What is microbial bio-terrorism
MICROBIAL FORENSICS: A POWERFUL DISCIPLINE FOR UNUSUAL BIOLOGICAL EVENTS
The main goals of medical B education are then the identification of the triggering agent, the determination of the source of the infection and the differentiation between a natural and an artificial occurrence. In contrast to investigating natural outbreaks, in the event of unusual events, forensic aspects must be considered and implemented from the outset. The "microbial forensics" that will then be used is a new scientific discipline. With the help of genetic typing techniques, a “genetic fingerprint” of the special outbreak strain is created and compared with reference profiles stored in databases. In the ideal case, this enables the examiner to identify the strain and to make statements about its origin.
The overview article underlines the importance of microbial forensics in the elucidation of unusual biological events and describes the skills already built up in the field of medical B protection as well as the challenges for further development.
For this purpose, a large number of molecular typing methods have been established at the Bundeswehr Institute for Microbiology in order to detect the genetic differences between different pathogen strains. The typing data of several hundred strains of different species were recorded in a database (BioNumerics).
The greatest challenges in further development are to create comprehensive and supranationally available databases with internationally standardized typing data and to implement quality management in the typing procedures.
Biothreats are currently associated with asymmetric warfare scenarios and non-state actors rather than with state-run bio-warfare facilities. In such scenarios, the deliberate release of a biological agent will most probably remain undetected until a cluster of cases will suggest an unusual outbreak of disease. Major goals of medical bio reconnaissance are rapidly to identify the causative agent, to trace the source and to differentiate between natural and deliberate outbreaks. In contrast to the investigation of overt natural outbreaks, forensic aspects have to be considered and implemented quite from the beginning when unusual outbreaks are to be investigated. If a biothreat agent is detected, it may be necessary to implement microbial forensics, a relatively new discipline that is mainly concerned with taking molecular fingerprints of biothreat agents by means of sophisticated molecular typing techniques. Its objective is to identify and trace back a particular strain by comparing it with the reference fingerprints stored in a typing database.
This overview underscores the importance of microbial forensics for the reconnaissance of unusual biological events, describes the respective capabilities that already have been developed in the Bundeswehr and outlines the challenges towards further developments. A broad spectrum of sophisticated typing techniques has been established in the Bundeswehr Institute of Microbiology. The typing data of several hundred strains of various species was fed into the BioNumerics database.
In the near future, the major challenges in microbial forensics will be the development of large databases with internationally standardized typing data and the implementation of quality management.
Today's risk assessment for attacks with biological (B) agents against military facilities or operations is no longer based primarily on the means of delivery (warheads, aircraft) that were typical of the earlier state BW weapons programs. Rather, scenarios are more likely in which the agent is deployed by terrorist groups using simple or makeshift devices. Such attacks have a small chance of being discovered the moment the agent is released. Rather, the medical service will initially be confronted with a cluster of illnesses. In such a situation, the first thing to do is to identify outbreak features that qualify the event as "unusual". In the event of an unusual outbreak, the possibility of deliberate biological agent release should be considered. In such a scenario, early diagnosis is crucial in order to interrupt the chain of infection and limit the number of serious and fatal courses. At the same time, forensic aspects must be taken into account in the diagnostic procedure and in the investigation of the outbreak, which, in the event of evidence of a B-agent, enable an unequivocal diagnosis in terms of reliable evidence with regard to the possible political and military consequences. This includes, for example, maintaining a complete “chain of custody”, thus excluding any mix-ups or manipulation of samples. This applies to human test materials in the same way as to animal samples, food, drinking water or environmental samples that are required in the context of the investigation of the outbreak. Since most of the potential B-agents also occur as natural pathogens of infections, a statement must be made in the further course as to whether the disease is natural or deliberately induced. For this purpose, new molecular differentiation methods are used, which under the term “microbial forensics” or “bioforensics” have established a new discipline in B-education.
Perception of biological risks and threats
Since the Convention on the Prohibition of the Development, Production and Storage of Bacteriological (Biological) Weapons and Toxin Weapons (B-Weapons Convention) came into force in March 1975, the production and storage of weapons based on microorganisms and other biological substances or toxins has been prohibited. The treaty has so far been ratified by 164 states, including the USA, Russia, Great Britain, Germany, France and the People's Republic of China.
