Defining The Microbial Forensics Program

Most would agree that based on past history and with current technology capabilities, the potential of biological weapons being used is greater than at any other time in history. Only a few individuals are needed with expertise and access to dual-use equipment (e.g., equipment used in the pharmaceutical or food industries) to inexpensively produce a number of bioweapons. As reviewed earlier, microorganisms and their toxins make particularly danger ous weapons because bioweapons can be grown from a single organism or cell; thus creation of large quantities can be accomplished readily and at low cost, starting with minimal amounts of starting material. Thus, even the smallest amounts of pathogens can be used to perpetrate a major terrorist event or biocrime. Furthermore, it can be difficult if not impossible to determine whether very small amounts of such materials have been removed surreptitiously from a facility that is authorized to possess pathogens. Most importantly, a biological weapon can be easy to conceal and, depending on the target, easy to disseminate. We must accept that it is impossible to guarantee that the government can deter or prevent all bioterrorist or criminal acts. Therefore, a proactive approach must be taken. In addition to the physical security measures being enacted, science can be effective as a deterrent and for identifying perpetrators. One major scientific approach to improve investigative capabilities for attribution and deter the use of pathogenic agents in an illicit manner is the development of a strong, scientifically rigorous microbial forensics program.

Microbial forensics can be defined as a scientific discipline dedicated to analyzing evidence from a bioterrorism act, biocrime, or inadvertent microorganism/toxin release for attribution purposes. One may consider attribution solely to be the "fingerprinting'' of a pathogenic agent, but the unique identification of a microorganism may never be possible, because of the clonal nature of many microorganisms and, on a case-by-case basis, lack of population and phylogenetic data. More importantly, the ultimate goal of attribution is identification of the persons who committed the bioterrorist act or biocrime, intentionally or inadvertently. In addition to microbiological analytical tools, traditional forensic analyses, such as human DNA analysis, dermatoglyphic patterns, analytical chemistry, tool marks, and other techniques will be used to analyze a bioterrorist event or biocrime evidence.

There is nothing exceptionally unique about forensic microbiological evidence compared with other forensic evidence. Recognizing a crime scene, preserving a crime scene, chain of custody practices, evidence collection and handling, evidence shipping, analysis of evidence, and interpretation of results are carried out in the same general manner as for any forensic evidence, except that the evidence will be handled as a biohazard [even more so than with human immunodeficiency virus (HlV)-infected blood]. It is anticipated that the majority of microbial forensic evidence will fall into the class-characteristic category, with some of the data being very informative and some being rather limited. For example, the bacterium Bacillus anthracis demonstrates low diversity among the various strains of the species. If analyzed in the context of a biocrime, one may only be able to identify the strain, while substrain resolution may be difficult. If such data can be derived, for example, by development of a specified biomarker assay, then further resolution may be possible. In con trast, HIV is a rapidly evolving virus. It is likely that two samples with a recent common origin could differ at a few nucleotide bases within the genome. Not having an identical nucleic acid sequence between an isolate and a reference sample in itself is not considered exculpatory evidence. Phylogenetic trees/ networks are used to demonstrate relationships of those isolates that have a close or similar ancestry compared with those isolates that have less related histories (see the Forensic Case Examples section below).

Since an attack may be easy to carry out and difficult to prevent, law enforcement needs to enhance its capabilities to confront this new challenge. Law enforcement already has the infrastructure (i.e., its traditional role) for attribution and deterrence. Measures need to be developed and implemented to effectively carry out law enforcement's responsibility in the field of micro-bial forensics. The major components of a successful microbial forensics program may be listed as:

1. Detection and Identification are keys to thwart bioterrorism. To effectively carry out attribution, robust analytical techniques need to be developed and implemented. Assay development to enhance sensitivity and specificity and expand detection capabilities must be promoted. Analytical solutions may have to be developed quickly and effectively. These include DNA-based systems, as well as analytical chemistry and physical analyses (i.e., nonbiological evidence characterization), culture, immunoassays, and the use of bioassays in animals and tissue culture.

2. Information and Databases will play an important role in the microbial forensics endeavor. The quality and accessibility of rapidly expanding and evolving databases, such as those that contain bioagent genomic sequence data, need to improve. To achieve this, national databases on pathogen genetics and other biological data (and to include nonbiological evidence) need to be created.

There needs to be a relational database on those who have access to these pathogens so that threats can be deterred or traced back effectively to possible sources. Security measures are already being enhanced to restrict and control access to select pathogens and toxins.29 While such a database may deter some individuals from participating in microbiology research, the infrastructure will more likely protect the legitimate user so that exchange of scientific information can proceed for the betterment of society (such as developing therapeutics and better diagnostic assays). Another database needed is one that is encyclopedic in nature. There are many sources that contain scientific information, including but not limited to publications, presentations, websites, and genomic databases. It is difficult to access all these sites in an effective and rapid manner. Being able to place at one's fingertips all microbiology data and data on associated nonmicrobial forensic materials will greatly enhance the investigative capabilities of the microbial forensic scientist.

3. A strain repository to house pathogens and other appropriate near-neighbor microorganisms must be developed. The "near-neighbor" concept is intimately related with the methods used for detection and identification. Some methods of identification that are robust may lead to a broad class of near neighbors, while sophisticated methods may define near neighbors that are narrower. Well-characterized samples must be available to enable good-quality assay development. Assays cannot be adequately validated without proper samples and reference material. Bioinformatic interpretation of analytical results from evidence samples may be more limited without properly defined samples and controls. In addition, better control of access and dissemination of select agents for research and development can be executed.

4. New analytical methods and some existing methods need to be properly validated. Such validation tasks are not limited to laboratory procedures; they equally apply to tools to be used for data interpretation and statistical assessment issues. Moreover, some biological crimes may require analysis by methodologies that may not have undergone the rigorous review process of that of standard operating protocols. A preliminary review process for such assays must be implemented (see Quality Assurance discussion below).

5. Quality assurance (QA) guidelines for microbial forensic laboratories must be established (some are already enacted due to public health regulations). One must employ high-quality practices to ensure that reliable results are obtained and to maintain public confidence.

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