness capability
Contingency planning capability
Joint exercise capacity
Readiness assessment and
medical countermeasure capacity
Communication and information sharing capability
Strategic, tactical and operational
decision making capability
4
Abstract
The forensic investigation of a B-terror incident
requires collaboration under stress of officers
and agencies that seldom interact under normal
circumstances.
The generic measures for a forensic response
plan includes central aspects to consider and how
to establish routines regarding preparedness,
collaboration, sampling, transportation, forensic
analysis and the construction of a joint statement
of witness. The focus is on roles, communication
and teamwork. Forensic awareness in first
responders, mutual exercises, exchange of knowledge and respect for each other’s competence
are essential.
The knowledge among the police and other
legal officials of what a veterinary forensic
pathologist can add to an investigation has
increased during the project and the handling
of real cases has improved with regards to chain
of custody, pre-necropsy x-rays, documentation,
evidence evaluation and the structure of the
statement of witness. The knowledge of forensic
entomology and palynology has also increased
in the legal community as a result of various
lectures and workshops as well as improved
methodology.
Several new and/or improved analytical
approaches have been developed for PCR-based
analysis of crime scene samples that may contain
microbial agents.
In conclusion; the forensic investigation
of real B-incidents and the everyday handling
of veterinary forensic pathology cases have
significantly improved as a result of the project.
There is an increased interest in forensic ento
mology and palynology. In addition, new and
more robust PCR-based analytical methods have
been developed.
Contents
Deliverable according to
Grant Agreement
Abstract4
Harmonization of forensic response plans.
Bridging Statement
6
Description of Deliverable
Link to EU CBRN Action Plan
6
Other relevant Actions
6
Contribution towards
overall objective of AniBioThreat
6
Task leader
6
Task partners
6
Authors of this report
6
The original task was expanded to include four
subtasks:
• Forensic response plan (generic measures)
(4.3.1)
• Improved forensic sampling and DNA
analysis (4.3.2)
• Veterinary forensic pathology (4.3.3)
• Forensic entomology, palynology and
taphonomy (4.3.4)
• Microbial forensics and epidemiological
investigations (4.3.5)
The deliverable consists of a short report
summarizing Task 4.3 and five appendices
describing the results of 4.3.1, 4.3.2, 4.3.3, 4.3.4
and 4.3.5, respectively.
Appendix 4.3.1 comprises the forensic response
plan mentioned in the Grant Agreement.
Aim6
Background6
Methodology8
Results and Discussion
9
Conclusion9
Future Outlook and
Recommendations10
Publications from Task 4.3
10
Acknowledgements10
References11
Appendix11
Bridging Statement
A forensic investigation of a suspected B-incident
puts high demands on the traceability (chain of
custody) and quality of the evidence and thus
also on the work processes, sampling and analysis
methods both at the crime scene and at the
involved laboratories. Since several authorities
need to cooperate (e.g. police, forensic authorities
and specialized laboratories), a coordinated
response plan (operational routines), efficient
cooperation and transparent legal documentation
are key issues.
Link to EU CBRN Action Plan
H. 42
Each Member State should ensure that first
responders receive training on forensic awareness in a CBRN crime-scene.
Other relevant Actions
H.30, H.43, H.44 and H.45
Contribution towards
overall objective of AniBioThreat
Identification of generic measures in a forensic
response plan is instrumental for efficient, safe
and legally solid investigations of suspected
animal bioterrorism incidents in the EU (4.3.1).
Improved sampling and analysis techniques make
it possible to extract more forensic information
from questioned materials, and information of
better quality (4.3.2, 4.3.4).
Establishment of forensic veterinary pathology
and the development of harmonized, scientific
methods for evidence evaluation and legal
documentation (statement of witness) are
instrumental for a successful court process (4.3.3).
Task leader
Swedish National Laboratory of Forensic Science
(SKL)
6
Task partners
• National Veterinary Institute (SVA)
• University of Lund (ULUND)
main Authors of this report
•
•
•
•
•
•
•
Birgitta Rasmusson, SKL/ULUND
Cecilia Vahlberg, SKL (4.3.1)
Johannes Hedman, SKL/ULUND (4.3.2)
Therese Ottinger, SVA (4.3.3)
Anders Lindström, SVA (4.3.4)
Mats Forsman, FOI (4.3.5)
Gunnar Andersson,SVA (4.3.5)
Aim
• To identify forensic generic measures for
suspected animal bioterrorism incidents
based on Swedish experiences (4.3.1)
• To improve the analysis of impure and
low-level DNA samples through pre-PCR
processing (4.3.2)
• To establish and develop forensic veterinary
pathology, especially with regards to
communication with the legal system,
evidence evaluation and the statement of
witness (4.3.3)
• To develop forensic entomology, palynology
and taphonomy in a veterinary setting (4.3.4)
• Investigate the prerequisites for robust
evaluation of evidence in for genetic
comparisons in microbial forensics (4.3.5)
Background
4.3.1 General (including 4.3.1)
Forensic evidence may be crucial for the conviction,
or clearance, of a bioterrorism suspect in court.
It may also provide intelligence information to
guide the on-going investigation, link the incident
to earlier attacks and find the origin of the
biological material. Both “classical” forensic
evidence (DNA, fingerprints etc.) as well as
”intrinsic” forensic evidence (from the microbial
agent and its matrix) must to be secured and
Fig.1. The four subtasks of 4.3 (and 4.2) address various steps in the forensic investigation of a suspected B-incident.
analyzed in a quality-controlled system.
Moreover, the results of the analyses should be
reported so that they are correctly understood
and used by the legal authorities and ultimately
the court. The forensic process “from crime
scene to court” in bioterror cases requires close
cooperation between many authorities and
experts, and is often performed under intense
pressure. As these events are (fortunately) rare;
the operational procedures need to be set in
advance and practiced regularly.
The Swedish National Laboratory of Forensic
Science (SKL) is part of the Swedish Police. The
role of SKL as a national forensic expert authority
includes establishing cooperation, coordination
and harmonization of the forensic process in
CBRNE incidents, with a focus on laboratory
analyses. Together with the Swedish Police
Board (Rikspolisstyrelsen, RPS), SKL also participates in the development of the crime scene
work in CBRNE cases.
In this report, we present the generic measures
in a forensic response plan for B-incidents (4.3.1).
The measures are, with some modifications,
applicable also to C, RN and E cases. The results
are based on work in AniBioThreat as well as
several national projects. We also present results
from work done in collaboration between
ULUND, SKL and SVA to improve sampling and
analyses techniques in forensic investigation of
B-incidents (4.3.2, 4.2.4), and work at SVA aimed at
establishing forensic veterinary pathology (4.3.3),
entomology and palynology (4.3.4). The ongoing
work on the implementation of the Bayesian
approach to forensic evidence evaluation in
veterinary pathology and epidemiology and the
development of harmonized, more transparent
statements of witness are presented in 4.3.3 and 4.2.
4.3.2 Improved forensic sampling
and DNA analysis
Implementation of the polymerase chain reaction
(PCR) technology as a rapid microbiological
detection method for effective control measures
requires knowledge about the identity of PCR
inhibitors, the mechanism of PCR inhibition,
pre-PCR processing and how to quantify microorganisms in biological samples with real-time
PCR. The concept of integrating sampling,
sample treatment and the chemistry of PCR,
i.e. pre-PCR processing, is a general approach
to overcoming PCR inhibition and producing
samples optimal for PCR analysis.
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4.3.3 Veterinary forensic pathology
Every part of the forensic necropsy procedure
is open to question by the court. Consequently,
both the forensic postmortem examinations per
se and the reporting of the findings must be
conducted in a manner that will permit the
findings and results to be accepted and correctly
understood in a court of law.
4.3.4 Forensic entomology, palynology
and taphonomy
Forensic entomology utilizes the knowledge
of insect growth rates to establish a minimum
duration of the postmortem interval, i.e. the time
since death.
Palynology is the study of pollen. Pollen
analyses at a crime scene and on a corpse can
aid in establishing time since death. Taphonomy
denotes the transition of a corpse from fresh to
skeletonized material (decomposition). The
main driving forces are temperature and insect
colonization.
4.3.5 Microbial forensics and epidemiological
investigations
Epidemiological investigations aim at finding
the source and rout of transmission in order to
prevent further transmission and to reduce the
risk of further outbreaks. Microbial forensics
investigations is undertaken to determine
whether a crime was undertaken, find the
perpetrator and gather evidence for a criminal
trial. Whole genome sequencing offers new
possibilities for identification, characterization
and attribution of pathogens but work remains
to remove the bottlenecks in the investigation.
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Methodology
4.3.1 Forensic response plan (Generic measures)
• Identification of Swedish authorities and
laboratories/laboratory networks that may
be involved in the investigation of an animal
bioterrorism incident
• Studies of the handling of veterinary cases in
Sweden; especially with regards to the work
at the National Veterinary Institute (SVA) and
legal cases
• Construction of a Handbook (4, part of
the national project Operational routines, see
Appendix 1) for the forensic handling of
CBRN cases including an animal bioterrorism
incident
• Consideration of the results from the projects
Operational Routines and Swedish Forensic
CBRNE Network in the perspective of a
B-incident in an animal facility
• Identification of generic measures (general
features)
• Communication of the generic measures to
the other participants in AniBioThreat via oral
presentation(s) and a written report (Appendix 1)
• Using the SAMBIO2013 exercise to test and
further develop the Handbook and the
forensic response plan
4.3.2 Improved forensic sampling and DNA analysis
• Literature studies
• Experimental: Methods for sampling and
sample treatment were developed and
compared. Additionally, the possibility to
improve DNA analysis of impure samples
by optimising the content of the PCR was
investigated (see Appendix 2 for more detailed
information)
• Scientific publications, PhD thesis (2011)
(Reference 5 – 9 and Appendix 2)
• Graduate course on PCR (reported in Task 6.1)
4.3.3 Veterinary forensic pathology
• Literature studies and construction of a
PhD study plan
• A survey (questionnaire) of the current status
of veterinary forensic pathology in Europe
• Initiation of a European network of forensic
veterinary pathologists
• Structured interviews to investigate
– how the current veterinary forensic
necropsy reports are understood and
used in the Swedish courts
– the transfer of knowledge and information
from veterinary science to law
• Adaptation of evidence evaluation using the
Bayesian approach (Reference 1– 3) to veterinary pathology findings and development of
improved templates for statements of witness
• Networking with law enforcement agencies
to improve the handling of veterinary
forensic cases.
