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Technologies required for strategic actionable net-centric biological defense systems consist of : 1) multiplexed multi-array sensors for threat agents and for signatures of the host response to infection; 2) novel vaccines and restricted access antivirals/bacterials to reduce emergence of drug resistant strains pre- and post-event; 3) telemedicine capabilities to deliver post-event care to 20,000 victims of a biological strike; and 4) communication systems with intelligent software for resource allocation and redundant pathways that survive catastrophic attack. The integrated system must detect all threat agents with minimal false positive/negative events, a seamless integrated broad-band communications capability that enables conversion of data to actionable information, and novel pre- and post-event treatments. The development of multiplexed multi-array sensors, appropriate vaccines and antibiotics, and integrated communication capabilities are critical to sustaining normal health, commerce, and international activities.

Different bacteria, viruses and toxins constitute a potential menace for people. The number of toxins that could be applied for a bioterrorist attack with real public health risk, though, is relatively limited. However, these natural toxins could cause difficult troubles. The objective of this paper is focused on the toxins of various origins that might be used as a biological weapon. To be used in such a way the toxin should be highly lethal and easily produced in large quantities. Our current knowledge on natural toxins is conducive to select the toxin list threatening public health. At present this list includes a few bacterial and plant toxins, as well as a set of toxins produced by algae and molds. Novel methods of toxin detection should be able to monitor the presence of many toxins at the same time.

Prior to the 1990 Iraq-Kuwait conflict it was well known that Iraq had developed weapons of mass destruction but the extent of its programs were unclear. After the Iraqi defeat in the ensuing Gulf war 1991 the UN Security Council authorized the creation of UN Special Commission for Iraq (UNSCOM) with the purpose of ridding Iraq permanently of weapons of mass destruction. Several conclusions can be drawn from more than ten years of biological weapons inspections in Iraq. Firstly, UNSCOM managed to get a rather clear picture of the past weapons programs. Secondly, it was not possible for Iraq to restart a substantial program with UNSCOM being present in the country. Thirdly, a full and final and complete account of the weapons program could not be established despite the use of the best intellectual and technical capabilities available at the time.

Epidemiological analysis by the WHO showed that the spread of infectious diseases in all Central Asia states, including Uzbekistan have worsen. Uzbekistan is one of the most populated Central Asian countries with 25 million people, approximately 70% of whom live in rural areas. Many infectious diseases are spread from animal sources. In Uzbekistan, rotavirus, coroavirus, parovavirus, herpes virus, enterovirus infections, and brucellosis, anthrax, foot and mouth diseases, salmonellosis, kolibacteriosis, plaque diseases and others are wide spread. Epidemiological monitoring and the measures systematically undertaken by the local health service ensure a stable level of morbidity for the first and second groups of infectious diseases. Natural breeding grounds for infectious diseases such as plague, anthrax, and brucellosis and others in the arbovirus-group infections like the Crimean-Congo hemorrhagic fever (CCHF), tick-borne encephalitis (TBE), and fever syndromes of unknown origin dictate the need for more in-depth and comprehensive study in a cross section of every region in Uzbekistan. In October 2003 within the Cooperative Threat Reduction Program (CTR) and the Biological Weapons Proliferation Prevention Program (BWPPP) sponsored by the U.S. Department of Defense, a program began to establish an Integrated, Secure and Sustainable Disease Surveillance System in Uzbekistan. Main goals of this project are: detect deliberate or accidental release of biological materials relevant to the bioterror threat; create, strengthen and integrate existing surveillance systems; facilitate integration of host nation scientists and institutes into the international scientific community; and to create a potential to integrate national surveillance systems into an international system. Key elements of the project are modern, standardized, reliable diagnostics methods, e.g. PCR-based diagnostics; improved communications, transport, and integration, e.g. computerization; data analysis and sharing. We plan to select optimal prototypes of worldwide known Laboratory Research Networks and contribute new sophisticated methods of available data analysis.

The Autonomous Pathogen Detection System (APDS) 1 is a stand-alone pathogen detection system capable of rapid, continuous, low cost environmental monitoring of multiple airborne biological threat agents. Its basic design comprises aerosol sampling, in-line sample preparation, multiplex detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. Its primary application is to warn civilians and emergency preparedness personnel of a terrorist attack, the same system could also have a role in protecting military personnel from biological warfare attacks. APDS instruments can be used at high profile events such as the Olympics for short-term, intensive monitoring or more permanent installation in major public buildings or transportation nodes. All of these units can be networked to a single command center so that a small group of technical experts could maintain and respond to alarms at any of the sensors. The APDS has several key advantages over competing technologies: (1) the ability to measure up to 100 different agents and controls in a single sample, (2) the flexibility and ease with which new bead-based assays can be developed and integrated into the system, (3) the presence of an orthogonal, real-time detection module for highly sensitive and selective nucleic acid amplification and detection, (4) the ability to use the same basic system components for multiple deployment architectures, and (5) the relatively low cost per assay (<$2 per 10-plex or $0.20 per assay) and minimal consumables.

