Microbial Forensics: Forensic Investigation and Bioterrorism
Introduction
Microbial pathogens bear adaptive mechanisms of growth and survival in order to naturally cause disease. Malicious individuals may manipulate this feature in the microbe’s genome to deliberately cause disease in an individual or group. Microbial forensics refers to the rapidly evolving process of investigating the association of a causative agent through microbial analysis with the particular individual or group affected. Analysts measure molecular variations between related microbial strains, using this data to infer origin, relationship or transmission route of that strain. Through multiple molecular analytic techniques, such as molecular phylogeny, whole genome sequencing, microarray analysis, and DNA fingerprinting, the infectious agent can be rapidly identified. These procedures are carried out in the context of a criminal investigation to isolate a type of organism and evidence of genetic engineering or evolution and attribute it to a causative agent. The innovative discipline of microbial forensics provides unique causative evidence, however due to the variability of microbial subjects; validation of investigatory protocols is critical to the attribution of this evidence. This essay attempts to outline the complexity and evidentiary value of the discipline through exploring the implications of microbial forensics in Australia and its limitations.
Brief history
Practical microbial forensic investigation began when the Federal Bureau of Investigations’ Hazardous Materials Response Unit formed in the 1990s. It was established to support bioterrorism investigations such as the 2001 anthrax attacks through providing specific attributive evidence acceptable in the U.S. legal court. Until recently in Australia, microbiology was rarely applied in forensic application. Since the anthrax attacks, the Australian Federal Police (AFP) has worked to find interdisciplinary research solutions to bioterrorism between microbiology and forensic investigation. The AFP had virtually no microbiology experience and the microbiology community had little forensic investigation experience. Consequently, partnerships have been promoted between the AFP and specialised microbial laboratories. The Australian Chemical, Biological, Radiological and Nuclear Data Centre was established in order to record “suspicious substance incidents” and enhance Australia’s capabilities in bioterrorism mitigation.
Recent developments
The AFP now practice microbial forensic investigations in processing exhibits and collecting evidence in collaboration with other scientific organisations to thoroughly process microbial samples to withstand strict legal scrutiny. There are now protocols established that guide packaging, decontamination, chain of custody, transport, security and storage of microbial samples. The AFP focus on the capability to analyse samples at the scene to provide early identification and further target investigation. The deployment of portable detection devices and AFP mobile laboratories in 2006 allow rapid on-site screening to rural regions suspected of an incident. In 2007, the national regulatory scheme for biological agents of security concern legislation was produced. This reviewed a list of biological agents divided into risk groups and proposed a register of entities that handle the biological agents of risk, enhancing Australia’s management of ‘Biocrime’ and reducing the risk of incidence. In history, Australia has received bioterrorism threats, including the 1984 foot and mouth disease hoax in Queensland. While there is no public record of bioterrorism incidents in Australia, the AFP target threats and hoaxes with the same focus.
Sample collection
The collection and preservation of microbial samples from a crime scene is a critical element of successful attribution following the investigation of a biological event. An excerpt from an article by Paul Kirk in 1953 outlines the importance of these methodologies; “Wherever he steps, whatever he touches, whatever he leaves, even unconsciously, will serve as a silent witness against him…. It is factual evidence…Only human failure to find it, study it, and understand it can diminish its value”
Across the multidisciplinary approach to microbial forensics, standard operating procedures in sample collection, protection, preservation and analysis of microbial samples are produced to minimise the “human failure”. A sampling plan should be developed from the assessment of the scene that outlines the purpose, potential hazards and required protection, compliance with all regulations, necessary skill and qualification of personnel, equipment required and considerations for the mitigation of contamination. The strategy for sample collection can be “targeted” in which the area sampled is thought to be the site of contamination or “probability” sampling, where random locations are sampled. In all methodologies, a forensic investigator must action sampling techniques in a manner that avoids degradation and contamination. Microbial evidence may present as clinical such as “Swabs, Faeces, Urine and Hair” or environmental as “Air and liquid filtrates, water and soil”.
