Appendix I

Invited Paper: BioWatch Improvements Within Existing Biological Detection Architecture

Disclaimer:

This white paper was prepared for the September 18-19, 2017, workshop on Strategies for Effective Biological Detection Systems hosted by the National Academies of Sciences, Engineering, and Medicine does not necessarily represent the views of the National Academies, the Department of Homeland Security, or the U.S. government.

Author and Affiliation

Name: George J. Dizikes, Ph.D.
Title: Director, Knoxville Regional Laboratory
Current Affiliation: Tennessee Department of Health
Former Affiliation (1996-2015): Illinois Department of Public Health

INTRODUCTION

The first time I heard about BioWatch, I was with the Illinois Department of Public Health at its Chicago laboratory. It was a few weeks before a planning and informational meeting on the BioWatch program was to be held in Diamond Bar, California. Unknown to me at the time of this meeting, which was at the end of January 2003, was the considerable amount of work already done by staff at several of the national laboratories, the Laboratory Response Network (LRN) of the Centers for Disease Control and Prevention (CDC), the U.S. Environmental Protection Agency, the Department of Homeland Security, and the Federal Bureau of Investigation, among others. This work was in response to the heightened awareness of bioterrorism threats sparked by the 2001 anthrax letter attacks. During his 2003 State of the Union Address, which occurred around the time of the Diamond Bar meeting, President George W. Bush announced that the federal government was “deploying the nation’s first early warning network of sensors to detect biological attack.”¹

While this network would be the most ambitious of its kind, it was not the first. A year earlier, at the 2002 Winter Olympics in Salt Lake City, a prototype of BioWatch, the Biological Aerosol Sentry and Information System (BASIS), was deployed at both indoor and outdoor sites throughout the Olympic venues (Imbro, 2003). Like BioWatch, BASIS utilized dry filters through which air was drawn to trap aerosol particles. Unlike BioWatch, filters were automatically changed throughout the day before collection. These filters were then transported to a laboratory where they were tested by real-time polymerase chain reaction (PCR) for the presence of specific DNA sequences representing biological agents of concern. By providing a series of filters, BASIS could, theoretically, determine a more precise estimate of when an agent may have been released, or at least when it was trapped by the filter. With BioWatch, a single filter is collected once every 24 hours, and a new filter assembly is then manually installed. At issue here is that a biothreat agent could be deposited on a filter anytime during the 24-hour period. Unlike the situation with BASIS, there is no way that finer resolution of this event can be achieved, unless the entire filter assembly is collected on a less-than-24-hour schedule. Under certain high-threat situations, this has been done, but cost considerations are usually the deciding factor. Processing of filters and interrogation of DNA sequences by real-time PCR is similar for both systems. Interpretation of PCR results would also be similar, but subsequent actions based on presumptive positive results may have differed based on the differences between an Olympic setting with international attendance and an American city.

The rationale for an early warning system like BioWatch is to provide response authorities with information concerning the presence of a biothreat agent before the public shows signs of infection (Shea and Lister, 2003). This “win-

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¹ See https://www.c-span.org/video/?174799-2/2003-state-union-address.

dow of opportunity,” between when an agent is released and when people become symptomatic and seek medical assistance (days to weeks depending on the agent), is a time when prophylaxis with antibiotics, antivirals, or vaccines may still be effective. Along these lines, a phrase heard at the Diamond Bar meeting, which some thought would become the motto of BioWatch, was “Detect to Treat.” Others, however, considered such a relationship between laboratory results and public health policy to be overly simplistic and potentially disastrous. Within the next few weeks, by the time the push packs of equipment and supplies began showing up at public health laboratories, the epidemiologists and decision makers within these public health departments were looking to the BioWatch program for additional guidance in interpreting and acting on a positive laboratory result. In the more than 14 years since the inception of BioWatch, these questions still loom large. In addition, during this time, the basic design of the BioWatch biological detection architecture has not changed significantly.

