6 Detection and Measurement of Biological Agents
6 Detection and Measurement of Biological Agents
6 Detection and Measurement of Biological Agents
The previous chapter was devoted to an analysis of what the committee feels is the
most probable course of events in a terrorist attack involving a biological agent--a covert
attack that, after a period of hours to weeks, will result in victims widely distributed in
time and location. Because the biological agents being discussed in this report do not
immediately produce effects, the first indication of an attack with a biological agent may
be the recognition of an unusual distribution or number of cases of disease, long after the
initial aerosol or solution has been dispersed or degraded. An important part of this
detective work is laboratory analysis of clinical samples, most often blood from a sick
patient. The previous chapter alluded to the possibility of new developments in such
diagnostic testing that might significantly decrease the time needed to arrive at a definitive
diagnosis. The present chapter examines those developments in more detail.
The chapter also examines the application and utility of these developments in the
detection of biological agents in the environment. There will be no fire and rescue teams
responding to a 911 call in an incident involving covert release of a biological agent, and
thus little use for the sort of rapid detection devices that are so important in responding to
chemical releases. Public health surveillance systems and the rapid analysis of
information from those systems may in time provide an indication of when and where the
biological agent was released, but unless there is a continuing source of agent, testing the
release site at that point will probably be useful for forensic purposes only (testing may
also be helpful in guiding clean-up after an attack with spore-forming agents like anthrax
that can survive in the environment for years). This is far different from the battlefield
scenario of military units facing an enemy with an arsenal of identified biological
weapons. Monitoring the environment for those agents and providing these at-risk troops
with the means to rapidly identify contaminated air, water, food, and equipment would
literally be vitally important. To the extent that similar high-risk situations can be
identified in the civilian environment (the President's State-of-the-Union Address? The
Superbowl? A soggy package labeled "anthrax"?), there may be a civilian need for
monitoring and detector technology as well. For this reason, although the committee does
not believe these situations will be frequent enough to merit a major investment for
civilian use, the chapter concludes by summarizing current R&D efforts on environmental
detection by military and other laboratories.
The classical approach to microbial detection involves the use of differential metabolic
assays (monitored colormetrically) to determine species type in the case of most bacteria,
or the use of cell culture and electron microscopy to diagnose viruses and some bacteria
that are intracellular parasites. Samples taken from the environment, such as soil and
water, and most clinical samples must be cultured in order to obtain sufficient numbers of
various cell types for reliable identification. The time required for microbial outgrowth is
typically 4—48 hrs (or two weeks for certain cases, such as Mycobacterium tuberculosis).
Furthermore, bacterial culture suffers from an inherent drawback: cells that are viable may
not be culturable, because they possess unanticipated nutritional requirements as a result
of genetic mutation. The following few pages lay out some general approaches being
taken to eliminate these drawbacks of the traditional methods and provide some examples
of efforts to apply them to detection of potential biological weapons. Biodetection is a
very large and active field which merits a study all by itself, and for that reason the rest of
the chapter is deliberately confined to technologies and research that has focused on the
agents of central concern to this report. The interested reader is referred to any or all of
the following general reviews: Turner et al. (1987), Janata (1989), Wolfbeis (1991),
Taylor and Schultz (1995), Van Emon et al. (1996), Rogers et al. (1995), Kress-Rogers
(1997). Boyle and Laughlin (1995) provide a history of the U.S. military biodetection
program, and Boiarski et al. (1995) described a large number of biodetection technologies
being explored by the U.S. military at that time.
Probe Technologies
Probe technologies include those based on: nucleic acids, antibody/antigen binding,
and ligand/receptor interactions.
Nucleic acid-based probes capitalize on the extreme selectivity of DNA and RNA
recognition. Nucleic acid probes, engineered single strands of RNA or DNA, bind
specifically to strands of complementary nucleic acids from pathogens. These probes and
their binding can be detected directly or by tagging with an easily detected molecule that
provides a signal. The design of the probe can be highly specific if there is a good fit to a
pathogen-unique region of the target nucleic acid, or it can provide more generic
identification if there is a fit with a region of nucleic acids conserved among several
related pathogens. The sensitivity of these hybridization assays for bacteria is between
1,000 and 10,000 colony-forming units; improved sensitivity is an important area of
research. Since the reaction is in real time, the time-consuming part of the method relates
to sample preparation and the time required to detect the signal.
