Sampling Strategies and Technologies
SAMPLING AND IDENTIFICATION
Substances that are believed to be biological weapons agents can be extracted for analysis from samples collected with surface wipes, vacuum filters, air filters, and liquid reservoirs and agar plates with air-impacted material, or from bulk solid or liquid materials that have been exposed. First responders to an incident are likely to use portable collection, detection, and identification devices or kits for the rapid characterization of those agents. Generally, the samples are hydrated and introduced to the detector kits to obtain a colorimetric or electronic display for rapid identification. The committee considered post-attack sampling as a source of data that would inform the assessment of the extent and degree of contamination, identify morphological changes in the substance, and monitor the effectiveness of decontamination. All of those phases of identification require confirmatory data and analysis. None should be limited to rapid identification systems.
Confirmatory procedures can be done by mobile on site laboratories or by the Centers for Disease Control and Prevention’s (CDC) Laboratory Response Network (LRN). It is important to consider the goal of the sampling, and to match it to the proper procedure. For example, polymerase chain reaction (PCR) sampling can provide information about whether DNA from an agent of concern is present, but it can not provide information on whether that agent is alive or growing in a facility.
In 1999, the LRN was established to respond to acts of biological and chemical terrorism. The LRN system has expanded significantly since its inception, and
it now consists of 120 state and local public health, veterinary, and military laboratories and international laboratories of those types, that normally perform public health analyses. Collectively, the facilities are equipped to respond quickly to acts of biological terrorism, emerging infectious disease, and other public health threats (CDC, 2004). The LRN national network of laboratories includes:
Federal—These include laboratories at CDC, the U.S. Department of Agriculture, the Food and Drug Administration, and other facilities run by federal agencies.
State and local public health—These are laboratories run by state and local departments of health. In addition to being able to test for Category A biological agents, a few LRN public health laboratories are able to measure human exposure to toxic chemicals through tests on clinical specimens.
Military—Laboratories operated by the Department of Defense, including the U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID) at Fort Detrick, Maryland.
Some laboratories are designated as national, reference, or sentinel, according to the tests they perform and how they handle infectious agents to protect workers and the public.
National laboratories include the CDC and USAMRIID. They have unique resources to handle highly infectious agents and the ability to identify specific agent strains.
Reference laboratories, sometimes referred to as “confirmatory reference,” can perform tests to detect and confirm the presence of a threat agent. Reference laboratories are intended to provide local authorities with effective laboratory support on a timely and responsive basis, so that they do not need to rely on CDC support for all cases.
Sentinel laboratories represent the thousands of hospital-based laboratories that are on the front lines. Sentinel laboratories have direct contact with patients. In an unannounced or covert terrorist attack, patients provide specimens during routine patient care. Sentinel laboratories could be the first facility to spot a suspicious specimen. A sentinel laboratory’s responsibility is to refer a suspicious sample to the right reference laboratory.
Bacillus anthracis has been identified as an agent used by bioterrorists. Its spores cause disease readily, and it is resistant to most adverse environmental conditions, such as extremes in temperature, sunlight, and drying (Bohm, 1990).
B. anthracis spores can live for decades without loss of viability (Turnbull, 1990). But its unique colony morphology and relatively fast growth in culture make its identification straightforward. In contrast, plague bacterium (Yersinia pestis) and smallpox virions are more fragile and susceptible to damage. PCR testing can be used definitively for positive identification of the agents of concern in this report. However, PCR testing does not differentiate live and dead agents; it merely provides DNA evidence that an agent is present. Consequently, in the case in which viability is important and the ability to grow samples in culture becomes important, the choice of sampling and preservation methods is crucial.
Specific sampling protocols should follow a General Sampling Plan, described later, which is developed by stakeholders, laboratories, and representations of public health and law enforcement agencies. The plan should allow for several factors that can complicate sampling for identification:
Suitability of the antibody, DNA, or RNA probes against a variety of agents and strains in terms of their specificity, microbial stability, and handling requirements (refrigeration).
Background false alarms caused, for example, by more than 200 microoganisms that have been collected in single-office environments, and many of them could be mistaken for biothreat agents because of preliminary information that could suggest that B. anthracis is present when the agent is in fact B. subtilis or B. cereus.
Background suppressions caused by other substances (oils, cleaning solutions) and high background biological loads can compromise the functionality of samplers and sensors.