• Lectures, workshops and seminars
• Scientific publications PhD thesis (2015)
4.3.3 Forensic entomology,
palynology and taphonomy
• Experimental:
– development of improved methods
for estimation of time of death
– methods for insect collection at crime
scenes
– studies of insect colonization of carcasses
– detection of forensically important insect
species using molecular biology
– application of Bayesian statistics to
forensic entomology and taphonomy
• Workshops and presentations at conferences
• Scientific publications and other reports
(Reference 10 – 14 and Appendix 4)
4.3.5 Microbial forensics and epidemiological
investigations
• Writing of scientific paper concerning
microbial forensics
PhD projects included in or related to 4.3
• Johannes Hedman, SKL/ULUND: DNA
analysis of PCR-inhibitory forensic samples,
ULUND in collaboration with SKL (Nov 2011)
(5) (4.3.2)
• Therese Ottinger, SVA : Development of
forensic veterinary pathology from crime
scene to court, SVA in collaboration with
SKL and SLU (4.3.3)
• Ronny Hedell, SKL: Statistical modeling to
make forensic evidence evaluation more
effective. Chalmers University of Technology
in collaboration with SVA, SKL and ULUND
(reported in 4.2)
Results and Discussion
Results
The results of 4.3.1, 4.3.2, 4.3.3, 4.3.4 and 4.3.5
are presented in Appendices 1– 5, respectively.
Discussion
The work on and related to the forensic response
plan (4.3.1) has had direct positive effects on the
forensic investigations of B-incidents. There has
been a radical increase in forensic awareness in
those agencies that may become involved in a
suspected incident and a sustained interest in
forensic issues. The laboratory collaboration
and forensic handling of CBRN incidents have
become more efficient and the results are of
better quality both from a scientific and a legal
perspective.
The routines for handling animal carcasses in
order to keep the chain of custody, documentation,
evidence evaluation and the structure of the
statement of witness have all improved as a
9
result of the project. The quality of the forensic
investigation as a whole is also better as a result
of networking and bridging activities. Positive
factors include better background information,
more precise questions, better handling of
carcasses before and during transport and
increased knowledge of what a veterinary
forensic pathologist can add to an investigation.
The knowledge, interest and appreciation
of forensic entomology and palynology have
increased among legal authorities during the
course of the project.
The work on improved forensic sampling and
DNA analysis has resulted in several new and/
or improved analytical approaches to problems
encountered in PCR-based analysis of crime
scene samples which contain microbial agents in
small amounts or in “problematic” matrices, e.g.
matrices with high amount of PCR inhibitors.
The need of microbial forensic databases
have been described in order to obtain a better
preparedness for investigating bioterrorism
incidents. The current limitations are the access
to high-quality reference databases of relevant
genome sequences and statistical tools for
evaluating sequence based evidence in the
context of relevant scenarios. (See Sjödin et al
and Appendix 5.)
Conclusion
The work performed in Task 4.3 has significantly
improved the preparedness and practical forensic
handling of a suspected B-incident in a veterinary
setting all the way from “crime scene to court”.
The importance of forensic awareness in first
responders (EU CBRN Action Plan H42), of
obtaining forensic evidence of high quality and
how the results should be reported (H44),
collaboration between different agencies (H45)
and the construction of an emergency response
plan (H30) have all been addressed in the
forensic response plan (4.3.1).
10
The work on forensic veterinary pathology
(4.3.3) and entomology/palynology (4.3.4) have
addressed H42, H44, and H45.
The project on PCR has addressed H44 in
depth (obtaining evidence of high quality).
Future Outlook and Recommendations
The recommendations in the forensic response
plan should be communicated within the EU
to help harmonize the national response plans
(operational routines).
Remaining problems at a national level include
issues around decision-making, legislation,
responsibilities and external communication.
In addition, the collaboration and forensic
awareness of first-responders needs to be further
developed.
An active network of veterinary forensic
pathologists in the EU should be set up.
Evidence evaluation in veterinary medicine and
microbial forensics should be further developed.
In both situations the development of a theo
retical framework together with databases of
relevant reference data are important.
The concept of PCR-processing should be
generally implemented in the development of
improved methods for diagnostic PCR within
the EU.
Acknowledgements
The framework of the EU-project AniBioThreat
(Grant agreement: HOME2009/ISEC/AG/191)
with support from the Prevention of and Fight
against Crime Programme of the European
Union European Commission – DirectorateGeneral Home Affairs. This publication reflects
the views only of the authors, and the European
Commission cannot be held responsible for any
use which may be made of the information
contained therein.
The participants in the Swedish national
projects Forensic Network and Operational
Routines are gratefully acknowledged.
Publications from Task 4.3
References
1.
Aitken, C. G. G. and Taroni, F. (2004). Statistics and
the Evaluation of Evidence for Forensic Scientists.
2nd ed. Wiley, Chichester.
2.
Buckleton, J. (2005). A framework for interpreting
evidence. In: Forensic DNA Evidence Interpretation
(J. Buckleton, C.M. Triggs, S.J. Walsh eds.), 27–63.
CRC Press, Boca Raton, FL.
3.
Nordgaard, A., Ansell R., Drotz, W., and Jaeger, L.
(2012) Scale of conclusions for the value of evidence.
Law, Probability and Risk 11(1):1-24[1] [4]
4.
Handbook: Operativa rutiner för forensisk mottagning
av CBRN-prov (In Swedish, in preparation)
5.
Hedman J: DNA analysis of PCR-inhibitory forensic
samples: Doctoral thesis, Lund University; 2011.
6.
Kumar Krishnamoorthy A: Composing an inhibitor
tolerant PCR buffer. Project report, Lund University
2011.
7.
Lindsten H: Applying Real Time PCR in Forensic DNA
Analysis. Candidate thesis, Lund University 2012.
8.
Kumar Krishnamoorthy A: Pre PCR processing of
forensic samples. Master’s thesis, Lund University
2012.
9.
Norén L, Hedell R, Ansell R, Hedman J: Purification of
crime scene DNA extracts using centrifugal filter
devices. Submitted to Investigative Genetics 2012.
Peer-review
•Hedman J, Knutsson R, Ansell R, Rådström P,
Rasmusson B. Pre-PCR Processing in Bioterrorism
Preparedness - Improved Diagnostic Capabilities for
Laboratory Response Networks. Biosecur Bioterror
2013; In press.
•Andersson, G., Lindström, A. and Sundström, A.
Bayesian networks for evaluation of evidence from
forensic entomology. Biosecur Bioterror 2013;
In press
•Norén L, Hedell R, Ansell R, Hedman J. Purification
of crime scene DNA extracts using centrifugal filter
devices. Investig Genet 2013;4:8.
•Disney, R.H.L., Garcia-Rojo, A., Lindström, A. and
Manlove, J.D. Further occurrences of Dohrniphora
cornuta (bigot) (Diptera, Phoridae) in forensic cases.
Submitted to: Revista Espanola de Medicina Legal.
•Fremdt H., Szpila K., Huijbregts H., Lindström A.,
Zehner R., Amendt J. 2012. Lucilia silvarum Meigen,
1826 (Diptera: Calliphoridae) – a new species of
interest for forensic entomology in Europe. Forensic
Science International.
•Gale, P., Stephenson1, B., Brouwer, A., Martinez, M.,
de la Torre, A., Bosch, J., Foley-Fisher, M., Bonilauri, P.,
Lindström, A., Ulrich5, R.G., de Vos, C.J., Scremin, M.,
Liu, Z., Kelly, L. and Muñoz, M.J. Impact of climate
change on risk of incursion of Crimean-Congo
haemorrhagic fever virus in livestock in Europe through
migratory birds. J Appl Microbiol. 112(2):246-57
• Sjödin,A., Broman, T., Melefors, Ö., Andersson, G.,
Rasmusson, B., Knutsson, R., Forsman. M. The Need
for High-Quality Whole-Genome Sequence Databases
in Microbial Forensics. Biosec.Bioterror 2013 In press.
10. Lindström, A. and Andersson, G. 2013 . Experimental
Design, Statistics, and Computer Modeling In:
International Dimensions and Frontiers of Forensic
Entomology. Forthcoming.
11. Andersson, G., Lindström, A. and Sundström, A.
Bayesian networks for evaluation of evidence from
forensic entomology. Biosecurity and Bioterrorism.
Book chapters
12. Disney, R.H.L., Garcia-Rojo, A., Lindström, A. and
Manlove, J.D. Further occurrences of Dohrniphora
cornuta (bigot) (Diptera, Phoridae) in forensic cases.
Submitted to: Revista Espanola de Medicina Legal.
•Hedman J, Lövenklev M, Wolffs P, Löfström C,
Knutsson R, Rådström P. Pre-PCR processing
strategies. In: Nolan T, editor. PCR Technology,
Current innovations. 3rd ed: CRC Press, Boca Raton,
USA; 2013.
13. Fremdt H., Szpila K., Huijbregts H., Lindström A.,
Zehner R., Amendt J. 2012. Lucilia silvarum Meigen,
1826 (Diptera: Calliphoridae) – a new species of
interest for forensic entomology in Europe. Forensic
Science International.
•Lindström, A. and Andersson, G. 2013 . Experimental
Design, Statistics, and Computer Modeling In:
International Dimensions and Frontiers of Forensic
Entomology. Forthcoming.
14. Gale, P., Stephenson1, B., Brouwer, A., Martinez, M.,
de la Torre, A., Bosch, J., Foley-Fisher, M., Bonilauri, P.,
Lindström, A., Ulrich5, R.G., de Vos, C.J., Scremin, M.,
Liu, Z., Kelly, L. and Muñoz, M.J. Impact of climate
change on risk of incursion of Crimean-Congo
haemorrhagic fever virus in livestock in Europe through
migratory birds. J Appl Microbiol. 112(2):246-57
PhD thesis
•Hedman J. DNA analysis of PCR-inhibitory forensic
samples: Doctoral thesis, Lund University; 2011.
ISBN: 978-91-7422-283-8.
appendix
Appendix 1:
Forensic Response Plan
(Generic measures)(4.3.1)
Appendix 2:
Improved forensic sampling
and DNA analysis (4.3.2)
Appendix 3:
Veterinary forensic pathology (4.3.3)
Appendix 4:
Forensic entomology,
palynology and taphonomy (4.3.4)
Appendix 5:
Microbial forensics and
epidemiological investigations.