WHERE WE ARE NOW: THE EXISTING BIOLOGICAL DETECTION ARCHITECTURE

Preanalytical Phase

As with other types of testing (environmental or clinical), there are three distinct phases to BioWatch: preanalytical, analytical, and postanalytical. The preanalytical phase of BioWatch is mainly composed of field operations. The field operators install air collectors at locations deemed suitable by the BioWatch Program Office, in coordination with the local jurisdiction, based on population coverage, weather patterns, and accessibility. The field operators also change out filters on a 24-hour basis and service the collectors, which have powered air pumps. Filters within their exchangeable holders are brought to the laboratory, which is usually associated with a city, county, or state public health department. Once at the laboratory, the field operators conduct a sample transfer protocol: transport containers are decontaminated, all holders are accounted for by entering unique identifiers into a computerized database called the Sample Tracking Tool along with other pertinent information (e.g., deployment date and time and collection date and time), and the holders are transferred to the testing area of the laboratory by means designed to minimize contact between field operators and laboratory personnel (e.g., via a passthrough). Field operators then receive empty filter holders from the previous day’s work and transport them back to their base of operations for inspection and reloading with new filters. Once ready for redeployment, the holders are then given new unique identifiers, which are entered into the sample management database.

The final preanalytical steps are performed by laboratory personnel: chain-of-custody forms are signed and copies are given to the field operators before their departure, plastic bags containing the filter holders are given one more de-

contamination with bleach, and the decontaminated bags are transferred to biological safety cabinets to begin processing.

Analytical Phase

Processing of the holders and filters is the first step of the analytical phase. All steps during the analytical phase are accounted for by entry of barcodes into another database, the laboratory data management system. To prevent cross-contamination, only one filter holder and filter is worked on at a time. A holder is opened, and its filter is removed and placed into a barcoded Petri dish. (Empty filter holders are set aside within the biological safety cabinet until the final results for the corresponding filters have been obtained later in the day. If everything is negative, then the holders are taken out for cleaning and recycling to the field operators.) A quarter of the filter is then cut and placed into a screw-cap microfuge tube with glass beads and buffer. The remaining piece of filter is secured into its Petri dish by sealing the lid with Parafilm®, and the Petri dish is placed aside for additional use or archiving. Once all filters have been processed in this manner, the tubes are shaken at a high rate of speed (bead beating) to disrupt material impacted on the filter and break open cells and viruses, thereby releasing their DNA. The resulting lysate from each tube is then subjected to differential filtration by means of centrifugation or vacuum assist to first remove large pieces of glass and other debris, collect and concentrate the DNA, and then wash the DNA into an appropriate buffer. These DNA preparations are then used for real-time PCR analysis, either individually or pooled in high-throughput settings. In any event, real-time PCR is performed for each organism of interest with the DNA from each filter, representing a specific collector site. In addition, a number of control reactions are set up to test for possible contamination and whether the DNA preparations contain inhibitors to PCR.

Standardized PCR reagents are used by all BioWatch laboratories, as well as some other government laboratories. Prior to their introduction, these reagents were extensively evaluated with regard to sensitivity, specificity, and other standard parameters of method validation. Initially, a single primer-probe set is used to screen for each agent. If a positive reaction is observed, several repeat real-time PCRs will then be performed on the DNA preparation in question, each with an additional, different primer-probe set for the suspected agent in order to challenge and enhance overall specificity. Only if the proper combination of primer-probe sets are reactive for a given agent will a positive result be considered. And only then will activities commence for the possible declaration of a BioWatch Actionable Result (BAR), along with its follow-on notifications and activities.