As a result, the risk perception for biological hazards decreased sharply in the West. Technically and scientifically skilled workers were laid off, reconnaissance institutions were closed and budgets were drastically cut. From then on, the focus was more on the threat posed by nuclear weapons. It was only when the state's B-weapons program, which was secretly continued and even expanded in the former Soviet Union, became known, and Iraq's B-weapons program, which was betrayed by a defector, led to increased risk perception again. After the anthrax attacks in the USA in 2001, the biological risks finally returned to the focus of government countermeasures. The US was insufficiently prepared for this type of attack. Triggered by the dramatic events of September 2001, a comprehensive concept for the monitoring, detection and defense of biological hazards was developed and research and development in biological protection was stepped up at great expense. In this context, “microbial forensics” was established for the first time.
Today, the risks emanating from biological agents are mainly associated with criminal (biocrime) or terrorist (bioterrorism) activities. A real threat situation arises from the interest of certain groups of people in biological warfare agents identified by the intelligence service and from individual incidents identified in this regard. After the elimination of the Saddam regime in Iraq and the fall of the former Soviet Union, there is no valid information about ongoing state biological weapons programs.
Examples of bio-terrorist and criminal activity detected include:
- Contamination of salad bars with salmonella by the politically and religiously motivated Rajneeshee community (1),
- Faked anthrax attacks in the late 90s in the USA (2) and after 2001 also in Germany,
- Attempts by the Aum Shinrikyo sect in Japan to use botulinum toxin, anthrax spores, Vibrio cholerae (pathogen of cholera) and Q fever pathogen (Coxiella burnetii) for bio-terrorist purposes (3),
- Killing of a Bulgarian dissident with the help of the plant poison ricin and
- 2001 letter attacks with anthrax spores in the USA.
There were also other documented but unsuccessful attacks.
Classic forensic methods such as the evaluation of fingerprints or the ballistic analysis of projectiles have been an integral part of criminal investigation for decades. Since the invention of highly specific molecular methods for analyzing the genetic information of perpetrators (DNA analysis) in the 1990s, forensic DNA examinations, for example of dander, hair, etc., have made an important additional contribution to the fight against crime. With the help of retrospective analyzes of suitable materials, even murders can be cleared up that sometimes took place 30 years ago, but have not yet been cleared up due to a lack of conclusive evidence. Analogous to the analysis of fingerprints, a match between the DNA profile (“genetic fingerprint”) and a profile stored in a database can lead to the unambiguous identification of the perpetrator.
But what if the crime was not committed with conventional tools or weapons, but with a living, highly pathogenic microorganism that aims to infect as many people as possible who then become seriously ill or die? Can the released microorganism provide clues as to its origin or even help to identify the person who deliberately released it?
The science that aims to answer this question is called “microbial forensics” or “bioforensics”.
Modern DNA-based typing methods enable the creation of genetic fingerprints of the microorganisms. By comparing the profiles, they allow B agents to be identified below the species level, that is to say at the strain or even isolate level. Techniques such as pulse field gel electrophoresis (PFGE) have been used for years to analyze disease outbreaks and to track the spread of a pathogen during an outbreak. This scientific discipline is called molecular epidemiology.
It was only in 2010 that the example of the EHEC / HUS outbreak in Germany demonstrated the value of molecular epidemiological methods for investigating the outbreak. For several years there have been dynamic typing databases for various hospital and food germs such as the "PulseNet" monitoring program of the CDC in Atlanta (USA). If necessary, the stored genetic fingerprints can be called up and compared with the own data of a current outbreak event. For a few years now it has also been possible to determine and analyze the complete genetic information of a microorganism in a very short time. In the context of the cited EHEC / HUS outbreak, for example, it was recently possible to determine the genetic information of the pathogen by using next-generation sequencing (NGS) technology and to more precisely determine the origin by comparing it with the genetic information of known EHEC strains define. Microbial forensics differs from molecular epidemiology insofar as the pathogen-specific data obtained must not only be scientifically accurate, but also secured in such a way that they are of evidence. The prerequisite for this is that the data was generated in accordance with strict quality management criteria. This applies to sampling and shipping as well as to the analysis itself.
