    Anthrax Detection: Agencies Need to Validate Sampling Activities
    in Order to Increase Confidence in Negative Results (31-MAR-05,
    GAO-05-251).

    In September and October 2001, letters laced with Bacillus
    anthracis (anthrax) spores were sent through the mail to two U.S.
    senators and to members of the media. These letters led to the
    first U.S. cases of anthrax disease related to bioterrorism. In
    all, 22 individuals, in four states and Washington, D.C.,
    contracted anthrax disease; 5 died. These cases prompted Congress
    to ask GAO to describe and assess federal agencies' activities to
    detect anthrax in postal facilities, assess the results of
    agencies' testing, and assess whether agencies' detection
    activities were validated.
    -------------------------Indexing Terms-------------------------
    REPORTNUM:   GAO-05-251
        ACCNO:   A20538
      TITLE:     Anthrax Detection: Agencies Need to Validate Sampling
    Activities in Order to Increase Confidence in Negative Results
         DATE:   03/31/2005
      SUBJECT:   Chemical and biological agents
    	     Disease detection or diagnosis
    	     Diseases
    	     Health hazards
    	     Infectious diseases
    	     Interagency relations
    	     Laboratories
    	     National preparedness
    	     Postal facilities
    	     Strategic planning
    	     Terrorism
    	     Test facilities
    	     Testing
    	     Disease surveillance
    	     Homeland security
    	     Anthrax

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    GAO-05-251

    United States Government Accountability Office

        GAO 	Report to the Chairman, Subcommittee on National Security, Emerging
      Threats, and International Relations, House Committee on Government Reform,
                                House of Representatives

    March 2005

    ANTHRAX DETECTION

    Agencies Need to Validate Sampling Activities in Order to Increase Confidence in
                                    Negative Results

    GAO-05-251

    [IMG]

    March 2005

    ANTHRAX DETECTION

    Agencies Need to Validate Sampling Activities in Order to Increase Confidence in
    Negative Results

                                     What GAO Found

    The U.S. Postal Service, Centers for Disease Control and Prevention (CDC),
    and Environmental Protection Agency (EPA) conducted several interdependent
    activities, including sample collection and analytic methods, to detect
    anthrax in postal facilities in 2001. They developed a sampling strategy
    and collected, transported, extracted, and analyzed samples. They
    primarily collected samples from specific areas, such as mail processing
    areas, using their judgment about where anthrax would most likely be
    found-that is, targeted sampling. The agencies did not use probability
    sampling in their initial sampling strategy. Probability sampling would
    have allowed agencies to determine, with some defined level of confidence,
    when all results are negative, whether a building is contaminated.

                                           5

    Sample analysis

    Source: GAO analysis of CDC, EPA, and USPS data.

    The results of the agencies' testing in 286 postal facilities were largely
    negative-no anthrax was detected. However, agencies did not use validated
    sample collection and analytical methods. According to the agencies,
    validated methods were not available in 2001. Thus, there can be little
    confidence in negative results. Validation is a formal, empirical process
    in which an authority determines and certifies the performance
    characteristics of a given method. Consequently, the lack of validation of
    agencies' activities, coupled with limitations associated with their
    targeted sampling strategy, means that negative results may not be
    reliable.

    In preparing for future incidents, the agencies have (1) made some changes
    based on what has been learned about some of the limitations of their
    sampling strategies, (2) made some revisions to their guidelines, (3)
    funded some new research, and (4) planned or conducted conferences
    addressing some of the issues GAO has identified. In addition, the
    Department of Homeland Security (DHS) has taken on the role of
    coordinating agencies' activities and has undertaken several new
    initiatives related to dealing with anthrax and other biothreat agents.
    However, while the actions DHS and other agencies have taken are
    important, they do not address the issue of validating all activities
    related to sampling. Finally, the agencies have not made appropriate and
    prioritized investments to develop and validate all activities related to
    other biothreat agents.