Postanalytical Phase

The postanalytical phase of BioWatch begins after all routine testing has been completed. For the vast majority of samples, a nonreactive signal will be

obtained with the initial real-time PCR screening, so a negative result will be reported through the LRN Results Messenger (LRN-RM); except for cleanup and paperwork, this will essentially end the day’s activities. However, if a reactive signal is obtained upon initial screening, the analytical phase continues with the DNA lysate being repeat tested with the full set of LRN real-time PCR reagents for the agent in question. An interpretative algorithm exists for each set of these confirmatory LRN reagents. If the results of this repeat testing do not fit the algorithm, then the sample is considered negative, and this result is reported through the LRN-RM, again ending the day’s activity. If, however, the algorithm is met, the sample is deemed presumptively positive for the targeted DNA sequences and, by inference, for the agent in question. It is at this point that significant postanalytical activities commence and the process of BAR determination begins.

Historically, presumptive positive results have been extremely rare and warrant considerable scrutiny and review. The first step in this process is to consult with the laboratory’s BioWatch director, who is the representative of the local public health jurisdiction, and the BioWatch staff. Various quality assurance measures will be undertaken, including a review of any recent work with the agent in question–either as part of a positive quality assurance filter, a proficiency testing filter, or work by either the BioWatch staff or personnel of the host laboratory. Recent results from the collector in question and nearby collectors will be reviewed, as will the results of periodic wipe tests within the BioWatch laboratory, which are conducted to determine possible contamination. Recent weather patterns will also be reviewed. If extenuating causes cannot be implicated in this presumptive positive result (or even if they can), then a series of consultations will be initiated–with the CDC and the BioWatch Program Office–to again review the circumstances surrounding the results in question. At this point, the laboratory may be advised to cut an additional quarter of the implicated filter and repeat the analysis using the full set of primers and probes for the agent in question.

Based on all available information, it will be the decision of the host laboratory authorities (i.e., the resident BioWatch laboratory director or designee) to complete the BAR Data Form, declare a BAR, and commence notifications and conference calls over the next several hours. The purpose of these conference calls is to provide situational awareness at a local and national level and recruit additional resources to further evaluate the implications of the BAR with respect to the larger national preparedness and response community. The ultimate question being investigated by these discussions is whether the BAR represents a public health emergency, warranting a local and national response, or are the circumstances surrounding the presumptive positive result explicable as natural phenomena and not requiring a spin-up of response activities. To date, all BARs have been interpreted as nonthreatening events.

A criticism of the BioWatch program has been, “How do we know it will work?” The standard method for determining the sensitivity of a test or system would be to compare how many times something was correctly identified with

how many times it was missed. The “problem” here is that there have not been any attacks on U.S. cities with biological agents since BioWatch was deployed, so we do not know how many times it worked and how many times it did not. And, if the anthrax letters are considered an attack, they occurred before BioWatch and were not an aerial release, which would not have been expected to be detected by the BioWatch collectors. Since no one is suggesting that a test attack on a city be undertaken, we are left to indirectly validate the system either in part or as a whole. The various components of the BioWatch system have been evaluated at the National and Department of Defense Laboratories. But, in my opinion, the most compelling demonstration of the sensitivity of BioWatch has been its ability to detect the spraying of Bacillus thuringiensis when applied as an insecticide to fields of crops and the detection of an intentional urban release of Bacillus amyloliquefaciens, a source of antibiotics and another biological pesticide (Garza et al., 2014).

HOW WE GOT HERE

Changes Made to Laboratory Operations

Since the inception of the BioWatch program, there have been changes made to the laboratory operations, but these have been mainly evolutionary rather than revolutionary in nature. Initially, changes were made to increase throughput to accommodate larger jurisdictions: block shakers accommodating an entire rack of tubes were introduced, replacing individual tube shakers for bead beating; 96-well vacuum manifolds were implemented for filtration steps, replacing individual filter/centrifuge tubes; and the screening protocol was changed to allow pooling of up to three DNA lysates for the initial real-time PCR screening step, thereby conserving reagents and amplification plate utilization. However, the follow-up to a reactive screen then requires that each of the DNA lysates comprising a pool be individually tested with the full LRN primer-probe set for the agent in question.

