Microbial Forensics
by Tanuj MalkaniDeliberately spreading disease among the enemy has been occasionally practiced over hundreds of years. But modern bioterrorism is more chilling than ever because of rapidly expanding knowledge about infectious diseases and biotoxins and their potential to wreak havoc in complex, interdependent societies.
If bioterrorists are ever brought to trial, the evidence against them will depend on the painstaking work of a detective in a lab coat.
Microbial Forensics is the emerging discipline in which researchers work to
track down the source of a pathogen, either in a criminal investigation of bioterrorism
attack or a study of naturally occurring disease outbreak, looking for molecular
microbial signatures. These signatures are detected by measuring the polymorphisms,
or variations, between microbial strains and are used to infer the origin, relationship,
or transmission route of a particular isolate.
These investigations require and use traditional investigative methodology,
established molecular techniques and new, advanced methods that still may be
under development. The new discipline of Microbial Forensics is thus a conglomeration
of an array of well established fields, such as microbial genomics, phylogenetics,
forensic informatics and classical microbiology. But unlike public health investigations,
microbial forensic investigation goes further to associate the source of the
causative agent with a specific individual or group. Microbial forensics will
be most effective if there is sufficient basic scientific information concerning
microbial genetics, evolution, physiology and ecology.
Microbial forensic analysis must encompass sample handling, collection, preservation, method selection, casework analysis, interpretation of results, validation, and quality assurance.
Since investigators must consider eventual prosecution and presentation of evidence in the courts, biocrime criminal investigations require careful controls and standards for validation and evaluation of technologies and the data they produce.
Recent Cases
Recently, microbial forensics has been used in cases such as the alleged transmission
of HIV from a Florida dentist to several patients. By sequencing viral fragments
from the dentist and infected patients, the prosecutors were able show that
he most likely infected at least six of his patients with HIV.
And in 1993, the Japanese Aum Shinrikyo cult – later responsible for releasing Sarin gas on a Tokyo subway – tried unsuccessfully to release a variety of pathogens, including the anthrax bacterium, Bacillus anthracis. Almost ten years later, Paul Keim, a genetics professor at Northern Arizona University, known for his work on the typing of bacterial strains, including those that cause anthrax and plague, got live cultures of the bacteria used in the Tokyo attack. Using DNA profiles, Keim's lab was able to identify the attack strain as one used in animal vaccinations, suggesting why the release had not resulted in health problems in the population in the release area.
In addition, Keim and colleagues at the Institute of Genome Research completed
the genome sequence for the Bacillus anthracis isolate taken from Ray Stevens,
the first anthrax letter victim. This was the first time researchers had used
whole genome sequencing for the forensic identification of a particular pathogen
strain.
But the recent anthrax attacks on the U.S. mail system raised a number of questions
like if an individual were charged with the attacks, would the court be willing
to convict him or her, using as evidence a variation in a genome sequence?
But will it be worth the effort?
Since investigators must consider potential prosecution and presentation of
evidence in court, biocrime investigations demand careful controls and standards
for validation and evaluation of technologies and the data they produce. Scientists
can easily evaluate new methods of detecting organisms implicated in a bioterrorism
attack, but taking the resulting evidence into a court is another matter. Any
microbial evidence, such as anthrax spores, that links to a suspect has to meet
stern standards.
Scientists at the annual meeting of the American Association for the Advancement of Science (AAAS) warn that such evidence will not be admissible unless researchers develop new molecular methods, such as genome sequencing, and adopt standardized methods and data.
But the problem is that we¹re not talking about a jury of our scientific peers, we¹re talking about lawyers, judges, juries. The consequences are not just having a paper rejected by a journal, but rather of sending someone to jail.
Also even if the anthrax perpetrator was caught, it might not be as easy to achieve a conviction, especially if the spores are part of the physical evidence. If PCR based tests on the spores found in the suspect’s possession were the same DNA signatures as the spores found in the letters, that’s fine for scientists. But if you take that into court, all sorts of questions arise: What does ‘same’ mean? Does it have to be 100 percent identical? Even if it’s 100 percent identical, does it really prove that it’s the same?
There are two main problems. First, although the technology for doing DNA based and other molecular analyses is widely used and universally accepted in the research community, the kind of rigorous validation and development of appropriate quality control standards for the use of this technology in forensics is still not well developed. Research is under way, but it might still be fairly easy for a defense lawyer to raise questions about accuracy and interpretation.
This is not necessarily due to weaknesses in the technology but rather to the fact that scientists had not thought about forensic uses and thus had not developed the validation and quality control guidelines appropriate for legal application. This should not be a difficult goal to reach, however, because rigorous validation and quality control guidelines have been developed for the use of biological technologies in hospital laboratories.
The second problem, which is currently being investigated, is the mutation
rate. If we want to show that the strain of bacterium that produced a spore
found in the suspect’s home or office is the strain used in the anthrax
attack and not a strain that was originally in the soil and was tracked into
the location, there is no problem. Different strains of Bacillus anthracis differ
enough at the DNA sequence level that even a partial genome sequence of the
two strains would be sufficient to make the distinction because there would
be a number of differences, what you might call a DNA sequence fingerprint.
If one is trying to locate the laboratory where the anthrax attack strain came
from, however, such distinctions would be more difficult since most laboratories
shared derivatives of the same strain.
Here, natural mutations over the past several decades that occurred in the strain due to numerous passages in the laboratory might be sufficiently abundant to allow the strain obtained from one laboratory to be distinguished from that obtained from another, but since the differences will probably be few in number, they will be less convincing than for strains isolated in different geographical locations.
How much difference is there? This is currently being investigated. Scientists believe that B. anthracis mutates very slowly, so it may be difficult to have a differentiation that holds up in court.
There are errors that occur during the DNA sequencing process (about 0.1% at present), but this problem can be solved by resequencing an area more than once. This issue has led scientists to consider other molecules besides DNA, such as those found in traces of the growth media used to cultivate the strain (if those could be detected), which might differ from one laboratory to another.
As part of the effort to deter biological terrorism and strengthen the law enforcement response to further such acts, the United States recently established a microbial forensic laboratory known as the National Bioforensics Analysis Center (NBFAC), which is part of the Department of Homeland Security and operates in partnership with the Federal Bureau of Investigation (FBI). The NBFAC provides a central facility to conduct analysis of evidentiary material. Although the NBFAC's infrastructure and capabilities draw on the best scientific resources available in the United States and on some resources internationally, the practitioners of the nascent field of microbial forensics recognize that there remain significant gaps in both science and operations that must be filled to establish a more readily responsive and effective system.
Clearly, biological weapons are assigned high priority in a country’s security, defense, counter proliferation, nonproliferation, intelligence and counterterrorist programmes, resources and policies. In order to strengthen its active defense against intentions, development, and use of these weapons, it is now time for the world community to establish a comprehensive forensic capability to effectively attribute biological weapons for investigative, intelligence, prosecutive, diplomatic, and policy purposes. Nevertheless, aggressive research programme are needed for forensic trace, micro chemical analysis, trace evidence analysis coupled with microbial forensics so that the discipline of microbiology and forensic science will grow and bear fruit for national security.
April 2006, Jeev
http://www.dce.edu/jeev
This work is licensed under a Creative Commons Attribution 2.5 License.