Growth and morphological changes over time of the agent in high humidity, closed areas, crevices, and mechanical spaces.
Redistribution of the agent by normal air circulation and turbulence and by the movement of personnel and equipment after an attack.
Uncertainties about persistence, cost, time, safety, environmental, and treaty concerns. It is difficult to perform exhaustive, conclusive tests in the presence of factors that affect the viability of biological threats. There is great uncertainty about the way different substances react to humidity, ultraviolet light, and temperature. For example, it is well known that the smallpox virus is not as susceptible to high humidity as are some other virus species. The 1969 outbreak of smallpox in the hospital in Meschede, Germany (Wehrle et al., 1970) demonstrated that, even though the index patient was isolated, 19 other patients and medical care personnel were infected over a 1 month period, and 4 died. The spread of the virus was attributed to the aerodynamic stack effect of the stairway between three floors and the leakage from an open window. One patient died who had been located in a room two floors above the index case. Fever and rash occurred as long as 33 days after the end of the presumed infectious period of the index case.
GENERAL SAMPLING PLAN FOR QUANTIFYING THE EXTENT OF CLEANUP
Sampling after an attack and after the initial identification is intended to provide data to define the extent of contamination and the risk for responders and building occupants. A general sampling plan for a contaminated facility can provide a roadmap for verifying the results of decontamination. Cleanliness criteria can be developed by a team that includes facility stakeholders; medical, public health, and environmental experts; decontamination technologists; laboratory analysts; and worker safety representatives. According to the building function and the acceptable risk derived from the team’s consensus agreement, the sampling plan should be reviewed by representatives of local, state, and federal agencies and by laboratory analysts; facility managers; structural engineers; and heating, ventilation, and air-conditioning (HVAC) engineers. The plan of action and milestones for sampling depend on the biological threats and contaminants present. Spores are hardier and more persistent than are vegetative cells, so spore sampling and retrieval conceivably could be more direct than would be possible if more fragile and dynamic vegetative material were the cause for remediation. Virions are more difficult to obtain and preserve for characterization because of their mechanical fragility and sensitivity to temperature and pH.
The systems engineering discipline can help define the sampling subsystems, interfaces, and tradeoff analyses in the general sampling plan. The subsystems definition will follow along the lines of bulk sampling, surface sampling, and air sampling with interfaces to training and guidance to the sampling staff, microbial analysis plans for the identified agents, data management, and sample archiving. Tradeoff analyses should weigh the different sampling objectives and form the basis for the selection of sampling methods, background characterization instruments, sampling schedule, analysis architectures, and sampling results as input to calculations for exposure risk assessments.
In the development of the plan, several other factors should be considered:
Sample handling protocols would provide procedures that ensure measurement of the physical distribution of the contaminant, and preserve viable material for culture inoculation (White and Fenner, 1994) or viral plaque assay (Litts, 2000; Sambrook and Russell, 2001) for identification and an assessment of virulence. A priori knowledge of the choice of assay will help dictate which wetting agents and elution techniques will be compatible with or detrimental to analysis. For example, salts (such as phosphate-buffered saline) can inhibit PCR efficiency. There are variations among vendor kits for sample cleanup (for example, Idaho Technology, 2004, Qiagen, 2003).
Laboratory capability will determine the ability to work with the proposed sample media (wipes, agar, bulk), analysis throughput, level of analysis, and storage.
Collection efficiency and the anticipated cost of the method would need to consider the number of samples to be analyzed and the need for on-site quantification.
Risk assessment utility would need to be identified for the sampling method to prove the usefulness to the building owner or the agency that has jurisdiction over the project.
Other potential uses of collected samples should be considered if they would require special handling. Special attention would be given to samples for use by law enforcement agencies if maintaining the chain of custody is important.
The general sampling plan should have clearly defined objectives; acceptance criteria for sample and analysis data; a calculation of the number and types of samples desired; and a microbial analysis plan and a risk assessment plan, each statistically rigorous. It is well accepted that several sampling strategies will be required to achieve different objectives (National Response Team [NRT], 2003). Potential sampling objectives might include the following:
Preliminary screening of a facility. The objective is to determine the extent of contamination and the viability of pathogens. Composite samples from large surface areas and air volumes are obtained to maximize the likelihood of finding contamination.
Identification of threat agents in bulk material. The objective is to determine qualitatively whether bulk material, such as dust in HVAC elements or powder in an envelope, is contaminated. Such sampling is also a tool for screening and evidence collection.