Task 4.3: Appendix 1
Task 4.3. Appendix 1. Forensic Response Plan (Generic measures) (4.3.1)
Cecilia Vahlberg and Birgitta Rasmusson Swedish National Laboratory of Forensic Science – SKL June 2013 Task 4.3: Appendix 1
WP4: EUROPEAN LABORATORY RESPONSE NETWORK (LRN) FOR ANIMAL BIOTERRORISM
THREATS TASK 4.3: FORENSIC RESPONSE PLAN AND IMPROVED FORENSIC SAMPLING AND
DNA ANALYSIS
Summary
Preparedness is crucial for rapid and correct handling of terror incidents involving microbial agents. Even a suspected incident, especially if not investigated correctly, may have severe impact on society, economy and public trust. Fortunately, real incidents directed towards animal facilities have so far been extremely rare, but the possibility cannot be ignored. Forensic awareness and to “start right” is crucial for the eventual prosecution and conviction of the offenders. Early mistakes in the investigation may have disastrous consequences for the legal process. It is also important to “finish right” and report the results in a way that is accepted and correctly understood by the legal system. There are many possible scenarios for intentional release of microbial agents and criminal intent may not be apparent from the start. The forensic investigation may require collaboration between officers and agencies that seldom interact under normal circumstances, e.g. local veterinarians and police officers. To optimize the process these officers and their respective agencies must practice working together before a real crises occur. The generic measures for a forensic response plan, presented in this report, includes central aspects to consider and how to establish routines regarding preparedness, collaboration, sampling, transportation, forensic analysis and the construction of a joint statement of witness. The focus is on roles, communication and teamwork. We emphasize the importance of forensic awareness for first responders, mutual exercises, exchange of knowledge and respect for each other’s competence. Remaining problems include issues around decision‑making, legislation, responsibilities and external communication. The collaboration and forensic awareness of first‑responders needs to be further developed. Many measures presented in this report can be applied to CBRNE‑incidents in general. 2
DNA ANALYSIS
Content
Introduction ................................................................................................................................................................4 Background ................................................................................................................................................................ 5 Aim ................................................................................................................................................................................... 9 Method........................................................................................................................................................................... 9 Related national projects and exercises ................................................................ 10 Results......................................................................................................................................................................... 13 Preparedness and forensic awareness .................................................................. 13 Communication, collaboration and operational routines ................................. 15 Investigation and forensic sampling ..................................................................... 16 Transportation .......................................................................................................... 17 Laboratory analysis ................................................................................................. 18 Discussion................................................................................................................................................................. 21 Conclusions.............................................................................................................................................................. 22 What we have accomplished ..........................................................................................................................23 Take-home message ...........................................................................................................................................23 References ................................................................................................................................................................ 24 Acknowledgements ............................................................................................................................................. 24 3
DNA ANALYSIS
Introduction
The vast majority of incidents involving microbial agents in a veterinary setting have “natural” causes. In some instances, outbreaks of disease in an animal facility may be caused by criminal negligence of varying degree, e.g. ignorance of regulations and security measures. Intentional release of microbial agents (B‑
terrorism) is fortunately rare; but it is a threat that should be recognized. The number, size and specialization of modern animal facilities, the extensive transportation of animals and feed and the potential huge negative impact on economy and public trust should be considered. B‑terrorism against animal facilities may hypothetically be performed to cause harm to society and instigate fear, get attention for a political cause (e.g. animal welfare), and/or to induce economical loss. Since animal facilities are potential targets for B‑terrorism, disease‑control authorities and officers, e.g. local veterinarians, should be vigilant in reporting any “irregularity” in the outbreak pattern as these may indicate an underlying crime. It is further important that these “irregularities” are swiftly and properly investigated. The Police should be immediately alerted in order to secure the scene for further investigations. Here, efficient and close cooperation both between the Police, disease‑control officers and experts on B‑agents and within the Police itself is required. When the investigation has been taken over by the Police, the facility (or part of it) will be regarded as a possible crime scene and a forensic investigation will be initiated. Compared to an “ordinary” crime scene; both ”classical” forensic evidence (DNA, fingerprints etc.) as well as ”intrinsic” forensic evidence (from the microbial agent and its matrix) need to be secured. There are many potential starting points and scenarios for a B‑terror incident and different disease‑control authorities, not familiar with the requirements of a criminal investigation, may already be active when the suspicion arises. Intense media coverage and both political and public pressure can be expected. Since B‑
terrorism (fortunately) is very rare, the involved officers and agencies will have little experience in handling such an incident. Together this poses challenges to the forensic investigation. 4
DNA ANALYSIS
To reduce the risk for error and unnecessary friction officers from agencies that might be involved in a B‑terror incident need to know other’s roles and competence beforehand and they should be used to work together. This can be achieved through education, study visits, various projects to establish joint operational routines and not least regular exercises. Knowledge, respect, communication, teamwork and preparedness are core factors. Forensic awareness among the first responders, other non‑police officials (subject experts etc.) and among decision makers is crucial for a successful forensic investigation. Simply expressed, forensic awareness is to keep an open mind to possibility of a criminal act, to know the basic forensic requirements, to “treat the scene as possible crime scene until proven otherwise”, to be aware of signs that distinguish a natural outbreak, accidental release and a possible criminal act, and to contact the Police immediately upon the slightest indication of criminal activity. This report describes important aspects to consider for a successful forensic investigation of a suspected B‑ terror incident, from a Swedish perspective. The results are based on experiences gained within AniBioThreat as well as the national projects Operational Routines and Swedish Forensic CBRNE Network, supported by the Swedish Civil Contingencies Agency (MSB). Background
The legal process for an “ordinary crime” encompasses a criminal investigation, prosecution, a court process and finally a verdict, see Figure 1. To successfully prosecute and convict an offender and/or clear the innocent it is important both to start right, and to finish right. The Police “owns” the crime scene and (in Sweden) the Police and/or the Prosecutor lead the investigation. Specially trained crime scene investigators (CSI:s) are responsible for the crime scene analysis and for securing materials and forensic trace evidence, e.g. DNA, fingerprints, fibers or shoe prints, from the scene. Some of these traces may be analyzed by local Police laboratories, especially in the major cities (e.g. fingerprints and shoe prints) but the majority (DNA, drugs, fibers, more “difficult” prints, and traces/prints from complex cases or severe/organized crime etc.) are sent to the Swedish National Laboratory of Forensic Science (SKL) for analysis. 5
DNA ANALYSIS
Sweden has around 10 million inhabitants and SKL annually handles around 50 000 crime cases plus an additional 50 000 analyses of DNA from suspects (buccal swabs). The forensic experts at SKL are rarely called to the crime scene. However all CSI:s are trained by SKL and SKL is often contacted by the CSI:s and/or the responsible investigator to discuss which analyses should be performed on the recovered material and in which order. In complex cases (more than one crime scene, high‑profile crime, many materials and/or and many different types of traces etc.), a special SKL case coordinator (a “generalist”) is assigned to the case. The “generalist” coordinates the work at SKL and acts as a liaison officer for the Police and Prosecutors office. He/she composes the final, comprehensive statement of witness, containing all forensic results. When applicable, forensic evidence may be combined (e.g. blood/ DNA and glass) and evaluated together. Figure 1. A schematic representation of the legal process for “ordinary crime”. The Police have well established routines for transportation and handling of forensic evidence. SKL is an accredited laboratory according to ISO 17025. Quality assurance of the crime scene work (ISO 17020) and the Swedish Police laboratories are well under way. Similar processes are in effect in other EU countries. Communication routines between the Police and SKL during case‑
work are also well established. Forensic analyses at SKL comprise both the analyses per se, and evidence evaluation using the Bayesian approach [1, 2]. A nine‑level scale is used to express the evidentiary value (likelihood ratio) in the statement of witness [3], see Figure 2. 6
DNA ANALYSIS
Figure 2. A schematic representation of the forensic process for ”ordinary” crime. Many scenarios may serve as starting points for a suspected bioterror incident, see Figure 3.