The main advantages of nucleic acid-based methods are universality (all living
organisms have DNA and/or RNA), specificity (every type of organism has some unique
sections of DNA or RNA), sensitivity (with amplification, very small amounts can be
detected), adaptability (base sequences common to several microbes, or even a whole
class of microbes, can be used as probes), and multiplex capabilities for a host of different
microbes (a sample can be probed for many different sequences simultaneously).
Disadvantages of this technology include difficulty in isolation and "clean-up" of DNA
samples, degradation of the nucleic acid probes, and interference from related sequences
or products. These are important obstacles to be overcome, even after specific and
accessible target sequences are identified and probes constructed.
Ligand-based probes were developed on the principle that every cell has cell-surface
proteins that bind other specific molecules. Ligands may be small or large, specific to a
particular microbial serotype or common to related groups, and bind with varying degrees
of affinity.
Transducer Technologies
Piezoelectric transducers rely on the use of certain crystals that produce an electric
charge when subjected to pressure. Subjecting those crystals to an electric current causes
them to vibrate at a frequency that is dependent upon their dimensions, including their
mass. Coating the surface of the crystal with, for example, antibody or nucleic acid probes
will alter that frequency, and more importantly, antigen binding or nucleic acid
hybridization will cause still more frequency change. There are some problems with
reproducing surface coatings, and the sensitivity is typically 105—106 cells. Specificity is
derived from the probe material. Piezoelectricity is the basis for several chemical agent
detectors using surface acoustic wave (SAW) technology, and it has been a popular
approach to biodetection in the recent past (Guilbault and Schmid, 1991; Guilbault, Hock,
and Schmid, 1992). Our inventory of active research shows only two such entries,
however: a NASA-funded contract at Southern University to develop a liquid-phase
crystal immunosensor, and a Naval Research Laboratory effort to develop an antibody-
based force amplified biological sensor (FABS). Both are still in the proof-of-principle
stage, the former using E. coli for prototype development, and the latter MS2 virus and B.
globigii.
Light absorption, or colorimetry, has also been used for transduction. A binding event
causes a color change that can be observed by the naked eye and/or quantified by
spectroscopic measurements. For example, colloidial gold bound to agent-specific
antibodies produces a red spot when "collected" by antigens in the sample. The "litmus
test" being developed by Charych and colleagues at the Lawrence Berkeley National
Laboratory is another colorimetric assay. Ligands that bind to specific viruses and toxins
are incorporated into a polymerized bilayer assembly that changes color when the agent
binds (Charych et al., 1996). This quick and simple test has a sensitivity of 108 virus
particles and 20 ppm for toxins. Although the sensitivity of colorimetric methods in
general is significantly less than that achieved with fluorescence, such methods are useful
where the agent is likely to be present in high concentrations.
Two variants of fluorescence being utilized in DoD research on bioagent detection are
up-converting phosphor technology (UPT) and the fiber optic evanescent wave guide
(FOWG). The former, whose development is being funded through DARPA
(Wollenberger et al., 1997; Wright et al., 1997; Cooper, 1998), uses a number of rare earth
compounds that, in crystal form, have the unique property of emitting a photon of visible
light in response to absorbing 2 or 3 photons of lower-energy infrared light of the proper
wavelength. Coating the crystals with antibody provides for a highly identifiable signal,
since no naturally occurring substances upconvert. Nine spectrally unique phosphors have
been synthesized to date, making it possible to simultaneously probe with as many as 9
antibodies. More phosphors are under development, although it seems likely that the
multiplexing limit will probably be closer to 9 than to 100. The Analyate 2000 fiber-optic
evanescent waveguide biodetector developed by the Naval Research Laboratory (Cao et
al., 1995; Anderson et al., 1996) also uses antibody probes, some of which are bound to a
glass optical fiber immersed in a capillary tube containing an aqueous solution of the
sample. Other antibodies, tagged with a fluorescent dye, are added to the sample, where
they bind to the target antigen. The antigen-labeled antibody complex then binds to the
immobilized antibody. Light from a near infrared diode laser travels through the fiber,
which contains it almost completely. The very small amount of light escaping, the
evanescent wave, excites the fluorescent tag, whose emission is sent back up the fiber and
detected via photodiode.