Determination of contamination of an article. The objective is to determine whether the surface of an article, such as a book or a telephone is contaminated. Typically, composite surface samples of large articles or individual samples of small articles are collected.
Extent and location of contamination (site characterization). After the hazardous contaminant is positively identified, further sampling is necessary to determine how far the contamination has spread and what pathogens are still viable. Sampling is performed to determine qualitatively and, if possible, semi-quantitatively, the extent and magnitude of contamination. Walls, floors, equipment, and air-handling systems should be sampled. Sampling results also should be used to establish exclusion zones for site control and decontamination.
Efficiency of decontaminations. Biological samplers are used with positive controls, such as spore strips and biological reservoirs (canisters, envelopes, open surface plates) that contain known concentrations of threat proxy microorganisms, along with environmental monitors, such as particle size distribution instruments and background chemical analyzers (gas chromatograph–mass spectrometer to ensure the absence of suppressants).
Sampling and analyses performed after an attack should be conducted in three phases, within the context of the general sampling plan.
Phase 1: Confirmation and contamination baseline. The specific nature of the contaminants—identification, microbial stage, and pervasiveness in the building environment—should be characterized and recorded to establish a baseline from which to determine appropriate decontamination approaches. Any substances that might cause suppression or false positives in microbial assays should be identified and quantified before cleanup and disposal actions are undertaken. The baseline determination could lead to gross decontamination of large concentrations of agent with high-efficiency particulate air (HEPA) vacuuming before large-scale remediation begins.
Phase 2: Assessment and characterization. As initial disposal of contaminated materials begins, cross-contamination and contaminant redistribution are inevitable. Regular sampling is necessary not only to follow the progression of the contaminant migration but also to test for contaminant attenuation, reaerosolization, morphological change, and growth potential. Analysis in phase 2 will inform the process of selecting the appropriate decontamination approach and bracket decontamination risk and expectation.
Phase 3: Decontamination effectiveness. Along with the use of positive controls, sampling in phase 3 will quantify the effectiveness of decontamination, verify the extent of residual contamination, and provide data for re-occupancy decisions.
Using Regular Sampling Intervals
Ideally, cost and laboratory support notwithstanding, regular sampling of the contaminated site should begin concurrently with specimen testing of exposed personnel and patients. The sampling schedule for analysis of—air, surfaces, machinery, HVAC, and electronics is necessary for several purposes:
Establishment of initial loading baselines
Analysis of agent decay rate and attenuation
Analysis of diffusion and reaerosolization
Analysis of redistribution of concentrations
Discovery of new incubation sites
(Presented solely to provide examples of the products available. No endorsement is implied.)
Redline Alert™ (Tetracore) is an immunochromatographic handheld ticket-type assay that uses lyophilized antigen to indicate by color change positive and negative responses to B. anthracis. The color change occurs within a few minutes.
BV™ Test (BioVeris Corporation) is a small test kit that is based on a sandwich immunoassay format. One antibody (specific to the pathogen) is immobilized onto microparticles and mixed with reagents to form a mixture that is transported to an electronic device to emit light. Test kits are available for anthrax, botulinum neurotoxins A, B, E and F, ricin, and staphylococcal enterotoxins A and B.
HANAA (Handheld Advanced Nucleic Acid Analyzer) (Lawrence Livermore National Laboratory) is a handheld unit that can test 4 samples at a time, based on PCR thermocycler techniques. The test amplifies agent-specific DNA fragments in less than 30 minutes.
Bio-Seeq™ (Smith Detection) is a handheld unit that can test 6 samples simultaneously, based on PCR thermocycler technology. It can give an identification in less than 30 minutes.
The sampling interval will depend on the morphology and projected stability of the agent. For example, vegetative cells will require more frequent sampling; spores and toxins might permit longer intervals. The biological agent might have been weaponized or altered in some way, such as by encapsulation. This paradigm will provide useful information for later decontamination selection and procedures. At least 3 identical samples should be taken at each site: the first for handheld kits/sensors for rapid identification, the second for on-site presumptive laboratory analysis with more sophisticated tools, and the third for confirmatory analysis at a designated LRN facility.
On-site rapid identification can be accomplished by one or several of sensors and kits for a small range of biological agents. Commercially available test devices (not endorsed, but listed in Box 9-1 for illustration) are available for many agents. Those devices use a variety of identification technologies and vary widely in their sensitivity and specificity.