Consequently, several different authorities e.g. local environmental protection officers, local veterinarians, central animal health authorities, local physicians and central human disease control agencies etc. may already be active when the Police are called to scene. Media attention will rapidly become considerable as well as the political pressure. Serious B‑ incidents are very rare but this also means that authorities are not used to detecting nor handling them. There is definite risk that the forensic process will get confused and complicated; which in turn may make prosecution of the offenders impossible. 7
DNA ANALYSIS
Figure 3. Examples of starting points for a suspected B‑terror incident. X, Y and Z represent different high security facilities to which the sample is transported. Forensic evidence must be correctly secured and handled to be eligible in court. Some EU countries, e.g. the UK, have very strict requirements of “chain of custody” (exact recording of the movement and handling of the material etc.). In countries with less formal rules, a defense attorney may still destroy the prosecutors case by questioning the handling of the material with regards to possible contamination, mix‑ups etc. When called to a suspected incident, the Police will rapidly secure the scene in order not to destroy evidence. Forensic awareness (“treat the scene as a possible crime scene until proven otherwise”) among the officers already active at the site (e.g. veterinarians, rescue personnel) will have prevented unnecessary access, unnecessary movement of objects etc. A log of everything that has been done at the site, when and by whom is a helpful measure. Microbiological samples that later will be used as forensic evidence may already have been collected when the Police arrive, or will be collected by the veterinarians and other officers at the scene together with the Police, see Figure 4. Knowledge about the requirements from the legal system with regards to forensic evidence, “chain of custody” etc. is essential for such sampling. 8
DNA ANALYSIS
Figure 4. Forensic analysis of the microbial agent in a B‑terror incident is performed by expert laboratories; here exemplified by the Swedish National Veterinary Institute (SVA). Sampling may be performed either by the Police or by veterinarians / microbiological experts at the site. Aim
The aim of task 4.3.1 Forensic Response Plan within the AniBioThreat project was to identify generic measures in order to respond to an animal bioterrorism incident, from a Swedish perspective. Method
•
The handling of veterinary cases in Sweden was studied; especially with regards to the work at the National Veterinary Institute (SVA) and legal cases (see also Appendix 3). •
The results from the national projects Operational Routines and Swedish Forensic CBRNE Network were considered from the perspective of B‑incidents in animal facilities. •
The insights gained in the above mentioned projects and the collaborative routines for field‑work was tested in a veterinary setting during the SAMBIO2013 exercise (April 2013). 9
DNA ANALYSIS
•
Evidence evaluation was applied to forensic veterinary medicine and a model for improved statements of witness was developed (results are reported in Appendix 3 and in Task 4.2). Related national projects and exercises
OPERATIONAL ROUTINES
Operational routines is a three year collaboration project (2011‑2013) ) funded by the Swedish Civil Contingencies Agency (MSB) with participants from the Swedish National Laboratory of Forensic Science (SKL), the Police, the Swedish Defense Research Agency (FOI) and the Swedish National Food Administration (SLV). The aim of the project was to develop collaborative methods for material handling and trace recovery from CBRNE‑contaminated materials. The project included collaboration with the Federal Bureau of Investigation (FBI) in the US. The initial plan was that project should encompass the entire forensic processes: forensic sampling at the crime scene, transportation, sample handling, analysis and evidence evaluation, see Figure 5. However, as the extent of this task became clear the focus in the project was limited to the first three processes, i.e. forensic sampling at the crime scene, transportation and handling of the materials. 10
DNA ANALYSIS
Figure 5. The initial scoop of the project Operational routines comprised the entire forensic process in a CBRNE‑related incident. It was later limited to the first three phases (forensic sampling, transportation and sample handling). THE SWEDISH FORENSIC CBRNE NETWORK
This network was established as a project (2011‑2012) funded by the Swedish Civil Contingencies Agency (MSB) and has been implemented since 2013. The core of the network (Figure 6, dark green circle) consists of central Police facilities (e.g. the national CBRNE‑specialists, the “bomb squad”) and those agencies that directly aid the Police in the laboratory‑based forensic investigation of CBRNE‑related crime, including the national high security facilities for C, B, RN and E. The aim of the Swedish Forensic CBRNE Network is to develop and strengthen the forensic expertise and competence within the CBRNE area to efficiently support the Police and other legal authorities in the investigation of an incident. The network functions as a platform for increased forensic awareness and preparedness by stimulating contact between authorities, educational activities and joint projects and exercises. Other agencies that may become involved in a CBRNE‑related crisis are associated to the network in various ways (Figure 6). SAMBIO2013
The exercise scenario in SAMBIO 2013 comprised an intentional release of a microbial agent (B‑terrorism). SAMBIO 2013 enabled several different authorities to practice working together. The exercise included both field work and a table‑top exercise and provided an excellent opportunity to test the 11
DNA ANALYSIS
routines for communication, collaboration and forensic sampling, which were developed in the project Operational routines. In addition, it gave SKL the opportunity to participate in the creation of both the scenario and the practical construction of the crime scene(s). Figure 6. Authorities that comprise and are associated to the Swedish Forensic CBRNE Network. Core authorities are found within the dark green circle. RMV = Swedish National Institute of Forensic Medicine, SKL = Swedish National Institute of Forensic Science, SLV = Swedish National Food Administration, FOI = Swedish Defense Research Agency, SMI = Swedish Institute for Communicable Disease Control, SVA = Swedish National Veterinary Institute. 12
DNA ANALYSIS
Results
Preparedness and forensic awareness
In Sweden, a suspected B‑terror incident may involve the rescue service, local veterinarians, the medical services, environmental agencies, forensic pathologists (human and veterinary), the National Veterinary Institute (SVA), the Police, the Prosecutors office, the National Food Administration (SLV), the National Laboratory of Forensic Science (SKL), the, the Institute for Communicable Disease Control (SMI), the Swedish Defense Research Agency (FOI), the Swedish Civil Contingencies Agency (MSB) and in some instances the Customs Office, the Coast Guard or the Military. All are experts in their respective fields, but their knowledge about each other and their degree of previous collaboration will vary considerably. Moreover, the collaboration within the authorities must function. One example is the Police, where CSI:s and CBRNE specialists, local, regional and central Police authorities (including the Secret Service), Police investigators and forensic experts etc. need to be able to work together. The rarity of the event, the external pressure and the potential danger of the agent involved, pose additional challenges to the investigation. Thus, to be successful, the work has to start long before an actual incident. The importance of “getting to know one other” (both personally and as agencies), developing joint routines and methods and performing regular exercises and cannot be over‑stressed. The Swedish Forensic Network, the project Operational Routines (and others), national CBRN exercises with “possible crime”‑ scenarios, AniBioThreat as a whole, including the exercise SAMBIO 2013, are all examples of such activities. Teamwork between experts from different agencies is essential both at the crime scene and in the laboratory, especially if forensic sampling needs to be performed on “hot” materials, in a high‑security facility. Communication, respect for each other’s expertise and an open mind are key issues. Adjustment of rules and regulations may be needed to achieve concurrency between authorities. The Swedish agencies listed above sort under four different ministries, and consequently there is a need for communication and collaboration also within the government. 13
DNA ANALYSIS
Legal issues that can cause clashes between apparently contradictory regulations during an on‑going crisis need to be identified and resolved. Moreover, agencies may have different routines for decision making and the expectations from society e.g. regarding external communication may vary considerably. This can cause a lot of friction during an incident. One example is the “ownership” of the scene and the investigation. As soon as a Police investigation is formally instigated (in Sweden), the scene “belongs” to the Police, and there will be a formal Police investigation going on in parallel with activities aimed at reducing the spread of disease, informing the public etc. The Police often want to reduce the information to the public as much as possible in the interest of the criminal investigation, while other authorities are expected to be very “open”. There may also be an acute need to inform the public to avoid the spread of rumors, fear, hoarding and violence. A related issue is the legal requirements of “confidentiality” (in Swedish “förundersökningsekretess”) in a police investigation. During an investigation forensic authorities e. g. SKL, only communicate the analyses results to the “direct customer” i.e. the Police investigator(s) and/or prosecutor(s) handling the case. This may also apply to results from a diagnostic expert laboratory (e.g. SVA or SMI) on e.g. the identity and origin of a bacterial strain. Diagnostic laboratories often have relatively little experience of handling results under “confidentiality” and may not recognize the risk that the results may harm the investigation significantly if communicated to the public, which includes the perpetrators. These “clashes” often become very apparent during an exercise, which illustrates the importance of such activities. Forensic awareness (“always treat a scene as a possible crime scene..”) in first responders encompasses a general awareness of the possibility (however slight) of a criminal intent, the ability to notice and react to anything “unusual” at the scene and the practical ability to do what is necessary in the situation (saving life, protecting property etc.) without unnecessarily destroying the scene (moving objects etc.) or forensic evidence (e.g. shoeprints) . The ability to make informed decisions, e.g. to be fully aware of both the positive and the possible negative impact of a certain action, decide what to do, and take responsibility for the decision later is central. 14
DNA ANALYSIS
Basic knowledge about crime, crime scenes and forensic awareness also makes it easier to distinguish between a natural outbreak, accidental release and a possible B‑terror attack. Before other officials from the Police or the Rescue service enter a scene, the first responders will do a basic check of the area to make sure it is safe to enter. Depending on the situation, this may be done by the Fire brigade, the Police, or others. The Police have well established routines for criminal investigations, crime scene work and securing of forensic evidence. It is therefore crucial to involve the Police as soon as possible upon the slightest suspicion of a criminal act. As in many similar situations, it is better to call one time to often than too late. Basic knowledge about Police work and personal contacts with Police officers during exercises etc. will make this a lot easier for e.g. local veterinarians called to an outbreak site. In a suspected B‑terror incident, involving for instance intentional release of Anthrax, Police CBRNE specialists, trained to work and sample material in “hot zones” will perform the registration of the scene and the sampling in the “infected” area. Their initial task is to thoroughly document the scene, so that everyone involved “gets the same picture” before any sampling is actually performed. Communication, collaboration and operational routines