Other changes involved the style of filter manifolds and filters, which have undergone change to provide greater uniformity and ease of operation. And, for greater safety, some laboratories use scissors rather than scalpels for cutting filters. Computerized laboratory information management systems to aid in specimen tracking and documentation of all laboratory steps were implemented and later upgraded. The LRN-RM for results reporting has been upgraded several times, but still remains somewhat cumbersome and time consuming. One of the agents that was originally in the screening panel, Brucella spp., is no longer included in testing, but all other members of the panel have remained constant for the past 14-plus years. However, in order to help evaluate the somewhat common environmental encounter with Francisella tularensis, a reflex panel of real-time primers and probes has been developed to distinguish between types pathogenic to humans and those that are nonpathogenic.

Over time, the original real-time PCR instruments have been upgraded with newer models by the same manufacturer. However, several years ago, an entirely different platform, the Bio-Rad Bio-Plex, was field tested at some of the BioWatch laboratories, including the one at Chicago. Rather than using real-time PCR, the Bio-Plex, which is a flow cytometer, evaluates end-point PCR reactions that are hybridized to DNA bound to microbeads of specific colors. These beads, and their corresponding DNA specificities, are identified through their unique color by the Bio-Plex, which also determines whether there are bound, fluorescently tagged PCR products. The advantage of the Bio-Plex is that the full set of PCRs for each agent can be evaluated simultaneously, thus shortening the assay time from that required for a two-step process of screening followed by confirmation, which is how the real-time assay is conducted. A down side to the Bio-Plex assay is the fact that end-point PCR is not semiquantitative in the way that real-time PCR is. Consequently, a source of potentially valuable information is missing when the implications of a BAR are being evaluated. Additionally, the PCR performed for the Bio-Plex is carried out in an open system, rather than the sealed plates used for real-time analysis, and such an open system is more susceptible to cross-contamination. In any event, after about a year, the Bio-Plex was abandoned and testing returned to a real-time platform. The exact reasons for this decision are still somewhat obscure and, perhaps surprisingly, the Bio-Plex was later used as the core analytical instrument in the short-lived Northrop Grumman Gen-3 instrument (more on this later).

Resuming analysis by real-time PCR, the original ABI Prism 7000 instruments were replaced by the Applied Biosystems 7500 Fast Dx, an instrument designed and manufactured to provide greater performance and durability than its predecessor. Other changes included reducing the number of cycles in a run from 45 to 40 to minimize unnecessary reactive artifacts at the extreme limit of PCR amplification. And the original LRN primer-probe sets used for initial screening were replaced with new sets from the government’s Critical Reagents Program (CRP). The CRP is also the source of the subspeciation reagents for F. tularensis. This change to CRP reagents was made to ensure greater supply reliability, improved performance, and increased uniformity throughout government-sponsored biological monitoring programs. Nevertheless, repeat testing of initially reactive DNA lysates is still performed with the full set of LRN primer probes as used for all other environmental or clinical testing conducted through the LRN-B.

Addition of Quality Assurance Measures

A fundamental and necessary change to the BioWatch system was the addition of a quality assurance program. As stated earlier, virtually all samples have proven to be negative for the threat agents being evaluated. The question then arises, to what extent can these negative results be defended as true negatives rather than suspected of being false negatives? The fact that there does not

appear to have been a bioterrorist attack on U.S. cities in the past 14 years supports these negative results. However, what about going forward?

The quality assurance program has implemented measures similar to those found in other quality systems (e.g., the International Organization for Standardization or Clinical Laboratory Improvement Amendments) to ensure the defensibility of results. These measures include regular (every 6 months) competency assessments of the testing staff, daily positive quality assurance filters with coded spikes simulating one or more of the test agents, regular (three times per year) proficiency testing challenges, annual internal audits, and semiannual site visits to provide an unbiased external audit of all laboratory activities. There is also a quality assurance program that applies to field operations.