Evidence is collected through the whole item, a portion of an item and swabbing a surface. An array of surface sampling methods, such as “swabs, wipes, vacuums, filters and aspirating needles,” are used to collect and detect microorganisms in order to locate contamination sources. However, due to the variability of the sample types, one method used to collect one biological sample may be ineffectual for another. In fact, there is little advancement in research on which methods and devices yield better “physical, biological and chemical” signatures. This is the reason microbial forensic investigators “collect various samples” with different methods and “preservation techniques” for use in an array of “analytical schemes”. The medium to which a pathogen is collected varies greatly with the technique used to stabilise the signature in question for analysis. For example, transport media for virus microbes contain compounds that inhibit further growth.
Analytical techniques
The variability of sample type within this discipline requires a large range of techniques for analysis. Techniques for forensic microbiology in Australia are similar to practices in public health and agricultural investigations. Samples are usually taken from the scene, amplified using polymerase chain reaction procedures and analysed with techniques such as microscopy, colorimetric assay and immunoassay. There is a current move towards using gas chromatography mass spectrometry to identify biomarkers at the scene of crime, however this requires further validation. Techniques such as Fourier transformed infrared spectrometry is applied in identifying non- hazardous samples. Typing assays to determine biological agent lineage and origin are accurate however require specialised knowledge and extensive validation for use in microbial forensics. The complexity of the circumstances in which microbes require sampling at a biological event emphasize the requirement for further research into appropriate sampling, transport and analysis strategies to accommodate the forensic scenario.
Principles of quality assurance
The novel nature of this field poses limitations in its use as a forensic technique as its practices currently require extensive quality assurance in the form of validation. Developing these protocols for analysis of new biological agents along with quality control measures, biohazard precaution and also validation under strict legal policies is a current endeavour. The Scientific Working Group on Microbial Genetics and Forensics establishes these guidelines for quality assurance. As this laboratory data is relied on heavily for prosecution, the quality management of the investigation requires extensive protocols however this may not always be available due to the novel nature of the field.
Given the complexity of a bioterrorism event, the current production of universal standard operating protocols that function as a template for all sample collection and analysis scenarios are unreasonable and could compromise the collection of critical evidence. If a case situation matches a template for sample collection, then the investigators actions could be better targeted, yet in most cases the combination of investigative techniques required for the variability of complex microbiological scenarios are mostly innovative.
Advantages, disadvantages, limitations
As threats of the malicious release of biological agents increase and Australia’s continued reliance on agriculture, unique challenges are posed to the forensics community in their investigation. The advancements in capabilities to sequence the whole genome of microbial strains and identify variations in their sequences has provided unique evidence in the application of bioterrorism. This is an unparalleled advantage in the investigation of emerging biological weapons using other forensic disciplines. The limitation of this discipline is the need of a rapid analytical technique to minimise time spent investigating the scenario, as this is not always possible during the identification of complex biological agents at the scene. Due to novel nature of the field and the lack of understanding about microbial forensics in traditional investigation, a major challenge for the ongoing investigation of outbreaks is that it becomes secondary to the public health response causing evidence that could be used to prosecute to be lost. In reality, the major disadvantage of microbial forensics is the lack of forensic experience and protocols in investigating biological agents, consequently, it has limited practical use in Australian forensic investigation.
Conclusion
Australia’s potential to process bioterrorism events through microbial investigation is hugely related to recent ground breaking advancements in molecular genetic advancements. However, our capability to process such forensics scenarios are inhibited by the novel nature of the field and the current endevour to validate a large spectrum of collection and analytical processes to adhere to strict quality requirements. Microbial forensics applies the analysis of biological events such as specific variations in an agent’s DNA, that infers attributable evidence unseen in traditional forensic practices. Currently in Australia, the processing of this evidence is reliant on collaboration with public health industries to form practical application in a forensic scenario.