Routines for communication between officials and agencies should be established and practiced well before an actual incident. In the project Operational routines, detailed routines for communication, collaboration and decision making for the Police/Secret service/Prosecutors office, the forensic experts at SKL and the CBRNE experts at high security facilities, working together on a CBRNE case were developed, see Figure 7. The Police investigator will inform the forensic experts at SKL about the incident. A case coordinator at SKL is appointed who is now responsible for the communication between the Police, the forensic experts at SKL and the CBRNE experts at the high security facilities. The case coordinator will contact the most suitable high security laboratory depending on the incident (in Sweden there a three such facilities, and the use of Danish facilities is being investigated). Factors that influence the choice of facility is what type of agent that is 15
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suspected, if more than one type of agent could be involved (e.g. B and C), the amount and type of secured material and where in Sweden the incident occurs. The manager at the high security facility is responsible for the initial contact with the case coordinator at SKL. This manager is also responsible for handling the request from the Police/SKL regarding the analysis of the contaminated materials. Figure 7. Flow‑chart describing the forensic investigation of a CBRN incident from a collaborative and decision‑making perspective. Investigation and forensic sampling
During a criminal investigation the Police is responsible for all the movements, documentation, analyses and sampling at the crime scene The Police and the Secret Service may also have specific intelligence information not available to other agencies. It is important to note that the Police is not ONE entity and that both local/regional and national forces may be active (and need to collaborate) during the various phases of the investigation. 16
DNA ANALYSIS
National Police CBRNE‑specialists that are trained to work in a “hot zone” will perform the crime scene investigation and the forensic sampling under the supervision and with advice from both CSI:s and (if necessary) forensic experts from SKL. Equipment for collecting and securing forensic evidence in a “hot‑
zone” was tested in the project Operational routines. The work resulted in routines for forensic sampling in a hot zone, including a sampling protocol. The work was done in collaboration between the Police and forensic experts at SKL. It is important to establish such routines also for other first responders, e.g. local veterinarians, at a possible crime scene. Moreover, all first responders should have access to the same type of equipment and materials to secure forensic evidence from a crime scene. Personal safety equipment was also evaluated in the project Operational routines. The equipment should protect the first responders but also make it possible to collect and secure the evidence material; which sometimes requires small, precise movements. The officers must also be able to transport (carry) the evidence in a practical way. Transportation
Contaminated (“hot”) material collected at a crime scene scene cannot be investigated in a normal forensic laboratory but should be transported to high security facilities (for microbial agents BSL3 or 4), see Figure 8. Routines for this were also developed in the project Operational routines. There are international regulations for the transportation of dangerous substances (ADR). In Sweden, the ADR‑S (MSBFS 2012:6 Transport av farligt gods) is applied. Transportation of dangerous substances performed by the Police, the Customs or the Coast guard can however be excluded from ADR‑S. A special transportation protocol is used to document e.g. who is responsible for the transportation and for receiving the material. In a CBRN‑incident, the Police will be responsible for the transportation of the material. The case coordinator from SKL is the liaison officer between the Police and the expert laboratories, and he/she will contact the high security facility to prepare for the arrival of the material. 17
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Figure 8. SKL is not equipped to handle B‑contaminated material. The “hot” material is instead transported directly to a high security facility. Specially trained forensic experts from SKL then join the B‑experts at the BSL‑3 or ‑4 facilty and together these officers secure both ”classical” forensic evidence (DNA, fibers etc), the microbial agent, and information from the agent matrix etc. Experts from other agencies, nationally and internationally, may also participate in the investigation. Laboratory analysis
Routines for the handling of CBRNE‑ materials at high security facilities have been developed in the project Operational routines. This includes guidelines for the collaboration of SKL and FOI in the event of the “unknown”, i.e a material which may contain C, B, RN or E or combinations thereof. Methods for forensic recovery and analysis at high security facilities were established for both ”classical” forensic evidence and forensic information from the agent and its matrix (“intrinsic” forensic evidence), see Figure 9. All methods and routines have been documented in a Handbook [4]. The handbook includes check lists, flow charts, equipment lists, (mobile and stationary), handling routines etc. The forensic experts from SKL are responsible for the analysis of ”classical” forensic evidence from the material e.g. DNA analysis, fingerprint analysis, IT‑forensics etc. The intrinsic forensic evidence, 18
which includes i.e. the nature and origin of the Task 4.3: Appendix 1
DNA ANALYSIS
material/microbiological agent, the production method, links to other seizures etc., is analyzed by the CBRNE experts aided by the forensic experts at SKL. All the steps in the operational routines, from the point where the high security facility is contacted by SKL, to the reporting of the analyses results, have been developed by practical testing using various scenarios and mock materials. The focus of AniBioThreat is microbial agents but the Police CBRNE specialists and the officers at the high security facility should also check the material for C, RN and E ‑substances. Un‑packing of the material transported from the crime scene is performed by a CBRNE‑expert together with a forensic expert from SKL or by a CBRNE expert under the supervision of a forensic expert. All the steps during the un‑packing are documented both in writing and by photography. The guidelines for the un‑
packing were developed through several practical exercises using mock materials. Figure 9. Practical routines for material handling and forensic analysis for CBRNE‑
contaminated material in high‑security facilities have been developed in the project Operational routines. 19
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Forensic experts in general analyses have little experience in performing forensic sampling and analyses outside their “home” laboratory. High security facilities provide an additional challenge, with the need for special routines and protective gear. CBRNE subject experts are familiar with high security facilities and with the handling of dangerous materials. However, they may not know about the special requirements for forensic analyses, including in which order and how various traces should be secured. Information, study visits, practical education, the construction of routines and teamwork exercises are essential to develop the joint expertise. Regular practice of the routines is important to uphold the team, find “bottlenecks” in the work flow and ensure that the forensic analysis performed at the high security facility is of the same quality as the analyses performed “at home”, see Figure 10a. Some exercises may be performed using a “fake” glove box, see Figure 10b. Much can also be learned by analyzing the investigation of real incidents. Figure 10a and 10b. Forensic experts from SKL preparing for an exercise at a high security facility (10a) and practicing document analysis using a ”fake” glove box (10b). The “heavier“ equipment (which need to be permanently stationed in the high‑
security facility), disposable materials and chemicals that are needed to perform ”classical” forensics at a high security facility have been identified, and check‑
lists were constructed for the supplies that will be brought from the forensic laboratory to the facility in case of an incident, using ready‑packed boxes. 20
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Following decontamination of the material, it can be transported to a regular forensic laboratory for analysis. However, some decontamination methods destroy forensic evidence. These issues were also addressed in the project Operational routines as well as in other on‑going projects. Discussion
The ultimate goal of the forensic investigation of a B‑terror incident is to bring the perpetrator(s) to justice and prevent further attacks. Our work on a national forensic network, operational routines and a forensic response plan (AniBioThreat Task 4.3) has provided direct positive results for the Swedish Police and especially for SKL. We have experienced a radical increase in forensic awareness in those agencies that may become involved in a suspected incident and a sustained interest in forensic issues. The laboratory collaboration and forensic handling of CBRN incidents has become more efficient and the results are of better quality both from a scientific and a legal perspective. Problems that have been identified and still needs to be addressed include the exact responsibilities of different authorities in a CBRNE‑related crisis. There is uncertainty regarding the decision‑making and there are legal issues which will need the aid of legal expertise. External communication during a police investigation is another topic with remaining uncertainties. Further work on evidence evaluation and the construction of comprehensive statement of witness need to be performed. The effect of decontamination on forensic trace evidence and which analysis that can/cannot be performed on “hot” materials is being further investigated. The importance of basic forensic knowledge and forensic awareness needs to be continuously highlighted, especially for first responders (veterinarians, medical officers, fire brigades, customs officers etc.). Guidelines on how to work in a possible “hot zone” with forensic awareness in mind should be established in collaboration between forensic experts and first responders. It should be possible to establish such routines without having to compromise on the performance of either authority. To conclude; a forensic response plan for CBRNE‑incidents need to include all actors and the whole chain of events, i.e. first responders, crime scene work, forensic sampling, transportation, handling of contaminated materials, forensic 21
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analysis, evidence evaluation and the construction of a statement of witness. Teamwork between experts is essential and it is absolutely crucial to perform exercises, in order to learn, to establish routines, to practice teamwork and to identify problems, e.g. bottlenecks in the analytical flow or cultural or legal clashes so that they can be resolved before an actual incident. Conclusions
•
Many possible scenarios (starting points) •
Even ”small” cases may be complicated to handle due to the many actors involved, the rarity of the event and intense external pressure •
Forensic awareness in first responders may make a huge difference for the end‑result •
The investigation of a CBRNE crime scene is led by a responsible investigator from the Police, the Secret Service or the Prosecutors office and all experts and expert labs act on his/her request •
It is advantageous to assign a special forensic case coordinator to CBRNE‑cases as a liaison officer •
Teamwork between trained forensic experts and B‑ experts is crucial •
Learn from each other and get to know one another before and incident occurs •
•
Construct and practice lab routines (flow charts)
Exercises are important to find “bottlenecks” and “points of friction” and will improve the workflow in real cases •
Necessary “heavier” forensic equipment should be stationed permanently in high‑security facilities. The forensic experts who will work together with the subject experts during the investigation can bring ready‑packed boxes with sampling materials, reagents etc. •
Aim for a comprehensive statement of witness that is accepted and correctly understood by the legal system, containing both ”classical” forensic results (DNA, fingerprints etc.) and the results on the microbial agent, its matrix etc. 22
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What we have accomplished