When BioWatch testing began, the program purposely avoided the use of positive control materials out of concern for cross-contamination and the possibility of issuing false-positive results. However, good laboratory practice requires that the positive performance of a test be evaluated on a regular basis. To assess possible cross-contamination of positive control material, an additional nucleic acid target is added to all positive quality assurance filters. This target is derived from a fish virus and would never be expected to be found on an air sampling filter collected from the field. If, on any given day, a positive signal is obtained on a collected filter which corresponds to the agent(s) spiked onto that day’s positive quality assurance filter, the DNA extracted from that collected filter is then tested for the fish virus nucleic acid by a specific real-time PCR assay termed the TAG assay. If the TAG assay is positive, then it is concluded that contamination from the positive quality assurance filter had occurred and that this is also the likely source of the other reactive signature(s). Of course, it could be that in addition to cross-contamination from the positive quality assurance filter, the agent(s) in question was also encountered in the field during the 24 hours of air sampling. To evaluate this possibility, another quarter of the “positive” filter would be cut, processed, and its DNA tested with the primer-probe sets for the agent(s) in question. This repeat cutting and testing of a positive filter is not exclusively reserved for situations of suspected cross-contamination by positive control material; quite often the follow-up to a BAR declaration will include this activity. The generally recognized division of a filter is up to two quarters for laboratory testing, one quarter for the FBI, and one quarter for archival purposes.

WHERE ARE WE GOING

The BioWatch program, while seemingly reactive to the events surrounding the anthrax letters of 2001, is in fact a proactive program for detecting an outdoor or indoor atmospheric release of several potential biothreat agents. The Biological Detection System (BDS) operated by the United States Postal Service (USPS) at its postal sorting facilities is actually the reaction to the anthrax let-

ters.² As such, the BDS only tests for anthrax and does so by scrutinizing letters–the source of the anthrax attack. The BDS may also be considered as one of the prototypes for an autonomous collection, detection, and reporting system. Around the time of the BDS, another automated system, the Autonomous Pathogen Detection System (APDS), had been developed by a National Laboratory. This system was capable of testing for bacteria, viruses, and toxins, and it was claimed that it could simultaneously test for up to 100 different agents.³ The APDS was piloted for a while in New York City, where there were concerns with false readings and excessive need for maintenance.⁴ This phase was followed by an open competition to develop and evaluate an automated BioWatch system, Gen-3, that was to replace all collectors currently in use with autonomous units and to expand BioWatch to additional cities. I was still with the BioWatch program in Chicago when Gen-3 was piloted by co-locating some collectors alongside standard filter collectors. By the time of deployment, only one of the developers was still being considered. While the instruments seemed to operate reliably, there was not a BAR to evaluate and there were serious questions about the acquisition process and long-term costs of operating the units (GAO, 2014).⁵ Following a Government Accountability Office review of the Gen-3 initiative, it was decided in April 2014 to cancel the project.⁶

A Reality Check: Some Drawbacks and Limitations

As has been described, the laboratory component of BioWatch is a highly routine, potentially monotonous process that is punctuated by rare moments of extreme excitement and activity. As with any routine activity, lapses in attention and procedural drift may occur, leading to unintended and unnoticed errors. The BioWatch quality assurance program helps to prevent and detect such errors, but constant vigilance and efforts to vary the staff’s job duties as much as possible are also an essential part of preventive actions.

It has been stated by the BioWatch program that, in all its years of operation, there have been no false-positive results out of millions of PCR assays. That is not to say that there have not been “false alarms,” but these have been authentic PCR signals generated from the organisms for which the primers and probes had been designed and intended to detect. It’s just that in all these cases the organisms were deemed to be naturally occurring and not a public health

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² See http://about.usps.com/news/state-releases/sc/2013/FAQ-BDS.pdf.

³ See http://www.globalsecurity.org/security/systems/apds.htm; https://str.llnl.gov/str/October04/Langlois.html.