Laboratory presumptive testing can follow sentinel (formerly Level A) laboratory procedures. The American Society for Microbiology (ASM) has agreed to take the lead in the development and dissemination of sentinel laboratory infor-
mation (ASM, 2004). The only agent specific guideline, for staphylococcal enterotoxin B, was published in January 2004.
Many of the new ASM guidelines for B. anthracis will rely on CDC guidance (CDC, 2001a). CDC recommends presumptive test procedures that include micromorphology by gram stain; microscopic observation of capsule; and routine culture for colonial morphology, hemolysis, motility, and sporulation. Confirmatory procedures include lysis by γ-phage, direct fluorescence assay, antimicrobial susceptibility testing, and the use of advanced technology tools such as time-resolved fluorescence, and PCR testing.
Human specimen samples from, and clinical observation of, people who are potentially exposed also can be part of the general sampling plan. CDC has established laboratory test criteria for the clinical diagnosis of plague (CDC, 2001b) that include clinical and laboratory conditions to be met for suspected, presumptive, and confirmed cases. CDC states (CDC, 2001a) that “serologic tests for potential exposure to B. anthracis are currently being evaluated and at this time their clinical utility is not known.”
Guidance developed by the International Commission on Microbiological Specifications for Foods (http://www.foodscience.afisc.csiro.au/icmsf.htm) to document the design of sampling plans for microbial pathogens in food also could be useful to consider in this context.
Close consultation with laboratory personnel is vital for planning effective sampling (Martyny et al., 1999). The type of material to be tested, the biological agents sought, information needed about the agents, and the expected results determine the appropriate collection method. Laboratory and field staff should discuss how much material is required to conduct particular assays, which wetting materials are to be used, the number of samples needed to obtain representative results, the number of samples the laboratory can handle in a day so that sample processing is not delayed beyond an acceptable holding time, and required sample storage and shipping conditions.
Samples to be tested for viable microorganism counts generally require overnight delivery and either must be chilled (maintained between ~4°C and 10°C) or kept at room temperature but protected from extremely high or low temperatures during transport. It must be determined whether samples should be sustained within the physiological pH range (7.0-7.4). In some cases, preservatives or an agent that neutralizes a biocide (sodium thiosulfate for chlorine in water samples) is added in the field to stabilize samples and limit changes before analysis. Some viral agents might need to be stored in an appropriate broth to determine virulence. Culture plates also can be inoculated at the collection site before samples are shipped, and convenient dipslides are available for some types of water testing.
Bulk samples can provide information about contaminated regions, microbial sources, and material for later correlation with diagnostic evaluation of people who have been exposed to the agent, although these samples should not be considered good estimators of actual or potential exposure. Bulk samples use portions of environmental materials (settled dust, sections of wallboard, pieces of duct lining, carpet segments, return air filters). It should be noted that, depending on the nature of the potential contamination, the samples should be sent to a laboratory equipped to handle pathogens of the appropriate biosafety level.
The objective of bulk sampling is to collect a portion of material small enough to be transported conveniently and handled easily in the laboratory that still represents the area or object being sampled (Martyny et al., 1999). Testing can be done to determine whether organisms have colonized the material and are actively growing and to identify surfaces where previously airborne contaminants have deposited and accumulated. Some infectious agents that are present in low numbers are difficult to culture from surface or air samples (to some degree because of low extraction efficiencies) and might be best identified from bulk sources (Burge and Solomon, 1987).
Settled dust or dust entrained in return-air filters might contain previously airborne biological particles that provide a more representative picture of exposure than would short-term air samples. Dust collected on return-air filters augments the assumption that collected particles would reflect the airborne materials to which building occupants had been exposed (Burge and Solomon, 1987). Filter deposits also can serve as a growth substrate if filters become damp. However, bulk samples cannot be used in place of air samples: Bulk samples do not accurately reflect past, future, or even current bioaerosol exposures. Researchers who have collected parallel bulk and air samples have seen differences that reflect the presence of different biological agents on surfaces and in the air (Fox and Rosario, 1994).