•
A Swedish Forensic CBRNE Network of institutes and experts. •
Increased forensic knowledge and awareness. •
Operational routines for communication, forensic sampling, transportation and sample handling. •
CBRNE case coordinators and forensic experts on DNA, fingerprints, fibers, and IT from SKL trained to work in high‑security facilities together with experts on C, B and RN. •
Improved communication and better teamwork between agencies and single officers during real incidents resulting in more efficient forensic analyses of higher quality. Take-home message
•
Forensic science is multidisciplinary. •
CBRNE forensics requires collaboration with new parties compared to ordinary cases. •
”Techniques” are ‑relatively‑ easy to develop, teamwork requires effort and commitment. •
Start right: Early mistakes may have big consequences. •
Finish right: Evaluate the results (evidence) and report in a way that is understood correctly by the legal system. 23
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References
[1] Aitken, C. G. G. and Taroni, F. (2004). Statistics and the Evaluation of Evidence for Forensic Scientists.2nd ed. Wiley, Chichester. [2] Buckleton, J. (2005). A framework for interpreting evidence. In: Forensic DNA Evidence Interpretation (J. Buckleton, C.M. Triggs, S.J. Walsh eds.), 27–63. CRC Press, Boca Raton, FL. [3] Nordgaard, A., Ansell R., Drotz, W., and Jaeger, L. (2012) Scale of conclusions for the value of evidence. Law, Probability and Risk 11(1):1‑24[1] [4] Operativa rutiner för forensisk mottagning av CBRN‑prov (In Swedish, in preparation). Acknowledgements
The participants in AniBioThreat Task 4.3, 4.2, 5, 6, SAMBIO 2013, Operational Routines and Swedish Forensic CBRNE Network and the Swedish Civil Contingencies Agency (MSB) are all gratefully acknowledged. // 24
Task 4.3, Appendix 2. Improved forensic sampling and
DNA analysis
Johannes Hedman, Arun Kumar Krishnamoorthy, Jenny Schelin, Birgitta Rasmusson and Peter Rådström Aim
The aim was to improve diagnostic PCR analysis of impure and low‑level DNA samples through using the concept of pre‑PCR processing (1, 2). Background
In a bioterrorism crisis, decision makers need accurate and timely information about the nature of the biological agent. The source is often complex biological samples with low levels of target nucleic acids, i.e. cells, spores and viruses. A great analytical challenge are the extraneous PCR‑inhibitory substances that pollute biological samples from the environment, impairing amplification and reducing the analytical limit of detection (3). Additionally, the concentration of target nucleic acids is often low, making an optimal limit of detection crucial for efficient analysis. Following sampling of surfaces, air or water, a heterogeneous and dilute sample must be converted into a concentrated, amplifiable nucleic acid extract. A rapid analysis process, with a high level of automation, is needed for high throughput diagnostic PCR. Pre‑PCR processing (3, 4) is an integrated concept where the issues of analytical limit of detection and simplicity for automation are addressed in all analytical steps leading up to detection and analysis of specific DNA fragments, i.e. sampling, sample treatment and the chemical composition of PCR (Figure 1). The overall aim of pre‑PCR processing is to streamline the analysis process, enabling a high throughput, and maximize the amplifiability of complex biological samples. The sampling method should maximize target uptake and minimize uptake of PCR‑inhibitory substances. In sample treatment there is a trade‑off between yield and purity, as extensive purification leads to DNA loss. A cornerstone in pre‑PCR processing is to apply DNA polymerase‑buffer systems that are tolerant to PCR inhibitors, thereby increasing the analytical success rate and lowering the need for time‑consuming and costly sample treatment procedures where DNA is lost (5, 6). Task 4.3: Appendix 2
DNA ANALYSIS
Pre-PCR processing
Sampling
Sample
treatment
PCR
Detection Assessment
<
<
of results
and analysis
Decision <
making
Figure 1. Overview of the steps included in the diagnostic PCR analysis process. The aim of task 4.3 was to improve PCR analysis by employing pre‑PCR processing principles. Methods
In this project, forensic DNA analysis was used as a model system to investigate general properties of diagnostic PCR analysis. Human DNA has been analysed throughout the project. On a molecular level, DNA of all organisms has similar properties, meaning that findings from one type of analysis can be transferred to analysis of DNA from other organisms. Real‑time quantitative PCR (qPCR) and multiplex PCR with capillary electrophoresis detection were used. The forensic human DNA profiling kit PowerPlex ESX16 was applied in several studies. There, fluorescence peaks represent the amount of generated specific amplicons. When comparing different sampling and sample treatment methods, the total sum of peak heights was used as a relative measure of the level of amplifiable DNA in the samples. The total sum of peak heights was normalised against the volume of DNA extract for each sample. Methods for sampling and sample treatment have been developed and compared. Additionally, the possibility to improve DNA analysis of impure samples by optimising the content of the PCR has been investigated, and PCR‑
inhibitory reference materials were developed as tools for quality control. The reference method for sampling was swabbing using Selefa Trade cotton swab. The Chelex method (7) was used as a reference method in DNA extraction, and was used unless another method is stated below. Sampling using alternative cotton swabs
Sampling is a key step in all diagnostic PCR analysis processes. Preferably, the amount of target nucleic acids should be maximized and the amount of impurities, i.e. PCR‑inhibitory substances, from the background matrix should be minimized in sampling. We have compared and evaluated three types of cotton swabs: Selefa Trade cotton swab (used as a reference), Abstrichtupfer Swab (Sarstedt) and Sterile Cotton Tipped Applicators (1WC‑FDNA, Puritan). The swabs were used to swab dried saliva and blood stains, and the extract was amplified using the multiplex PCR kit PowerPlex ESX16 (Promega Corporation), containing 16 genetic markers (see reference 5 for more methodology details). No significant differences in the levels of amplifiable DNA were found between the reference swab and the two alternative swabs 2
DNA ANALYSIS
(Table 1). However, the Abstrichtupfer swab performed better than the Puritan swab. Also, the Puritan swab showed bad absorption of NaCl prior to swabbing, leading to smudging of the stain in swabbing. The cotton on the swab was quite loosely packed on the wooden stick, making it difficult to cut off the cotton for extraction. In total, the swab from Selefa Trade and the Abstrichtupfer are the best alternatives for future tests. Table 1. Comparison of three types of cotton swabs for sampling of dried saliva and blood stains on glass. The PowerPlex ESX16 kit was used in amplification. The P values refer to t‑tests between each of the alternative swabs and the reference swab (Selefa Trade). N=5 Swab type Cotton, Selefa Trade Puritan 1WC‐FDNA Abstrichtupfer swab Cotton, Selefa Trade Puritan 1WC‐FDNA Abstrichtupfer swab Sample Dried saliva, 10 µL Dried saliva, 10 µL Dried saliva, 10 µL Dried blood, 1 µL Dried blood, 1 µL Dried blood, 1 µL Normalised total sum of peak heights (average, in rfu) P value 75 195 ‐ 50 093 0,15 94 844 0,08 73 197 ‐ 28 408 0,17 39 021 0,20 Sampling using M-Vac
Cotton swabs are limited by the size of the objects/surfaces that can be sampled, and they work poorly for absorbing materials such as fabrics. Cutting pieces of fabric for analysis is another option, but large sample pieces are difficult to work with in a streamlined fashion. A possible sampling alternative for larger surfaces and absorbing materials is the M‑Vac sampling instrument (M‑Vac Systems Inc.), based on wet vacuuming of biological stains. We evaluated the M‑Vac system for sampling on inert surfaces and fabrics. M‑Vac gave significantly higher DNA concentrations from dried saliva stains on laminated wood, compared to cotton swabs (Table 2). On glass, M‑Vac and cotton swabs gave comparable results. Also, M‑Vac retrieved over twice as much DNA from saliva stains on towels (terry‑cloth) compared to cotton fabric (T‑shirt), showing that the absorption properties of the material are important for sampling. 3
DNA ANALYSIS
Table 2. Comparison between cotton swabbing and M‑Vac sampling. Selefa Trade cotton swab was applied. DNA concentrations were gathered using the qPCR kit Quantifiler Human (Life Technologies). Average DNA
concentration
Sampling method
Surface
(ng/µL)
P value
Laminated
Cotton swab (N=3)
wood
0,57
Laminated
M-Vac (N=3)
wood
1,14
0,02
Cotton swab (N=2)
Glass
1,20
M-Vac (N=2)
Glass
1,34
0,38
M‑Vac was successfully applied for retrieving DNA from the wearer of various clothes, including denim fabric, leggings and cotton T‑shirt. Complete forensic DNA profiles were generated from all tested clothes types. Partial DNA profiles were retrieved from an “offender” pressing his hand against the shoulder of a person wearing a shirt. M‑Vac could be a good alternative for sampling of stains that are too large to allow for efficient swabbing, and also for clothes and fabrics. DNA extraction through sonication
Quick and simple DNA extraction procedures have been developed and tested. Different leaching and extraction buffers have been investigated, specifically comparing deionized water and PBS. No significant differences were found between the tested buffers. A sonication procedure has been optimized and compared to the standard Chelex extraction (8). Inevitably a too strong sonication process leads to DNA degradation. In our set‑up, one minute sonication at 35 kHz and 56°C (Bandelin Sonora) gave the best results, but still slightly lower amplification according to the normalised total sum of peak heights (rfu) than the Chelex extracts (not significant, Table 3). Five minutes sonication gave significantly poorer amplification (lower allelic peaks, rfu). Table 3. Comparison of Chelex extraction and sonication, 35 kHz at 56°C, for extraction of swabbed dried saliva stains (10 μL saliva). The PowerPlex ESX16 kit was used in amplification. The P values refer to t‑tests between each of the alternative swabs and the reference. N=2 Extraction method Chelex extraction Sonication 1 min. Sonication 5 min. 4
Normalised total sum of peak heights (rfu) 277439 227614 36683 P value ‐ 0,27 0,01 Task 4.3: Appendix 2
DNA ANALYSIS
DNA extraction using TE-4 buffer and heating
Chelex extraction is a quick protocol with good yield, as no DNA transfers are included. The Chelex beads serve to chelate positive ions that would otherwise catalyse DNA degradation at the elevated temperatures needed for lysis (7). However, Chelex has a pH of around 11, which is not optimal for polymerase activity or for DNA storage. We evaluated a protocol where a TE‑4 buffer (pH 8.0, 10 mM Tris, 0.1 mM EDTA) was applied instead of the Chelex beads. This gave a considerable improvement when amplifying impure crime scene samples (moist snuff tobacco). The TE‑4 extracted samples allowed for amplification in the presence of a three times higher amount of inhibitor compared to Chelex. Evaluation of DNA loss when pelleting cells
Many DNA extraction protocols involve pelleting of cells by centrifuging prior to lysis. Pelleting is a powerful way of concentrating the sample, and also serves to rid the sample of water soluble PCR inhibitors. However, by removing the supernatant, DNA in solution will be lost. This has previously not been investigated. We evaluated the DNA loss from pelleting by extracting dried saliva stains (24 h) and analysing both DNA from the pellet and from the supernatant. By quantifying the two sample fractions, we found that the pellet fraction contained on average over 30 times more DNA than the supernatant. For these fairly newly dried stains, pelleting did not lead to any significant DNA loss. However, for older stains with a higher level of extracellular DNA, pelleting could lead to significant DNA losses. DNA purification using centrifugal filter devices