⁴ See http://www.homelandsecuritynewswire.com/dr20140430-dhs-cancels-acquisition-of-biowatch-s-generation-3-technology.

⁵ See http://www.homelandsecuritynewswire.com/dr20140430-dhs-cancels-acquisition-of-biowatch-s-generation-3-technology.

⁶ See https://www.afcea.org/content/dhs-cancels-biowatch-gen-3-acquisition.

threat. In some ways BioWatch shares the same limitations that surround the use of a police radar detector. A radar detector is really just a microwave receiver; it can respond to the presence of police radar, but it will also respond to an automatic door opener at a nearby shopping center. What is required in both cases is additional information to evaluate the signal and arrive at an appropriate risk assessment.

The laboratory work of BioWatch is conducted by individuals who are essentially guests at a host laboratory. As such, the facilities allocated to BioWatch may not be ideal and their improvement or upgrade may not be a very high priority for local and state government budgets. Additionally, interactions between the computer/information technology component of BioWatch and the host IT structure may, at times, seem to be at odds and with cross purposes. For the BioWatch laboratory to function successfully, it is essential that there be a lead worker with excellent managerial and interpersonal skills. It is also essential that a good working relationship be established between the BioWatch lead and the representative(s) of the host laboratory with whom he or she interacts. Beyond this, it is important that these two individuals, while representing the laboratory, establish good working relationships with other members of the BioWatch Advisory Committee, and that the anticipated events following a BAR be fully exercised before there is an actual event.

A Modest Proposal

As stated earlier, the goal of BioWatch is to become aware of the purposeful release of a bioterrorism agent before the public becomes symptomatic. Following this window of opportunity is the realm of syndromic surveillance, which has the potential to alert the public health community to the early emergence of a disease. Even though syndromic surveillance operates in a more delayed time frame compared to BioWatch’s potential, its advantage is that it can alert the medical community to new, novel, or unexpected diseases. As currently designed, BioWatch can only find what it’s looking for. The holy grail of a biological detection system would be the virtually instantaneous detection and identification of any agent posing a biological threat. An even more ambitious system would be one that could detect and identify the presence of any threat–biological, chemical, or radiological. Unfortunately, a universal detection system, while appealing, is likely beyond any foreseeable capabilities, even going out 10 years. In my opinion, select agents and toxins (especially select agents) pose the most immediate and greatest threat of clandestine release. Many chemical agents, by their nature, make their presence known almost immediately, and radiological agents are more difficult to obtain than biological agents that can be grown from the environment and transported with little risk of detection.

The question may then be: how does one maximize the focused role and potential benefits of BioWatch? When an agent is released, the clock begins; and, as mentioned before, the countdown to symptoms is likely a matter of only a few days. Since BioWatch filters are usually collected once every 24 hours,

and several hours are required for a field worker to complete a collection route and deliver filters to the laboratory, a release early in the collection cycle means that more than a day will have already passed even before testing begins. Add to this the time from when the first filter of the day arrives at the laboratory to when the last screening result is known (5 to 6 hours), plus the time to run the confirmatory PCR (1 to 2 hours), and one can see that more than 32 hours may have already passed. Attempts to reconstruct events at the time of release for purposes such as either plume modeling or the tracking of potential contacts are further hampered by the uncertain timing of the release.

One way to improve the resolution of when a release occurred (or, rather, when the collection of positive material occurred) would be to collect a series of sequential filters, as was the case with BASIS. However, if each filter is tested, then reagent costs will increase dramatically. To avoid this, the unit could have one filter which collects aerosols for the entire 24 hours, while a second holder contains a cassette which exposes filters sequentially for a predetermined period of time (e.g., 1 to 4 hours each). Alternatively, if there were no 24-hour filter, a piece of each of the sequential filters could be combined to make a pooled DNA preparation. In any event, if the 24-hour filter or pool tested positive, then each of the individual sequential filters could be tested to determine when the positive material was encountered.