Midwest Research Institute has begun the development of an autonomous biological agent detector system based on the enhancement and integration of proven technologies. The design concept incorporates a high volume air to liquid sampler, fluid conditioning and concentration systems, and organism and toxin detection platforms. More than 20 threat agents identified by the US Centers for Disease Control are addressed using an integrated microfluidic system. Organisms are detected using a combination of advanced multiplexed PCR techniques. Protein toxins are detected using amperometric transduction of an immunoreaction. Core technologies from established organizations are used. The device is designed to be used in an urban area. The 0.3 m device has a targeted purchase price of US $ 25,000 in quantities of 3,000 units with annual total operating costs of US $ 10,000.

Demonstration of system prototypes in realistic user environments is a critical element in the maturation of detection-based systems employed for defense against Weapons of Mass Destruction (WMD) attacks. The United States Department of Homeland Security utilizes structured efforts called Domestic Demonstration and Application Programs (DDAPs) to overcome the diverse barriers associated with moving laboratory technologies into useable, end-toend systems. In this talk, specific examples, drawn principally from the major DDAPs concerned with the chemical and biological defense of transportation facilities, will illustrate the key issues and payoffs. System demonstrations and prototype deployments play important roles for the diverse participants in the development process, including technologists, commercial suppliers, end users, and government funding agencies. The deployments identify environmental and operational problems that impact system utility. The involvement of users is key to determination of realistic operational concepts and requirements. The prototype deployments also provide incentives for private sector investments in detector development and in other enabling technologies. In some cases, prototype deployments have evolved directly into full-scale, operational systems. These initial operational systems have then provided the basis for subsequent technology changes to improve operability and reduce costs. Two DDAPs that have already grown into operational, deployed systems are the Biological Aerosol Sentry and Information System (BASIS) for wide-area, detect-to-treat, biological defense and the Program for Response Options and Technology Ehancements for Chemical Terrorism (PROTECT) system for subway, detect-to-warn, chemical defense. The PROTECT program has been succeeded by the PROACT program which is focused on the biological defense of airports and BASIS is the foundation for the nation’s BioWatch defense system. The importance of DDAPs and other demonstration programs is underscored by their relative growth as a fraction of the US Department of Homeland Security Science and Technology effort.

Conducting validation studies of qualitative biothreat identification assays is a new field compared to validation of standard clinical laboratory techniques or bioanalytical methods. The technical challenges in biothreat assay testing include such things as (a) effects of various bacterial or viral concentration techniques; (b) residual matrix-associated amplification inhibitors; and, (c) differentiating live from dead cells. The determinations that must be made in conducting qualitative assay validation studies are: (1) specificity rate, (2) sensitivity rate, (3) false positive and false negative rates, (4) system suitability testing, (5) robustness, (6) linearity, (7) range, (8) precision and (9) limit of detection. The Association of Analytical Communities (AOAC) requires that each assay validation is completed with a Package Insert review ensuring validation parameters are accurately reflected. Upon completion of validation, a “Quality Policy Certification,” will be issued for each assay and published in the AOAC journal. The focus of the project presented in this paper is to provide the Department of Defense (DoD) and Department of Homeland Security (DHS) with a well-executed plan leading to the successful validation of assays for biothreat agents. The project will hopefully show the need for a Validation Center which can provide thorough and well-designed validation studies for qualitative assays and instrumentation. Validation that qualifies the technique/instrument to sample type, outcome expectations (forensic or force protection) and reproducible detection limits with false positives, false negatives and confidence calculations would also be included. In addition to the main laboratory (Validation Center) executing the validation study, five additional laboratories would also be required to complete validation testing.

Although banned by the BTWC in 1972, biological warfare agents continue to be a threat to military and public health. Countermeasures can only be effective if rapid detection and reliable identification techniques are in place. TNO Prins Maurits Laboratory, and Delft University of Technology and Bruker-Daltonik (Germany) are developing an bioaerosol alarm detector, based on fluorescence pre-selection, Aerosol Time-of-Flight Mass Spectrometry (ATOFMS) and Matrix-Assisted Laser Desorption/Ionization (MALDI). Using this combination, mass spectra were obtained from single biological aerosol particles.

Cell Physiometry tools will be described, having applications in cell diagnostics and cell separations, that can be directly exploited in biodetection technologies. These tools employ dielectrophoresis (DEP), using microelectrode arrays energized by broad band frequency generators. Biochemical labels, beads, dyes or other markers and tags are not required. Extensive and validated studies have shown that different cell types, cells at various stages of maturation or proliferation, and cells exposed to toxic agents, exhibit characteristic DEP signatures associated with their distinctive morphologies and cellular structures. The microelectrode array, sample chamber and fluidics are located with on-board electronics on a microscope stage. The electronics is used to control voltage signals to the electrode arrays, and images of the DEP responses of the cells are captured using an image processing tool. Further details are provided elsewhere. The technology is inherently fast and inexpensive. Particles, such as cells and bacteria, are characterized or sorted as a flux, introducing a high degree of parallelism as opposed to the serial analysis of events using conventional cell sorting instruments.