Purification of DNA extracts inevitably leads to DNA loss (9). This is often overlooked, and several common purification methods have not been systematically evaluated with respect to DNA recovery. We have investigated the performance of two commercially available filter devices with respect to their DNA recovery rates and their performance in purification (10). Applying Microsep 30K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30K retained 62 to 70% of the DNA. The increased purity following filter purification counteracted some of this DNA loss, leading to improved amplification for blood on denim (Amicon Ultra 30K and Microsep 30K) and saliva on envelope (Amicon Ultra 30K). In total, Amicon Ultra 30K performed better than Microsep 30K, through higher DNA recovery and better ability to purify impure extracts. To maximize the chance of obtaining positive DNA results, the level of purification employed for a certain sample type should be correlated to the inhibitor‑tolerance of the applied PCR amplification system. Excessive purification not only leads to DNA loss, it also costs money and lowers analytical throughput. 5
DNA ANALYSIS
Development of PCR-inhibitory reference materials
Generally, inhibitor‑tolerance is investigated by adding one specific compound in purified form to the PCR reactions, e.g. humic acid or haematin. However, real biological samples are much more complex, containing several compounds in an unknown mixture. We came up with the concept of making reference materials for common PCR‑inhibitory sample matrices, mimicking the background of true samples. We have developed reference materials for common forensic sample matrices such as cigarette butts, chewing gum and moist snuff (11). The purpose of the reference materials is to provide a standardized but still real‑life like background for PCR‑based DNA analysis, for investigation of PCR‑inhibitory effects and quality control of new reagents and kits. Evaluation of PCR facilitators to counteract inhibition PCR facilitators are compounds that can be added to the PCR to improve amplification. We made a thorough literature study of PCR facilitators that can counteract inhibition, for a complete review see reference (1). We then evaluated various PCR‑facilitating substances in a qPCR set‑up. Among gp32 protein, L‑carnitine, trehalose and others, trehalose gave the most promising results, rescuing amplification in impure crime scene DNA samples that otherwise completely blocked the reactions (12). Blending facilitators has been shown to improve amplification of impure extracts (13, 14). However, careful optimisation of the ingoing substances is needed. We evaluated a number of blends, and all of them gave negative effects on amplification even for pure samples. Using one or two facilitators, e.g. trehalose and BSA, is therefore advised. Elevating the pH and using tricine instead of the normal tris buffer has showed some positive effects (15) but must be investigated further. Conclusion Improved awareness among Laboratory Response Networks (LRN:s) regarding pre‑PCR processing is important, as ineffective sample processing may lead to false‑negative or ambiguous results, hindering the decision making process in a bioterrorism crisis. Here we have shown that there is a lot to gain in terms of analytical throughput and limit of detection in PCR based analysis by optimizing sampling, sample treatment and PCR chemistry. Implementing these new methods in LRN:s is the next step, following the present project. Several scientific papers based on the findings of this project are in preparation. Publications
Hedman J, Knutsson R, Ansell R, Rådström P, Rasmusson B. Pre‑PCR Processing in Bioterrorism Preparedness ‑ Improved Diagnostic Capabilities for Laboratory Response Networks. Biosecur Bioterror 2013; In press. 6
DNA ANALYSIS
Hedman J, Lövenklev M, Wolffs P, Löfström C, Knutsson R, Rådström P. Pre‑
PCR processing strategies. In: Nolan T, editor. PCR Technology, Current innovations. 3rd ed: CRC Press, Boca Raton, USA; 2013. Hedman J. DNA analysis of PCR‑inhibitory forensic samples: Doctoral thesis, Lund University; 2011. ISBN: 978‑91‑7422‑283‑8. Norén L, Hedell R, Ansell R, Hedman J. Purification of crime scene DNA extracts using centrifugal filter devices. Investig Genet 2013;4:8. Supervision of students
The following BSc/MSc students were supervised within the diagnostic PCR project: Arun Kumar Krishnamoorthy: Composing an inhibitor tolerant PCR buffer. Project report 15 ECTS, Lund University, 2011. Arun Kumar Krishnamoorthy: Pre PCR processing of forensic samples. Masterʹs thesis 30 ECTS, Lund University, 2012. Harwati Lindsten: Applying Real‑Time PCR in Forensic DNA Analysis. Candidate thesis 15 ECTS, Lund University, 2013. Vanessa Las Heras: Advanced qPCR optimization. Project report 15 ECTS, Lund University, 2013. References
1. Hedman J, Knutsson R, Ansell R, Rådström P, Rasmusson B. Pre‑
PCR Processing in Bioterrorism Preparedness ‑ Improved Diagnostic Capabilities for Laboratory Response Networks. Biosecur Bioterror 2013;In press. 2. Hedman J, Lövenklev M, Wolffs P, Löfström C, Knutsson R, Rådström P. Pre‑PCR processing strategies. In: Nolan T, editor. PCR Technology, Current innovations. 3rd ed: CRC Press, Boca Raton, USA; 2013. 3. Rådström P, Knutsson R, Wolffs P, Lövenklev M, Löfström C. Pre‑PCR processing: strategies to generate PCR‑compatible samples. Mol Biotechnol 2004;26(2):133‑146. 4. Hedman J, Rådström P. Overcoming inhibition in real‑time diagnostic PCR. Methods Mol Biol 2013;943:17‑48. 5. Hedman J, Nordgaard A, Rasmusson B, Ansell R, Rådström P. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles. Biotechniques 2009;47(5):951‑958. 6. Hedman J, Nordgaard A, Dufva C, Rasmusson B, Ansell R, Rådström P. Synergy between DNA polymerases increases polymerase chain 7
DNA ANALYSIS
reaction inhibitor tolerance in forensic DNA analysis. Anal Biochem 2010;405:192‑200. 7. Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR‑based typing from forensic material. Biotechniques 1991;10(4):506‑513. 8. Kumar Krishnamoorthy A. Pre PCR processing of forensic samples. Masterʹs thesis, Lund University 2012. 9. Miller DN, Bryant JE, Madsen EL, Ghiorse WC. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl Environ Microbiol 1999;65(11):4715‑4724. 10. Norén L, Hedell R, Ansell R, Hedman J. Purification of crime scene DNA extracts using centrifugal filter devices. Investig Genet 2013;4:8. 11. Lindsten H. Applying Real Time PCR in Forensic DNA Analysis. Candidate thesis, Lund University 2012. 12. Hedman J. DNA analysis of PCR‑inhibitory forensic samples: Doctoral thesis, Lund University; 2011. 13. Zhang Z, Kermekchiev MB, Barnes WM. Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq. J Mol Diagn 2010;12(2):152‑161. 14. Horakova H, Polakovicova I, Shaik GM, Eitler J, Bugajev V, Draberova L, Draber P. 1,2‑propanediol‑trehalose mixture as a potent quantitative real‑time PCR enhancer. BMC Biotechnol 2011;11:41. 15. Kumar Krishnamoorthy A. Composing an inhibitor tolerant PCR buffer. Project report, Lund University 2011. 8
Task 4.3. Appendix 3. Development of Veterinary Forensic Pathology
from Crime Scene to Court (4.3.3)
Therese Ottinger, SVA Every part of the forensic necropsy procedure is open to question by the court. Consequently, forensic postmortem examinations must be conducted in a manner that will permit the findings and results to be used in a court of law. The aim of 4.3.3 was to develop the statement of witness in veterinary forensic pathology, to introduce evidence evaluation using the Bayesian approach (1, 2) and to investigate and improve communication with the legal system. During the first months extensive literature studies of veterinary forensic science, human forensic pathology, forensic science, evidence evaluation and veterinary pathology were undertaken. During month 6‑8 a research plan for PhD studies was developed and accepted by the Swedish University of Agricultural Sciences. Three studies were planned and scientific publications are in progress. A survey of the current status of Veterinary Forensic Pathology in Europe has been conducted. A questionnaire was sent out to 134 pathology laboratories in Europe and a few other countries. The response rate was 54% and the results are currently being processed for a planned paper. The aim of the study was to review the status of veterinary forensic pathology in Europe and to initiate and establish a network between laboratories in different countries. A second, recently started study, will investigate how the current veterinary forensic necropsy reports are understood and used in the Swedish courts and the transfer of knowledge and information between different disciplines, i.e. from veterinary science to law. To achieve this we have initiated a collaboration with researchers at the Department of Thematic Studies ‑ Technology and Social Change at Linköping University, Sweden and we have performed systematic interviews with veterinary pathologists and selected representatives of the Swedish legal system. The results will be analyzed and reported in a scientific publication. Task 4.3: Appendix 3
DNA ANALYSIS
Methods of evidence evaluation based on the logical approach have been tested on pathology findings. New templates for statement of witness are being developed and will be tested on the customers, i.e. police, investigators, prosecutors etc. A workshop in evaluation of evidence in combined pathology and toxicology cases was carried out (AniBio Threat task 1.1 appendix 10). Since forensic veterinary pathology is a small and fairly new discipline there is a lack of reference databases which poses a problem. The process is being documented for a planned publication. A large part of the project concerns networking and interoperability with law enforcement agencies and officials. A close collaboration with the animal welfare group of the Stockholm county police has been established. The aim is to enhance and speed up the logistics and “paperwork” of forensic veterinary cases. We believe better communication between the pathologists and the investigating police will improve the legal documentation by the police and veterinarians both and legal process as a whole. In autumn 2012 we participated in the education of the animal welfare police, prosecutors and the Stockholm county administrative board veterinarians. The aim was is to enhance knowledge of various animal‑ or veterinary‑ specific issues in an investigation of crime involving animals. In April 2013 representatives of the Prosecutors office educated veterinary pathologists on the role of the pathologist as an expert witness and a participants in a criminal investigation. Knowledge of veterinary forensic science is essential in order to recognize signs of abuse in animals and the subject has been recognized as a powerful tool in the work to recognize and prevent domestic violence. Research shows a strong link between animal abuse and intrapersonal violence. Our project on improving the forensic investigation on crime against animals has caused both societal and media interest. The project is currently being presented at a nationwide series of seminars on domestic violence, arranged by the County administrative boards. 2
DNA ANALYSIS
The handling of forensic veterinary pathology cases at the Swedish National Veterinary Institute (SVA) has been improved during the course of the project based on knowledge obtained from in depth literature studies and national as well as international networking and bridging activities. The routines for handling the carcasses in order to keep the chain of custody, for documentation of identity and of the lesions, have all been updated. Use of pre‑necropsy radiology e.g. computed tomography and X‑ray has increased. The quality of the investigations as a whole has been improved as a result of the bridging activities with the law enforcement agencies. Positive factors include better background information, more precise questions, better handling of carcasses before and during transport and increased knowledge of what a veterinary pathologist can add to an investigation. References
[1] Aitken, C. G. G. and Taroni, F. (2004). Statistics and the Evaluation of Evidence for Forensic Scientists.2nd ed. Wiley, Chichester. [2] Buckleton, J. (2005). A framework for interpreting evidence. In: Forensic DNA Evidence Interpretation (J. Buckleton, C.M. Triggs, S.J. Walsh eds.), 27–63. CRC Press, Boca Raton, FL. [3] Nordgaard, A., Ansell R., Drotz, W., and Jaeger, L. (2012) Scale of conclusions for the value of evidence. Law, Probaibility and Risk 11(1):1‑24 // 3
Task 4.3. Appendix 4. Forensic entomology, palynology and taphonomy
(4.3.4)
Anders Lindström, SVA A main activity in forensic entomology has been education of crime scene investigators, Customs officers and forensic pathologists. Through this, an awareness of insects and pollen as evidence material has been created. Case work on homicides has also contributed to an increased awareness of insects and pollen as evidence as well as taphonomic changes of human remains in the establishment of a postmortem interval. Forensic entomology is a rapidly evolving discipline that utilizes the knowledge of sarcosaprophagous insect, mainly blowflies, growth rates to establish a minimum duration of the postmortem interval, i.e. the time since death. Pollen.