While a system such as that just described will improve resolution of when a release occurred, it will not improve the window of opportunity for treatment by reducing the time from release to reporting a positive result. The only way that situation can be improved upon with the current system hardware would be if filters were collected and brought back to the laboratory and tested on a more frequent schedule than once every 24 hours. For example, if filters were collected every 4 hours, then the maximum elapsed time from the collection of a threat agent on a filter to its identification would be reduced by 20 hours to 12 hours rather than 32–a considerable improvement–but operating costs will also dramatically increase. Another way to test filters (or some other collection device) more frequently without the need for additional field or laboratory staff would be to deploy autonomous detection systems that phone or radio results to the health department. As discussed earlier, such systems have been field tested, but with mixed results. Also, these devices are highly specialized with a limited potential for distribution. Consequently, they are initially quite expensive and still require regular service and maintenance.

When the Gen-3 autonomous detector was being field tested in Chicago, I had the opportunity to become familiar with some of its internal operations. Several things struck me:

1. I consider the cyclone collection system, with its ability to collect samples into a liquid state and for variable lengths of time, to be vastly superior to the dry filters used by the portable sampling units currently used in the field.

2. The liquid handling, DNA extraction, and PCR modules have to be custom designed and built in limited quantities, which means they will be expensive.
3. A few years earlier, the Bio-Rad Bio-Plex had been used by some of the BioWatch laboratories. It had been abandoned in favor of real-time PCR, but it was now occupying the bottom of the Gen-3 unit serving as the detection unit for the PCR products. While in use at the Chicago laboratory, we had been advised by Bio-Rad not to move the instrument because of the sensitive alignment of its laser optics. Would this sensitivity also pose a problem for Gen-3 use?
4. The Gen-3 units required heating and air conditioning to maintain optimal temperature and relative humidity for proper operations under outdoor deployment intended for extremes from Midwest winters to Arizona summers. Would this really work, and what about a power failure?

My thought here is that perhaps we are asking too much of an autonomous system–that it be universally deployed, rugged, durable, and cheap. The most successful autonomous detection system that I am aware of is the USPS BDS. It has very high specificity, with no alarms ever during its extensive deployment history. It is designed to operate in an indoor, controlled environment and check for a single agent, B. anthracis, which is arguably the most likely agent that will be used in a bioterrorism attack. And, it is based on a widely used and essentially off-the-shelf PCR system made by Cepheid. At the same time, the BDS has the ability to detect and communicate positive result within a few hours of its encounter with anthrax.

Perhaps a unit like the BDS could be designed using basic off-the-shelf components (e.g., Cepheid GeneXpert or bioMérieux BioFire technology) for use in indoor settings such as train and subway stations, pedways, and airports. Like the current BDS, the system could just test for B. anthracis, or it could be multiplexed for a few additional agents. These indoor settings would provide better environmental control and greater security than most outdoor locations, while offering better access to power and communication networks. And, these high-traffic venues would benefit more than most outdoor settings to the rapid notification of a bioterrorism event. Several years ago there was a full-scale exercise of a biological release in the downtown subway system in Chicago. By the time a BAR would have been declared, the entire downtown area of Chicago would have been contaminated, requiring an indefinite, extensive quarantine with financial losses and cleanup costs in the billions of dollars, not to mention the enormous amount of potential morbidity and mortality among the citizenry. In addition to operating well within the window of opportunity, rapid detection and notification of an indoor release could also lead to measures minimizing the spread of the agent, thereby reducing its overall impact. Such measures are impractical with outdoor releases.