Typing methods and databases
The laboratory analyzes used in microbial forensics include the cultivation of the pathogen, a series of phenotypic analyzes such as biochemistry, mass spectroscopy and electron microscopy (for example to determine additives or the type of preparation that can be aerosolized). However, nucleic acid-based typing methods form the core, with which differences in the base composition can be determined in defined variable gene segments of the pathogen genomes. Just as humans differ genetically, there are also genetic differences between different strains of the same bacterial or virus species. Depending on how finely differentiated the method is, one can detect genetic differences not only between strains, but also between individual isolates of the same strain. Common methods are, for example, multilocus sequence typing (MLST) (4), multilocus variable number of tandem repeat analysis (MLVA) (5) or the analysis of single nucleotide polymorphisms (SNPs), clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) (6) as well as genome sequencing (7) and microarrays.
However, the procedures have different levels of differentiation. The gold standard for outbreak investigations is currently the high-resolution MLVA. The methods mentioned are also well suited for the various pathogen species to different degrees. For this reason, a separate range of methods is used for each species, which is also geared to the gene loci specifically found in this species (Fig. 1).
The future of molecular typing is already looming. In the US, the cost of whole genome sequencing has already fallen below $ 200. In Europe, too, costs will fall accordingly. At the price level reached in the USA, whole genome sequencing will largely replace the methods that have been used up to now. The comparison of different genomes will then primarily take place on the computer through in-silico analyzes, whereby the criteria of the typing methods used so far can still be used.
The establishment of a global and comprehensive typing reference database occupies a key position in the successful development of microbial forensics. Not only genetic typing information is stored there, but also further information about the individual genotypes such as the geographical occurrence of the strains, origin or distribution in laboratories. In order to be able to assign an outbreak strain and identify it with a probability of success, a representative variety of all genetic variants of a species must be stored. The prerequisite for the establishment of such databases is the international exchange of data, methods, living pathogens and DNA between research institutions. Another prerequisite is the standardization of the analysis methods used. At present, many of the data sets in the individual countries are incompatible with one another because they were developed using different methods. So in the future it will be an essential goal of microbial forensics to harmonize the methods and develop uniform analysis algorithms.
The creation of an international database is therefore not only a technical, but also a political challenge for the international community in order to enable the free exchange of the required data and materials. Communication between scientists, political stakeholders and federal authorities plays a crucial role in this. Naturally, however, possible dual-use aspects stand in the way of the free exchange of logs and information. It is therefore necessary to implement rules and controls without creating unnecessary obstacles.
From this point of view, microbial forensics is a new science that makes use of various established methods and sciences in a purposeful manner. This includes microbial gene and genome analyzes, phylogenetic examinations, classical forensics, bioinformatics, computer science as well as traditional microbiology and epidemiology.
Development of skills in microbial forensics in medical B-protection
In cooperation with national and international typing experts, various molecular typing methods for B-protection-relevant pathogens such as Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella spp. and Burkholderia mallei / pseudomallei etc. established. The range of methods (Fig. 1) includes modern analysis methods such as the above-mentioned MLST, MLVA, SNP analysis and next-generation high-throughput sequencing methods such as 454 pyrosequencing technology (Roche Diagnostics) or Illumina sequencing technology. Newly developed software makes it possible to create zoomable genomata for the direct visualization of sequence differences and for comparative analyzes at the genome level (Fig. 2).
InstMikroBioBw has one of the most comprehensive collections of human and animal pathogenic microorganisms in Europe. The strain collection has been continuously expanded since the late 1980s and today comprises several thousand bacterial and virus isolates. This particularly valuable strain collection is a basic requirement for developing and validating new analysis methods. In addition, the regular collection makes the institute a sought-after and efficient cooperation partner.
By applying the typing methods listed above to the institute's collection of strains, genetic profiles of a large number of pathogens relevant to B protection have already been created and stored in a database (Tab 1). The complex analysis software BioNumerics (Applied Maths, Belgium) serves as the database platform (Fig. 3). By integrating all essential bioinformatic analysis tools, from simple sequence analyzes to complex cluster analyzes to the de novo assembly of entire genome sequences, BioNumerics is the ideal analysis and evaluation tool. The network capability also allows a quick and uncomplicated exchange of data between individual project partners.