Forensic palynology is the discipline of pollen studies applied on forensic work. Although pollen can be used as trace evidence, it has in this project mainly been employed for establishing a postmortem interval. Since different plants flower at different times the flora of air borne pollen varies over the season. By matching samples of pollen from the nasal cavities and sinuses of a deceased person with pollen samples collected throughout the season a reasonable time frame for time of death can be established. Task 4.3: Appendix 4
DNA ANALYSIS
Taphonomy is a paleontology term that describes the biological, chemical and physical transitions of a fresh corpse or cadaver ultimately to a fossil. Forensic taphonomy is mainly concerned with the transition from fresh to skeletonized. Since this is a natural process the rate varies but the main driving forces are temperature and insect colonization. The decompositional changes to human remains follow a sequence and a rough estimate of time since death can therefore be calculated. We have also made efforts to combine different disciplines to gain new insights. A research paper and a book chapter have been produced on the combination of Bayesian statistics and forensic entomology and taphonomy. Workshops and meetings
•
•
•
•
Several workshops within the AniBioThreat network have contained lectures and/or posters on forensic entomology, forensic palynology or forensic taphonomy. Numerous lectures and workshops for crime scene investigators, Customs officers and forensic pathologists. Meetings of the AFFA group (Arbetsgruppen För Forensisk Arkeologi (Working group for forensic archaeology)) where a combined field and theory course for crime scene technicians is held. During the course archaeologists, osteologists, forensic pathologists and a forensic entomologist (AL) lecture on various aspects of excavating a grave and sampling evidence. The course ends with a field excercise where two buried pigs are exhumated. European Association for Forensic Entomology, Torún, Poland (2012). Posters
•
•
•
2
European Association for Forensic Entomology conference 2012: o First report of Hydrotaea similis Meade, 1887 (Diptera: Muscidae) breeding in a human cadaver. A. Grzywacz, A. Lindström, M. J. R. Hall o Bioterrorism and forensic entomology. A. Lindström o First finding of Calliphora subalpina and C. loewi (Diptera, Calliphoridae) breeding in a human cadaver. A. Lindström o An unususal use of forensic entomology in a dismemberment case. A. Lindström, A. Stenius, K. Nyberg ASM Biodefense and Emerging Diseases Research Meeting 2012: o Bioterrorism and forensic entomology. A. Lindström European Wildlife Disease Association, Vlieland, The Netherlands, September 13‑16, 2010: o Pedicle Fly‑Strike by the blow fly Lucilia illustris in Swedish Roe Deer (Capreolus capreolus). S‑O. Nielsen, A. Lindström Task 4.3: Appendix 4
DNA ANALYSIS
Publications and manuscripts
•
•
•
•
•
Lindström, A. and Andersson, G. 2013 . Experimental Design, Statistics, and Computer Modeling In: International Dimensions and Frontiers of Forensic Entomology. Forthcoming. Andersson, G., Lindström, A. and Sundström, A. Bayesian networks for evaluation of evidence from forensic entomology. Biosecurity and Bioterrorism. Disney, R.H.L., Garcia‑Rojo, A., Lindström, A. and Manlove, J.D. Further occurrences of Dohrniphora cornuta (bigot) (Diptera, Phoridae) in forensic cases. Submitted to: Revista Espanola de Medicina Legal. Fremdt H., Szpila K., Huijbregts H., Lindström A., Zehner R., Amendt J. 2012. Lucilia silvarum Meigen, 1826 (Diptera: Calliphoridae) – a new species of interest for forensic entomology in Europe. Forensic Science International. Gale, P., Stephenson1, B., Brouwer, A., Martinez, M., de la Torre, A., Bosch, J., Foley‑Fisher, M., Bonilauri, P., Lindström, A., Ulrich5, R.G., de Vos, C.J., Scremin, M., Liu, Z., Kelly, L. and Muñoz, M.J. Impact of climate change on risk of incursion of Crimean‑Congo haemorrhagic fever virus in livestock in Europe through migratory birds. J Appl Microbiol. 112(2):246‑57 Supervision
3 MSc students and one a crime scene investigator (BR, diploma work) were supervised within the project: • Cajsa‑Viktoria Lidman (2011): Estimating time of death from accumulated degree‑days by evaluating the decomposition state of human remains. • Beatrice Fält (2011): Risk of disease transmission from pathogen contaminated carcasses in water using B. cereus as a model of the Anthrax disease causing agent B. anthracis • Anna Stenius (2010): Molecular genetic determination of forensically relevant Calliphora and Cynomya species. • Birgitta Rickardsson (2011): Insamling av insekter vid brotts‑ och fyndplatser (in Swedish) // 3
Appendix 5: 4.3.5 Microbial Forensics and epidemiological investigations. Gunnar Andersson (SVA), Mats Forsman (FOI) Epidemiological investigations and Microbial forensics investigations have many similarities but differ in their scope. The former typically aims at finding the source and rout of transmission in order to prevent further transmission and to reduce the risk of further outbreaks. The latter is undertaken to determine whether a crime was undertaken, find the perpetrator and gather evidence for a criminal trial(1). In epidemiological investigations evidence is largely based on attribution type‐matching. Techniques based on antigenic profiles and resistance to bacteriophages are gradually being replaced by DNA based methods such as pulse field gel electrophoresis (PFGE) and multiple‐locus variable‐number tandem repeat analysis (MLVA). More recently traditional typing methods are largely being replaced by sequence‐based attribution methods including whole genome sequencing (2) which is becoming the state of the art in microbial forensics (1). Three interrelated stages have been identified in the microbial forensic investigation (Figure 1): (i)
(ii)
(iii)
Identification of the biological agent Characterization of the event as intentional/unintentional Attribution to a specific perpetrator Identification of agent(s)
Characterization of event
(Intentional Unintentional)
Attribution to perpetrator
Epidemiological
investigation
Microbial forensics
investigation
Treatment and prevention measures
‐Type of agent (Bacterium, virus etc)
‐ Genus/species
‐Contageous disease?
‐Microbial resistance?
‐Availability of vaccines?
In case of overt attack:
Public health preparedness
actions needed
‐Type of agent (Bacterium, virus etc)
‐ Genus/species
‐Contageous disease?
‐Microbial resistance?
‐Availability of vaccines?
Prevent further transmission
Tracing source of infection/contamination and determine transmission routes
‐ Comparison of outbreak strain to strains from suspected sources
Natural or deliberate origin
Overt: Caracterization of outbreak strain regarding
suspicious/unexpected features.
‐Signs og genetic manipulation
‐Unexpected antibiotoc resistance patterns
‐Unexpected virulence determinants.
‐Etc.
Not applicable
With exception for criminal
neglectance, violation of food
legislation, fraud etc.
Who is responsible?
Finding the perpetrator
‐Identifying of potential sources of weapon
strains and outbreak strains through
sequence comparison.
‐Comparison with strains linked to perpetrator.
Assessing the weight of evidence
Evaluating of what the observed similarities
and differences means in relation to the relevant questions and hypotheses.
Figure 1. Stages in epidemiological‐ and forensic microbiology investigations. Based on (1). Task 4.3: Appendix 5
For each stage the need the is an increasing demand of evidence in form of reference data regarding typing patterns or sequence data from strains of different origins in combination with an understanding of the pathogen’s population genetics. Many of the questions asked during the first two stages of a microbial forensics investigation are and an epidemiological investigation are similar (Figure 1) and the two investigations are may be conducted in parallel. The attribution step is unique to microbial forensics although also an investigation on non intentional events may reveal crimes such as fraud or violation of food legislation. At this stage, in addition to more “traditional” forensic analyses of recovered materials from the crime scene ( e.g. analysis of human DNA, fingerprints, and fibres)22, detailed analyses are conducted of the attack strain. In the case of a fully covert attack the event will be detected when signs of disease appear in exposed animals or humans and the identification of the pathogen responsible will be part of the animal or public health response before the initiation of the microbial forensic investigation (1). In contrast during a covert attack where the perpetrator announces the attack, or the authorities act on intelligence information, identification of the organism will also be a part of the forensic investigation (Figure 1). In this stage knowledge of the pathogen’s identity will be important for the scopes of the epidemiological investigation as well as the microbial forensics investigation. The characterization of the event will determine whether the outbreak is intentional or unintentional in origin. Also this is important for the scope of both kinds of investigations, for different reasons (Figure 1). Anomalies in various epidemiological characteristics, such as antibiotic resistance patterns, geographical occurrence of the disease, transmission routes, and general outbreak intensities and dynamics, have been proposed as potential indicators of deliberate release of a biological agent(1). In this context whole genome sequence analysis has the power to reveal or predict unexpected or previously unknown features of the organism, such as antibiotic resistance or unusual virulence determinants. Whole genome sequencing is already being adopted in modern molecular epidemiology investigations (2) including the German outbreak of Shiga toxin producing E. coli in 2011. The attribution stage aims to find the person(s) responsible for the attack and to collect legally valid evidence that can be used to convict the perpetrator(s) in a court of law. The forensic investigation will involve a detailed investigation of the weapon, i.e. the microbial organism as well as classical police methods including the collection of witness statements and intelligence information and traditional forensic evidence like fingerprints. In this stage characterization of outbreak strains and comparison with reference strains are performed in order to target the investigation on particular suspects as well as to collect evidence to be presented in the court trial (1). For both purposes it is essential to evaluate the weight of the microbial evidence in the light of the relevant hypotheses. This requires an in‐depth understanding of the population structure of the species (or lineage) as well as access to databases of representative high quality (sequence) data from relevant microbial populations. Task 4.3: Appendix 5
Misunderstandings relating to probabilities have contributed towards several miscarriages of justice and this has called for the development of guidelines for interpreting and communicating statistical evidence (3) including DNA fingerprints. There are major differences between human and bacterial reproduction mechanisms, resulting in very different population structures. The different sexual and asexual reproduction mechanisms will lead to differences in how genetic similarities and differences can be investigated. Thus in order to enable robust evaluation of the value of evidence for the results of genetic comparisons, in relation to the relevant scenarios and hypotheses there is a need for high‐quality databases (whole genome sequence) that represent the global population structures of important threat agents in combination with a further development of the statistical foundations of microbial forensics and evidence evaluation. References 1. Sjödin A, Broman T, Melefors Ö, Andersson G, Rasmusson B, Knutsson R, et al. The Need for High‐
Quality Whole‐Genome Sequence Databases in Microbial Forensics Biosecurity and Bioterrorism: Biodefense Strategy, Practice, and Science. 2013;In press. 2. Carriço JA, Sabat AJ, Friedrich AW, Ramirez M. on behalf of the ESCMID Study Group for Epidemiological Markers (ESGEM). Bioinformatics in bacterial molecular epidemiology and public health: databases, tools and the next‐generation sequencing revolution. Eurosurveillance. 2013;18(4). 3. Aitken C, P. Roberts, P., Jackson, G. Practitioner Guide No 1. Fundamentals of Probability and Statistical Evidence in Criminal Proceedings, . In: Law RSSsWGoSat, editor.: Royal Statistical Society; 2010. Title
“Bio-preparedness measures
concerning prevention, detection
and response to animal
bioterrorism threats”
Acronym
AniBioThreat
Total cost
€7.003.992,26
FINANCEs
With the financial support from
the Prevention of and Fight against Chrime
Programme of the European Union.
European Commission –
Directorate-General Home Affairs.
Grant Agreement Nr
HOME/2009/ISEC/AG/191
Start date of the project
1 October 2010
Duration
3 years
COORDINATOR
National Veterinary Institute
SVA, Sweden
www.anibiothreat.com
Bridging security,
safety and research
The aim of the project AniBioThreat
is to improve the EU’s capacity to counter
biological animal bioterrorism threats
in terms of awareness, prevention and contingency.
The project will contribute to create
a safer and more secure world.
To succeed, we need to carry on a borderless dialogue.
AniBioThreat builds bridges across boundaries
dividing countries, competencies, and disciplines.
In our work, we strive to be Collaborative,
Learning, Efficient, and Alert,
to be a Robust organization.
Keep it CLEAR!
With the financial support from the Prevention of and Fight against Crime Programme of the European Union
European Commission – Directorate – General Home Affairs

Deliverable 4.3

Transcription

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