My recommendation for outdoor (and nonautonomous indoor) collection begins with replacing the dry filter collection method with a liquid-based cyclone-type collector. It should be possible to design this unit as a module that can be rapidly exchanged by a field worker. Since aerosols would be introduced into a liquid phase, there is flexibility in the choice of liquid. For example, a disinfecting liquid could be used to ensure that all organisms collected are inactivated. Or, a buffer of some sort could be used to preserve the viability of organisms. Additionally, a Peltier cooling device could be incorporated into the unit to help preserve organisms. The cyclone unit could also be designed to provide discrete timeframes of collection (e.g., a separate tube every 4 hours). Starting with a suspension of potential biothreat agents, rather than a desiccated sample, several options present themselves:

1. Once at the laboratory, nucleic acids could be extracted using a commercial, automated platform, like the Roche MagNA Pure–some models of which have been validated by the LRN for their assays. An automated platform would dramatically speed the extraction phase, while providing better containment and less chance for operator error. Essentially, one or two people could perform the entire laboratory portion of BioWatch, even for large jurisdictions, especially if a pipetting robot were also used for preparing PCR master mixes. This pipetting robot would also provide greater fidelity and speed of operation. Currently, there is little to no automation in the BioWatch laboratory, but changes here could speed operations, improve performance, reduce the potential for errors, and reduce personnel costs. And, while robotic instruments are not inexpensive, over time they are less expensive than manual methods with recurring personnel costs.
2. In addition to being subjected to the standard real-time PCR assays, the material in suspension can also be investigated by any number of additional laboratory procedures, without relying on equipment specially built to be installed in an autonomous detector. (Although it is nearly 17 years old and does not address some newer technologies [e.g., next-generation DNA sequencing], see North American Technology and Industrial Base Organization [2001] for an extensive review of collection, detection, and identification technologies, as well as issues of general consideration surrounding biological detection systems.) An advantage to having the BioWatch laboratory located within an established public health laboratory is that many of these facilities participate in the CDC’s Advanced Molecular Detection initiative, and in doing so have acquired sophisticated equipment that can be shared with BioWatch for the identification of pathogens. Some of the additional laboratory procedures that can be envisioned include the following:

3. 1. Antibody-based identification or enrichment of specific organisms or classes of organisms may be done either by magnetic bead separation or flow sorting. Once enrichment has been achieved, other identification methods may be employed, such as next-generation or chip-based DNA sequencing or mass spectrometry (e.g., matrix-assisted laser desorption ionization time of flight). Some of these methods are general, in that they will present information that can be used to identify an unanticipated pathogen.
   2. DNA sequencing may be successful even on an unenriched population of bacteria if it utilizes specific PCR-based amplification, or if interfering sequence data can be eliminated in silico through information processing. However, in the case of PCR-based sequencing, one is again rather restricted to finding only what one is looking for.
   3. Organisms may be cultured from the suspension. While this is not a rapid process, there is information available from a pure culture of a threat agent that may not be readily obtainable from any other source (e.g., antibiotic resistance patterns). So, while culture would not be the primary method for identification, it promises to provide information unobtainable from desiccated, dead organisms.
4. Since a series of collection tubes would be available, a pool of these could be made which would provide the starting material for a 24-hour screen. Alternatively, the cyclone collection unit could be designed to provide continuous as well as sequential sampling. In any event, if this pool were positive, then individual tubes representing shorter, sequential periods of time could be analyzed to better define the time of release and collection.

In conclusion, I estimate that, by utilizing a cyclone collector and laboratory robotics for sample processing and PCR analysis, the time from sample receipt to results reporting could be halved. And, laboratory staff could also be reduced to a similar degree. Moreover, the aqueous suspension provided by the cyclone collector can be the starting point for additional laboratory analyses. Because of the level of automation at the laboratory, it may be feasible to institute more frequent collections from the field, without incurring proportional increases in personnel costs. The greatest improvement in this area would occur when collections are made every 12 hours, rather than every 24 hours. More frequent cycles of collection then provide only incremental improvements. A 12-hour collection cycle coupled with laboratory robotics could reduce the maximum time from collection to detection and reporting from greater than 32 hours to approximately 16 hours. This improvement may really be the difference between life and death for thousands of people, should an attack occur. And, this change would be the first significant, sustained improvement to turnaround time since the beginning of the BioWatch program.

REFERENCES

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