Hybrid Technologies
There are some detection devices in which there is no clear division of probe and
transducer. Methods based on physical properties and separation are good examples: mass
spectrometry and gas or liquid chromatography. Mass spectrometry (MS) is a major
analytical technique in which materials to be analyzed are converted into gaseous ions or
otherwise characteristic fragments. The fragments are then separated on the basis of their
mass-to-charge ratio. A display of this separation constitutes the mass spectrogram. MS is
an extremely sensitive, selective, and rapid technique. Quantities of chemicals as small as
10—18 moles can be detected within milliseconds in highly purified samples, and MS has
demonstrated detection of 106 cells. In a field environment, or whenever samples are
heterogeneous, the constituents must be separated before they can be reliably identified, a
task accomplished in a variety of ways, including gas chromatography (GC), high
performance liquid chromatography (HPLC), or the use of two mass analyzers (one to
perform the separation and a second to produce the mass spectrum of the resulting
analytes).
For example, the Biological Integrated Detection System (BIDS) continuously samples
ambient air and determines the background distribution of aerosol particles. Aerosol
particles with diameters in the 2 to 10 micron range are concentrated and analyzed for
biological activity, as indicated by the presence of adenosine 5´-triphosphate (ATP). Flow
cytometry then separates and concentrates bacterial cells, and antibody-based tests are
conducted for specific agents. At present, the system includes tests for the bacteria
responsible for anthrax and plague, botulinum toxin A, and staphylococcal enterotoxin B.
Much less expensive point detectors are available as prototype "One Step Hand-Held
Assay" devices. These instruments are currently produced by the Navy Medical Research
Institute (NMRI) at Bethesda, Maryland, (similar devices have recently become
commercially available through Environmental Technologies Corporation) and are based
on antigen capture chromatography. Eight different devices are used to assay liquid
samples for the presence of Y. pestis, F. tularensis, B. anthracis, V. cholerae, SEB, ricin,
botulinum toxins, and Brucella species, respectively. A color change provides a positive
or negative indication within 15 minutes. The sensitivity of these assays varies from an
order of magnitude below a fatal dose (ricin) to more than an order of magnitude above
the infectious dose (anthrax). These devices are strictly screening assays, and the analyses
are subject to error from the introduction of other contaminants. Therefore, positive
results need to be confirmed with standard microbiology assays, conventional
immunoassays, or genome detection via polymerase chain reaction (PCR) technology.
Both NMRI and USAMRIID at Ft. Detrick, Maryland, have deployable field laboratories
that can perform these additional confirmatory assays (and assays for 15 to 20 other
potential agents). However, the confirmatory assays do not yield results as quickly.
Detectors with higher sensitivity than those presently available will be needed to detect
biological aerosols at minimally hazardous concentrations.
Potential Advances
The perceived need for faster, surer results for timely detection of hazardous
biologicals in the environment has spawned a large and growing number of research
programs. Biological detection is the largest single category in the committee's inventory
of relevant technologies (Appendix B). Space does not allow discussion of each, but most
of the devices are variations on a small number of approaches that were described in the
previous section on patient diagnostics. As in the case of chemical detectors, the
underlying approach largely determines the sensitivity, selectivity, versatility, and
reliability. Application to detection of biological agents in the environment differs from
patient diagnostics primarily in the increased need for portability, ease of use by
nonscientists, speed, and methods for collecting and preparing the sample. The following
pages first describe the main approaches to sampling the environment for biological
agents. We then consider some current research and development on new and better
devices for detecting, identifying, and quantifying biowarfare agents and how they might
meet needs of civilian medical personnel in domestic terrorism scenarios. We conclude
with recommendations for prioritizing R&D in this area.