In order to position InstMikroBioBw as an efficient and competent partner in the field of molecular pathogen typing / bioforensics in the European network, a permanent working group “Molecular typing and bioforensics” was set up in 2010 under the direction of Priv.-Doz. Dr. Holger Scholz furnished. The primary goal is to expand the international network of the institute with other institutions that are active in the field. At European level, the institute is represented as an important partner in the European Biodefense Laboratory Network (EBLN) of the European Defense Agency (EDA). The aim of the joint project of 12 countries is to harmonize and standardize analysis procedures and to set up a jointly usable database for pathogens relevant to protection. By pooling resources and working closely with different countries, European capabilities in the field of medical B protection are to be improved. At the transatlantic level there is a cooperation with the working group of Prof. Dr. Paul Keim at Northern Arizona University, Flagstaff, Arizona. Paul Keim is one of the leading specialists in the field of molecular typing and bioforensics in the USA and was instrumental in investigating the letter attacks with Bacillus anthracis spores in 2001. In a project "Technology and Data Integration with the Bundeswehr Institute of Microbiology" funded by the Department of Homeland Security (DHS) since 2010, typing methods are to be aligned and a common database established.
Another cooperation for the comprehensive typing of Yersinia pestis, the causative agent of the plague, exists with the National Center of Infectious Diseases Natural Foci (NCIDNF), Ulan Bator, Mongolia. The typing of Mongolian plague strains is unique and provides new insights into the evolution of this pathogen, which is important from the perspective of medical B protection (8, 9).
Conclusions and Outlook
While Europe lacks a common strategy to counter biological risks and microbial forensics is still in its infancy, the US is different. In the report on the National Research and Development Strategy for Microbial Forensics, a concept for bio-terrorist threat prevention is presented in an impressive manner. The purpose of this National Strategy is to direct and bundle the research efforts of the US government to promote microbial forensics in order to provide the United States with the best scientifically proven ability to support categorization in the event of a potential or actual biological attack. This strategy includes a research agenda for bioforensics and also aims to promote cross-agency coordination and training of decision-makers, as well as scientific and technical personnel in this area.
US investments in research infrastructure are also impressive. Shortly after the anthrax attacks, for example, the construction of the National Bioforensic Analysis Center (NBFAC) for USD 143 million was initiated. NBFAC has 150 employees when fully operational and has an annual budget of $ 50 million. A similar approach would be desirable at European level. However, government investments of the magnitude mentioned are hardly to be expected here. The establishment of bioforensics can therefore only succeed if the military and civil institutions active in the field of B protection join forces to form a B protection network. They have to standardize their methods and develop the genetic typing of microorganisms for forensics by implementing a quality management system. All areas of the health system that carry out outbreak reconnaissance and biohazard prevention tasks are also required, because the focus of the probable scenarios will be patients. The Bundeswehr Institute for Microbiology can make valuable contributions to this and already has the most developed repertoire of methods in Germany. Comprehensive government research funding programs should be devoted to this problem.
- Torok TJ, Tauxe RV, Wise RP, et al .: A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars. JAMA 1997; 278: 389-395.
- Moran GJ: Update on emerging infections from the Centers for Disease Control and Prevention: bioterrorism alleging use of anthrax and interim guidelines for management- United States, 1998. Ann Emerg Med 1999; 34: 229-232.
- Olson KB: Aum Shinrikyo: once and future threat? Emerg Infect Dis 1999; 5: 513-516.
- Schouls LM, Reulen S, Duim B, et al .: Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: strain diversity, host range, and recombination. J Clin Microbiol 2003; 41: 15-26.
- Marsh JW, O’Leary MM, Shutt KA, et al .: Multilocus variable-number tandem-repeat analysis for investigation of Clostridium difficile transmission in Hospitals. J Clin Microbiol 2006; 44: 2558-2566.
- Pourcel C, Salvignol G, Vergnaud G: CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 2005; 151: 653-663.
- Read TD, Salzberg SL, Pop M,, et al .: Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis. Science 2002; 296: 2028-2033.
- Kiefer D, Dalantai G, Damdindorj T, et al .: Phenotypical Characterization of Mongolian Yersinia pestis Strains. Vector Borne Zoonotic Dis. 2011; Oct 24. [Epub ahead of print]
- Riehm JM, Vergnaud G, Kiefer D, et al .: Yersinia pestis Lineages in Mongolia. PLoS One. 2012; 7 (2): e30624.
Source: Military Medical Monthly 2012/4
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