Bulk samples are cut or otherwise aseptically removed from a source and placed in clean, new or sterilized containers, usually sterile jars for dry items and sterile bottles for water samples. Sealable plastic bags are useful for samples of ventilation duct lining, ceiling tiles, wallpaper, and similar materials. To preserve the integrity of samples and avoid cross-contamination, paper bags can be placed in plastic bags with a packet of desiccant material to keep the sample dry. The amount of sample to collect and the manner in which to remove and transport it will depend on the sample type and the analytical methods to be applied.
Samples of loose material, such as carpet dust for antigen detection, can often be collected with a vacuum device fitted, for example, with HEPA filters and bags.
Depending on how the results will be used, individual areas can be sampled separately or samples can be combined (during collection or in the laboratory). Making composite samples from large areas reduces the number for analysis and can improve the likelihood of detecting the material of interest. Sometimes a square meter of test surface is sampled or a prescribed area is vacuumed for a specified period. The results from the sampling are presented as the amount of biological material per gram of dust for that area. Alternatively, an entire room or building might be vacuumed and the results reported as the average concentration of biological agents in the dust collected for that unit.
Surface sampling can be done with wipes, swabs, and HEPA vacuum socks. It is preferred over bulk sampling when a rapid, less costly, and less destructive method of sample acquisition is desired. Along with air sampling, surface sampling allows repeated measurements within the sampling phases mentioned earlier. Because of the three-dimensional nature of surfaces amenable to contamination (walls, ceilings, floors, furniture, equipment, duct work), surface sampling can be used to analyze the initial spatial distribution and the dilution of substance, the redistribution and reaerosolization of contaminants during consequence management, and the effectiveness of decontamination. The concentration and character of biological substances on surfaces depend on many factors, such as particle or droplet size, precipitation rate, and surface affinity. The surface material, history of exposure to moisture, exposure to ultraviolet light, temperature, indoor air circulation, and exposure to background chemicals are additional factors.
The consequence management and decontamination activities resulting from the B. anthracis attacks on government and commercial facilities in 2001-2002 have provided a basis for discussion of the merits and uncertainties of surface sampling. The remediation resulted in the issuance of interim guidance for environmental sampling from CDC (CDC, 2002a) and the U.S. Postal Service (USPS).
The preface of “Comprehensive Procedures for Collecting Environmental Samples for Culturing Bacillus anthracis” (CDC, 2002b) states that:
Currently, no occupational or environmental exposure standards exist for anthracis spores. In addition, there are presently no validated sampling and analytical methods specifically for B. anthracis in environmental samples. Data are lacking on collection efficiency of the sample collection media (swabs, wipes, filters, etc.) for typical porous and non-porous surfaces encountered in indoor environments (e.g., furniture, carpet, letters, clothing, ventilation system filters). The effect of varying concentrations of B. anthracis-containing particles and dust loading on sampling efficiency has not been studied. Further, the recovery efficiency of the analytical methods (efficiency of removal of B. anthra-
cis spores from the sample collection media) has not been adequately evaluated and limits of detection have not been established.
Providing that objectives are attainable, a general sampling plan can offer guidance for obtaining useful information. The CDC website and 13-page environmental sampling guidance are consistent with information presented to the committee about the successful sampling efforts by the Armed Forces Radiobiology Research Institute (AFRRI, Dr. Greg Knudson) for environmental testing at the Brentwood mail facility, and by the Bio-One solutions LLC (John Mason) for decontamination effort at the American Media, Inc. (AMI) building. The CDC guidance addresses the overall planning (training, safety, record keeping, and documentation); sampling strategy (bulk sampling, surface sampling with wipes or swabs, surface sampling by HEPA vacuuming, air sampling); sample handling (packaging and shipment, sample analysis, sample interpretation); and specific collection procedures, materials, and equipment (bulk sampling, surface sampling with wipes and swabs, surface sampling by HEPA vacuuming, air sampling).
Another important feature of the CDC publication is its emphasis on sample logging (location, time, date, area size, map of sample areas, person collecting) and chain of custody. It is clear that recommendations for procedures are evolving, with the need for peer review and consensus particularly for the threat substances with microbial features inconsistent with anthrax spores.
