Microbial Forensic Tools

Epidemiology and forensics have similar roles when investigating biocrimes. However, after the determination of the cause, doctors treat the patient. Forensic scientists proceed further with analyses. There are additional stringencies placed on the forensic investigation, which include maintenance of chain of custody of the evidence and more detailed analyses of the pathogen that was used as a weapon. If possible, the analyses may determine the strain or sub-strain of the organism and other features that may be unimportant to the public health sector.

While protocols exist for evidence collection and preservation, challenging samples will be encountered. Samples that include soil, mud, swamp water, large items that may not fit into biosafety containment hoods, and other types of materials must be considered. The BFAC and its partners must consider various scenarios and develop processes to handle a wide variety of samples and sample types, at times under the constraints of biocontainment.

Clearly DNA typing methodologies will figure prominently in the attribution of a pathogenic weapon. First, the DNA must be successfully extracted from a sample. Extraction procedures that are used for pristine samples may not be adequate for more challenging environmental samples. Efficiency of recovery may be low, and polymerase chain reaction (PCR) inhibitors may not be removed effectively. The BFAC and its partners will have to promote the development of better DNA extraction methods. Once DNA has been recovered, molecular biology tools and biomarkers exist to assist analyses; these include sequencing, microarray analyses, pathogenicity array analyses, single-nucleotide polymorphism (SNP) characterizations, 16S rRNA sequencing, variable number tandem repeat analysis, and antibiotic resistance gene characterizations. This list of analyses is impressive. However, more research is needed regarding the selection of the genomic region of pathogen agents to determine the severity of pathogenicity. Further, still more assays need to be developed for better resolution of strains and substrains of pathogens. Detecting bioengineered organisms is also extremely important. Inserting a toxin gene into a normally nonpathogenic organism is feasible. Not only can previously harmless organisms be endowed with virulence by genetic engineering, but certain virus vectors can also serve as carriers of harmful genes that could be inserted into a host's own cellular DNA. Analysis of the genome of an organism may be able to differentiate a bioattack from a natural outbreak. Toxins may be characterized by immunoassays, biofunctional assays, peptide or protein-based assays, or by mass spectrometry. In addition, trace levels of the DNA from the organism that produced the toxin may be present, and DNA-based assays could aid in attributing the source of the toxin.

As stated above, forensic investigations use all types of physical evidence to attempt to obtain information on the organism, the persons involved, the places involved, the processes used to develop or disperse the weapon, the instrumentation used, and/or the time of the criminal act. Other materials and methodologies may assist in an investigation of a bioterrorism event, and at times may be more informative than DNA-based tests. Traditional physical evidence collected at a crime scene will also be evaluated. In addition, a recent report by the American Academy of Microbiology34 described a number of non-DNA-based approaches to consider for the microbial forensic panoply of assays. These include:

1. The physical attributes, such as morphology and microstructure, acquired by the microorganism(s) during preparation for weaponization may be distinctive and provide information on attribution;

2. Isotope analyses can be used to approximate the age and source of the microorganisms;

3. Traditional physiologic methods (e.g., fatty acid composition, phage typing, serotyping) may provide information to further identify the microorganisms;

4. Remnants of growth media and media components adhering to the microorganisms may provide clues to the source of the weapons or methods used to prepare the material;

5. Stabilizers and additives in the preparation of a sample may be signatures that can provide leads about the perpetrator;

6. Incidental biocontaminants, such as environmental pollen and fungi, may give clues to the location and time of year the sample was prepared;

7. Bacterial endemism is the existence of unique strains of bacteria that may exist in only one location or rarely in other locations. Identification of such incidental bacteria coexisting in a weaponized sample may be used to geolocate the sample source or place of preparation;

8. Monitoring changes in the immunological response of a host to a pathogen, such as temporal IgG and IgM responses to epitopes of the microorganism, may indicate whether or not an individual has had long-term or recent exposure to the microorganism or toxin;

9. Immunoassays are often used as rapid detection methods and are especially applicable to field-deployable assays. Antibodies may also be specific to particular protein signatures that could further resolve the identity of a material.

Analysis of all pertinent physical evidence collected at a crime scene will assist in attribution. It is of tantamount importance to consider other tools besides solely DNA-based assays. Since microorganisms often propagate clon-ally, it may be difficult to differentiate some samples based on genetics, especially slowly evolving species. These non-DNA-based assays and others are part of the suite of potential approaches that a microbial forensic scientist may be able to use for attribution today and in the future.

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