Sampling
Sampling has to do with how the material that is to be tested is brought to the detector,
whether it comes from air, liquid, solid objects, surfaces, or from human tissue. There are
several issues that make sampling for biological agents challenging. The first issue is that
the sampling is normally targeted at living organisms; therefore, the technology must not
"harm" the sample. Secondly, because most detector devices require a liquid sample,
collection of airborne microbes must be extracted from an aerosol or particulate for and
concentrated in a liquid. Third, the target microbe is generally only one component of a
complex matrix of biological elements and chemical compounds that may affect the
detection process, so the sample must often be purified to some extent. Last, the sample
must be highly concentrated for a rapid analysis. Four general types of sampling devices
designed to accomplish one or more of these objectives are: (1) viable particle-size
impactors, (2) virtual impactors, (3) cyclone samplers, and (4) bubblers/impingers. Each
of these technologies is described below.
Two very important sampling issues must be addressed, regardless of the technology
employed. First, the environment in which the target microbe exists can significantly
affect the physiology of the microbe and with that the efficacy of the detection procedure.
Bacillus anthracis, the causative agent in anthrax, provides a simple example: in the
environment it exists as a hard, oval, inactive spore highly resistant to sunlight, heat, and
disinfectants, but in tissue, including blood, it germinates into a rod-shaped vegetative
bacillus actively proliferating and producing its characteristic toxins. Detection strategies
appropriate for one form of the organism may be entirely ineffective in the presence of the
other form. Less dramatic but equally important, components of the matrix in which a
microbe exists contribute significantly to the microbe's growth state and gene expression
in a way that is just beginning to be explored for most organisms. Detection strategies
focused on a specific structure or gene product can thus vary wildly, if sampling
conditions are not clearly specified.
Stand-off Detection
Sensitivity to infectious dose level is probably not important for early warning, since
an aerosol cloud intended to kill or incapacitate even one individual will certainly involve
concentrations far in excess of the infectious dose (later decisions about clean-up and
reoccupation of contaminated areas may need that level of sensitivity, but speed will be
less of an issue, and respiratory protection will allow use of more sensitive point
detectors). Specificity also may not be critical in the use of stand-off detectors. For
example, we may just need to be alerted to the presence of live biologicals. This is also
true for the control of contaminated environments, determination of decon efficacy, and
dynamic threat assessment (real-time assessment of a threat, including remediation).
Stand-off detection offers safe, real-time determination of microbial contamination.
Significant advances have been made with the use of lasers for the detection of
aerosolized agents by light-scattering characteristics, infrared and Raman spectroscopy,
and fluorescence, but these same methods can also be used to determine total microbial
contamination on objects (Powers and Ellis, 1998) and in situations where effective
sampling is impossible for reasons other than distance. The efficacy of these devices is
somewhat limited by the range at which the determination is desired (typically several
kilometers for military systems). Longer distances, of course, are more difficult, and the
necessity for prior intelligence, subsequent deployment, and then line-of-sight use of the
technology would seem to limit its utility in urban bioterrorism scenarios. Applications of
true stand-off detection would seem to be limited to monitoring predetermined, high-risk
sites or large public gathering places, such as stadiums, for aerosol clouds.
An alternative approach for long distance detection is the small model airplane-like
unmanned aerial vehicles (UAV) being developed by the Naval Research Laboratory and
Research International (Foch, 1998). These vehicles, ranging in size from a few inches to
a foot in size, may eventually carry on-board sensors and down-link data to ground-based
control. There is a weight and size limitation on the sensors that can be carried on-board,
but prototype vehicles have been successfully demonstrated in cities and inside buildings
as well as in outdoor terrain. Furthermore, they are reusable and easily transported. In the
event of biological agents being released in a building, such vehicles could locate "hot
zones" and monitor decon efficacy, reducing human exposure and risk.
Point Detection
Point detection refers to testing a sample that has been taken directly from the
environment suspected of harboring the target agent. Needs in this regard include not only
investigation of suspected sources of contamination but also monitoring the air/water
systems in buildings for general pathogen contamination or contamination by specific
biological agents. A number of the embryonic microbe detectors described above in the
section on patient diagnostics are being examined for utility in environmental detection as
well.
Most of the current R&D on detection of biological weapons employs nucleic acid- or
antibody-based probes combined with optical, most often fluorescence, transduction, or it
involves adapting separation-based technology like mass spectrometry.