Surface sampling plans should be reviewed and technical improvements should be identified and subjected to peer review. Skolnick and Hamilton (2004) have commented that the 2001-2002 procedures appear essentially “ad hoc,” lacking reference to earlier published works such as the NASA spacecraft testing activity for planetary protection (NASA, 1980). Different agencies recommend different collection procedures, and there are ambiguities in some documents. Areas of divergence include recommendation for swab or wipe material (Dacron or Rayon versus cotton), areas to be sampled (the guidance area is varied and generally too big to avoid overloading by surface debris), collection stroking (vertical, horizontal, rotational combinations), and choice of wetting agent (sterile water, phosphate-buffered saline). Skolnick and Hamilton pointed out several inconsistencies in swab-and-rinse assay procedures specified in interim guidance documents issued by the U.S. Postal Service and the CDC
There is disagreement about whether dry or wet swabs are more effective for surface sampling. The reviewed procedures provide no justification for the use of dry swabs in swab-rinse environmental testing. Moreover, the interagency Brentwood study leads Skolnick and Hamilton (2004) to consider the dry swab data unreliable. In Chapter 3 of this report, the committee cites problems with the use of dry swabs at the Wallingford Connecticut Postal facility, where their use led to a false negative. Subsequent sampling at Wallingford with wet wipes
showed positive results. The results and the reports by AFRRI and BioOne Solutions led this committee to conclude that dry-swab or dry-wipe surface sampling should be abandoned in favor of wet-wipe surface sampling. However, there are few quantitative data for the collection efficiency and biological viability of wet-wipe techniques.
Another area of disparity involves the use of detergent as an additive to the sample rinse to aid spore extraction. USPS did not incorporate its use in its swab-rinse procedure. The volume of rinse used to extract the swab also was different—CDC recommends a 3 milliliter (mL) solution; USPS used a 1.5 mL.
The fraction of the total extract volume inoculated onto culture plates was also differed. CDC used a 1 in 10 ratio; USPS used 1 in 15. Both methods cultured too little of total extract volume for use as a “rule out” assay that should be maximally sensitive to support a “zero” tolerance policy—a policy that itself should be reassessed (Skolnick and Hamilton, 2004).
The authors also question the number of culture plates inoculated per sample (CDC used 3 versus USPS used 1). The culturing of a single plate provides no measure of variation and is not considered good laboratory practice.
A new generation of wetted sponge sampler kits (Edgewood Chemical Biological Center [ECBC], 2004) offers potential improvements in wiping, handling, storage, and elution. The new kit should be tested and evaluated by experts for each class of pathogen (with taxonomic order, for a wide range of environmental backgrounds, and for their compatibility with different assay techniques). Buttner and colleagues (2001) have evaluated the collection efficiency of wetted swabs and wetted sponge wipes for removing B. subtilis spores from vinyl floor tile, and from soiled carpet. In trials with and without background contamination with P. chrysogenum the criteria for efficiency included the physical sampling loss (up to 7%) and the loss from the microbial analysis (about 25%). The resultant efficiencies were reported as varying from 67% for wet cotton swabs to 74% for the wet sponge wipe. Quantitative PCR analysis was compared with culture analysis, and there was, a discussion of the inhibition of microbial growth in the analyses attributable to non-biological contaminants in the carpet. However, the wetting agent, the volume of extraction rinse, and the amount inoculated onto the culture plate were different enough from those used in other investigations to make comparisons difficult.
The physical principles of particulate sampling are well established, and the adaptation to sampling for biological agents is rapidly maturing. Air sampling is particularly useful in the determination of biological load within a dynamic environment, where the circulation of the air and the presence of more than one contaminant are factors. Common to all aerosol sampling is the need for efficient
collection that aids quantification of the aerosol concentration—a critical parameter estimating exposure.
Several varieties of air sampler are available, classified by the way in which they deposit particles for analysis:
Slit samplers have rectangular impaction nozzles that deposit particles onto an agar-based medium for incubation or onto a glass slide or tape strip that is examined through a microscope. The collection substrate can be stationary, or it can move continuously or periodically under the slit.
Centrifugal samplers use a particle’s inertial behavior—in a radial manner for agar strip impactors and cyclone samplers. Centrifugal sampling also is used to partition particles into a liquid for later analysis.
Liquid impingement samplers use a process that is similar to solid-plate impaction, but inertia forces the particles onto a surface submersed in or washed with liquid. All-glass impingers are widely used because of the gentle nature of physical disruption.
Filtration samplers take advantage of inertial forces, interception, gravitational settling, diffusion, and electrostatic attraction to separate particles from an air stream and deposit them on or within a filter. The filters usually are held in inexpensive cassettes attached to portable pumps. Dry-filter units often use polyester felt filters for frequent retrieval, although impaction and desiccation can reduce the viability of some pathogens. Some of the newer gel filtration systems (Sartorius, 2004) offer the potential for increased viable yield.