The sensitivity of detection of nucleic acids can thus be greatly improved by nucleic
acid amplification. The polymerase chain reaction (PCR) takes time, and a major aim of
current research is to shorten the time to approach real-time amplification. Idaho
Technology's LightCycler, one of the fastest presently on the market (Wittwer et al.,
1997), can carry out 30 cycles in 6 minutes by using tiny glass capillary tubes for the
sample and high-velocity hot and cold air. RNA can be converted to cDNA by reverse
transcriptase (RT) and thus amplified by PCR. The time required for conversion to cDNA
is also a subject of active research. Several new amplification methods do not require heat
cycling. These include Transcription-based Amplification System (TAS) (Kwoh et al.,
1989), Self-Sustained Sequence Replication (Guatelli et al., 1996), and Strand
Displacement Amplification (SDA)(Walker et al., 1992).
In general, however, degradation of the nucleic acid probes and interference from
related sequences or products from the microbial environment significantly limit the
current application of this technology beyond well-equipped and experienced laboratories.
A single microbial cell can be detected in the laboratory from highly purified DNA by
these methods, but environmental samples have regularly failed to achieve this, usually
having a detection limit of 105 microbes. PCR detected 100 percent of spiked samples in
one study (Candrian , 1995), but only 15 percent of naturally infected samples.
Considerable effort is being made at Lawrence Livermore National Laboratory (LLNL) to
solve these problems and combine nucleic acid-based assays with antibody-based tests in
an automated field-deployable system (Mariella, 1998; Belgrader, 1998). Miniaturized
PCR units with significantly reduced cycling times have also been developed by a
partnership of USAMRIID, LLNL and the California biotech company Cepheid, Inc.
(Ibrahim et al., 1998; Belgrader et al., 1998; Northrup et al., 1998). The long-term goal of
this work is a hand-held instrument featuring disposable cartridges containing all
necessary reagents, reaction chambers, waste chambers, and microfluidics to extract,
concentrate, amplify, and analyze nucleic acids. Concurrent efforts at sequencing the
genes of possible biological warfare agents and identifying organism-unique probes are
under way at Army (USAMRIID) and Navy (NMRI) laboratories (Farchaus et al., 1998;
Higgens et al., 1998), LANL (Keim et al., 1997), LLNL (Andersen et al. 1996), the
University of Texas-Houston (Hoffmaster and Koehler, 1997), and Duke University
(Harrell et al., 1995), so piggybacking onto a commercial market that Cepheid estimates
at over $1 billion seems feasible.
The one-step hand-held tickets described above that are produced at NMRI and more
recently by Environmental Technologies Corporation are an example of immunoassay
technology combined with chromatographic transduction. The sensitivity of these simple
devices is much lower than that achieved in clinical laboratories, but they are inexpensive
and easy to use. For those reasons, they are probably the most logical choice for Hazmat
teams and other emergency responders seeking to test the contents of a suspicious
package for the presence of the dozen of so agents on the military threat list. The Analyte
2000 is another well developed (but not yet commercially available) immunosensor. The
Naval Research Laboratory developed this device, which combines antibody probes with
a fiber-optic waveguide transduction system. Other work is focusing on miniaturizing and
automating the testing process, incorporating the requisite antibodies with optimal
sampling and transducer technology, and producing antibodies against specific biological
agents and strains. Scientists at the University of Texas, Austin (Daugherty et al., 1998;
Georgiou and Iverson, 1998) are taking the last of these areas one step further, reducing
the size of anthrax antibodies to that fragment of the light chain actually binding the
antigen, identifying the relevant amino acids at the binding site, and making systematic
substitutions to achieve higher affinity and selectivity.
The previously described device under development at Utah State University for
detection of total pathogenic microbes (including spores) is an example of a ligand-based
probe with fluorescence-based transduction. A hand-held unit simply using fluorescence
to determine total viable microbes requires no physical contact with the samples and no
specialized expertise to use, but it can provide detection in seconds with a sensitivity of
~100 cells. In this respect, it is useful for determining contamination on objects and from
environments where it is difficult to obtain samples.
Three substantial R&D efforts are currently under way that focus on mass
spectrometry (MS) for identifying biological agents. DoD is close to fielding a truck-
portable Chemical Biological Mass Spectrometer (CBMS) and already has research under
way at Oak Ridge National Laboratory (ORNL) for a second-generation unit that is
lighter, faster, and more sensitive (Wayne Griest, personal communication to FJ Manning,
1/23/98). Although very expensive compared to most portable chemical or biological
detectors and dependent on a rapid and efficient separation system, the name underlines
an important advantage of this approach--the potential for a single instrument that will
detect both chemical and biological agents, industrial and naturally occurring as well as
military. Unlike many of the current test systems and detectors, such an MS-based
detector could be used in a whole gamut of Hazmat situations rather than as confirmation
of a hypothesis about a possible agent. The instrument's versatility would be limited only
by the size of the existing library of mass spectra.
Although MS has the potential to identify infective agents and recent advances have
significantly reduced the size of the device, libraries of unique signatures of agents have
not been determined. In addition, it is not clear that these signatures can be distinguished
in a natural environment containing signatures of large amounts of other microbes,
especially at concentrations near infectious-dose levels.
The committee does not see routine monitoring in the manner of smoke detectors (i.e.,
without some independent reason to suspect an attack) as either feasible in the foreseeable
future or worthy of a high-priority effort to develop that capacity, but there may be times
and places where pre-incident intelligence may suggest temporary deployment of existing
military monitoring systems.
Given the delayed effects of the biological agents, it is also difficult to envision many
situations that would demand highly sensitive biological detection by first responders. The
ability to determine total viable microbes present, total pathogenic microbes, and specific
viable pathogens will likely cover the needs presented by both overt and covert "events"
as well as provide monitoring and early warning. The ERDEC/EnViron virus detection
system might prove to be a useful complement to a ligand probe system for detecting total
pathogenic bacteria and handheld immunoassay tickets in a multistage approach
beginning with the very general and progressing to the highly specific as required. The
alternative might be a miniaturized mass spectrometer of the sort being developed at
Hopkins/Maryland or Oak Ridge to be a generic chemical and biological identifier.
Although prototypes are decreasing in size and weight, the real challenge lies in the
development of a library of unique signatures for biological agents in the presence of
large quantities of other microbial contamination and interferents, in addition to the
achievement of infectious-dose-level sensitivity.
In the area of patient diagnostics, there is a clear need for methods capable of detecting
infective dose levels (e.g., 10—100 cells or virions) of most biowarfare agents at a speed
that allows for effective therapeutic strategies to be administered (e.g., antibiotics,
vaccination, supportive therapy). Furthermore, these new methods must also be able to
detect "friendly" microbes that have acquired virulence factors by natural or genetic
engineering methods and those that have been microencapsulated to disguise their identity
(such as the detection of virulence factors or toxin production). Ideally, this technology
will be incorporated into a diagnostic system capable of identifying many more common
pathogens, assuring frequent use of the system and eliminating the need for clinicians to
make a specific request for a very seldom-used assay.
The committee therefore has identified the following research and development needs:
6-1 In the area of patient diagnostics, the Public Health Service should encourage federal
research agencies to leverage burgeoning commercial development of faster, cheaper,
easier assays of common pathogens rather than independently developing diagnostic
technology for the less common pathogens thought to be good candidates for
bioterrorism.
6-2 In the area of environmental detection, the Public Health Service should closely
monitor military biodetection R&D efforts for inexpensive or multipurpose biodetectors
that might be appropriate for purchase or loan by civilian agencies rather than
developing threat agent-specific assays from the ground up.
6-3 Both of these leveraging efforts will require the federal government to conduct or
support:
Basic research to identify characteristics which might be used to develop more
effective probes and/or enhance probe performance for known biowarfare agents and
especially genetically altered microbes. Understanding of microbial metabolism,
sporulation, toxin production and excretion, regulation of virulence factors, and
bacteriophage interaction are crucial in this respect. New approaches for preventive and
therapeutic strategies are also likely from this basic understanding.
Scenario-specific testing of detection performance and comparisons under standard
conditions for characterization of the sensitivity, specificity, reliability, response
constraints, and usability (ease of use, cost, robustness, useful life, response time, and
human effort and experience required).