Generally, two main considerations involve the choice of nutrient agar for direct culture analysis—particles are forced onto an agar plate for incubation—or the use of filters for the trapping particulate matter before physical disruption and subsequent culture or spore analysis (counting, morphology). For the substance under investigation, research will be necessary to resolve the tradeoffs among the several inertial impaction methods.
The collection efficiency for air sampling is generally divided into three components (Willeke and Macher, 1999):
Inlet sampling efficiency is a measure of the ability of a sampling inlet to entrain particles from the ambient environment regardless of particle size, shape, or aerodynamic behavior.
Particle removal efficiency is a measure of ability to separate particles from the sampled air stream and deposit them on or in a collection medium.
Biological recovery efficiency is a measure of ability to deliver the collected particles to an assay system without altering their viability, activity, physical integrity, or other essential characteristics.
There are many commercially available collectors whose properties and
The following information is presented solely to provide examples of the products available. No endorsement is implied.
Several collectors can be used to capture particles in a broad range of sizes. The devices have 450-1000 liters-per-second flow rates and can be used to sample the air in a medium-sized room in hours rather than the days required by low-flow-rate devices:
If analysis of particles in a specific size range is required for a smaller area, such as the inside of an air duct during decontamination, it is more appropriate to use a size-segregating cascade device:
specifications (particle size range, flow rate, collection media) must be matched to the sampling plan and objectives (Box 9-2). For example, it can take 40 hours for smaller particles—in the range of 0.5 micrometers (µm) to 1 µm—to settle; 10-µm particles will settle in a few minutes. Initial assessment and characterization of the severity of an attack requires information about the size range of particles, and that process requires a calculation of how much is resuspended, for example, by large-volume blowers.
FINDINGS AND RECOMMENDATIONS
General Centers for Disease Control (CDC) sampling guidance exists for Bacillus anthracis spores, but there is no official guidance for the collection of vegetative B. anthracis, plague bacteria, or smallpox virions.
Sampling protocols must be appropriate to the threat. Sampling for B. anthracis spores should be done according to published guidance from CDC and the Na-
tional Institute for Occupational Safety and Health. The CDC and the American Society for Microbiology should develop sampling and analysis guidelines for the other threat agents. Other agencies (such as the EPA and the FBI) that may be involved in sampling also should be consulted.
Surface sampling with dry wipes led to false negatives at the Wallingford postal facility and to inconlcusive results at the Brentwood postal facility.
Dry-wipe and dry-swab surface sampling should be abandoned in favor of wet-surface swipe techniques. HEPA vacuum surface sampling should be continued as complementary to surface swiping.
Different threat substances require different sampling protocols. The variety of collection approaches currently in use results in widely varying collection and extraction efficiencies, which hamper attempts to quantify the initial extent of contamination.
Sampling and analysis should be standardized. Research should assess the efficiency of collection and analysis for each type of biological agent. Unless the sampling efficiency is known, the amount of contaminant deposited cannot be estimated with confidence.
There is consensus within the federal government regarding the value of a general sampling plan to guide the use of various surface-, air-, and bulk-sampling methods.
The general sampling plan should be the result of the consensus of facility stakeholders; medical, public health, and environmental experts; decontamination technologists; laboratory analysts; and worker safety representatives. It should encompass three phases: (1) confirmation and contamination baseline, (2) assessment and characterization, and (3) decontamination effectiveness. Some sharing of expertise will be necessary for all groups to be well enough informed to come to consensus.
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Buttner, M.P., P. Cruz-Perez, and L.D. Stetzenbach. 2001. Enhanced detection of surface-associated bacteria in indoor environments by quantitative PCR. Applied and Environmental Microbiology 67(6): 2564-2570.
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Fox, A. and R.M.T. Rosario. 1994. Quantification of muramic acid, a marker for bacterial peptidoglycan, in dust collected from hospital and home air-conditioning filters using gas chromatography. Mass Spectrometry. Indoor Air 4: 239-247.
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Martyny, J., K.F. Martinez, and P.R. Morey. 1999. Source Sampling. Ch. 12 in Bioaerosols: Assessment and Control, J. Macher, ed. Cincinnati, Ohio: American Conference of Governmental Industrial Hygienists.
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