                     United States Government Accountability Office

    Contents

      Letter

    Results in Brief
    Background
    Agencies Were Challenged by Problems Associated with Sampling

    Activities The Five Activities Involved Many Variables That Can Affect

    Results The Sampling Results Were Largely Negative Lack of Validation
    Raises Questions about the Reliability of

    Negative Results Agencies Have Taken Some Steps to Prepare for Future
    Incidents Conclusions Recommendations for Executive Action Agency Comments
    and Our Evaluation

                                           1

                                          3 9

                                           17

                                         45 46

    56 68 79 82 83

    Appendix I Objectives, Scope, and Methodology

    Appendix II 	Information on Sampling Events in Facilities with Positive
    Results

    Appendix III 	Comments from the Centers for Disease Control and Prevention

    Appendix IV 	Comments from the Department of Homeland Security

             Appendix V Comments from the United States Postal Service 108

      Appendix VI 	Comments from the Association of Public Health Laboratories 110

    Appendix VII Comments from the Department of Defense

    114

    Tables

    Table 1: Agencies' Sample Collection Methods for Initial Sampling,

    October 2001 through April 2002 Table 2: Total Samples Collected and
    Sampling Events Table 3: Delivery Bar Code Sorting Machines Sampled,
    Wallingford

    Facility, 2001 Table 4: Agency Evaluations, October 2001 through February
    2002

                                         27 46

                                         52 65

    Figures

    Figure 1: Agency Sampling Activities 18 Figure 2: Three Sample Collection
    Methods 29 Figure 3: Laboratory Analysis of Samples in Preliminary and

    Confirmatory Tests 37 Figure 4: Primary and Wallingford, Connecticut,
    Facilities:

    Sampling Methods and Results 48 Figure 5: Test Results Were Largely
    Negative 50 Figure 6: Lack of Validation Can Affect Individual Activities
    and the

    Overall Process 61

    Abbreviations

    APHL Association of Public Health Laboratories
    ATSDR Agency for Toxic Substances and Disease Registry
    BSL biosafety level
    CDC Centers for Disease Control and Prevention
    CFU colony forming unit
    DBCS delivery bar code sorter
    DHS Department of Homeland Security
    DNA deoxyribonucleic acid
    DOD Department of Defense
    EPA Environmental Protection Agency
    FBI Federal Bureau of Investigation
    HEPA high-efficiency particulate air
    HHA hand-held assay
    HHS Department of Health and Human Services
    HVAC heating, ventilating, and air conditioning
    LRN Laboratory Response Network
    NBACC National Biodefense Analysis and Countermeasures Centers
    NCID National Center for Infectious Diseases
    NIOSH National Institute for Occupational Safety and Health
    NJDHSS New Jersey Department of Health and Senior Services
    OSHA Occupational Safety and Health Administration
    OSTP Office of Science and Technology Policy
    PCR polymerase chain reaction
    P&DC processing and distribution center
    RODAC replicate organism detection and counting
    TSWG Technical Support Working Group
    USAMRIID U.S. Army Medical Research Institute for Infectious Diseases
    USPS U.S. Postal Service

    This is a work of the U.S. government and is not subject to copyright
    protection in the United States. It may be reproduced and distributed in
    its entirety without further permission from GAO. However, because this
    work may contain copyrighted images or other material, permission from the
    copyright holder may be necessary if you wish to reproduce this material
    separately.

    United States Government Accountability Office Washington, DC 20548

    March 31, 2005

    The Honorable Christopher Shays
    Chairman, Subcommittee on National Security, Emerging Threats,

    and International Relations
    Committee on Government Reform
    House of Representatives

    Dear Mr. Chairman:

    In September and October 2001, contaminated letters laced with Bacillus
    anthracis, or anthrax spores,1 were sent through the mail to two senators,
    Thomas Daschle and Patrick Leahy, and members of the media. The letters
    led to the first cases of anthrax disease related to bioterrorism in the
    United States. The postal facilities in New Jersey and Washington, D.C.,
    that processed the senators' letters became heavily contaminated.2 Other
    mail routed through these facilities, as well as additional ones in the
    postal
    network, also became contaminated. Numerous federal facilities in the
    Washington, D.C., area-the U.S. Supreme Court, Walter Reed Army
    Institute of Research, Department of Health and Human Services (HHS),
    and main State Department buildings-were also later found to be
    contaminated.

    The mail for these federal facilities was believed either to have come in
    direct contact with the contaminated letters or to have passed through
    sorting equipment at the postal facility that processed these contaminated
    letters. In all, 22 individuals contracted anthrax disease in four states
    (Connecticut, Florida, New Jersey, and New York) as well as in
    Washington, D.C. Five of these 22 individuals died.

    The threat of bioterrorism had been recognized for a considerable time in
    the United States, as well as internationally. Long before the anthrax

    1"Anthrax" in this report reflects commonly used terminology. Technically,
    the term refers only to the disease caused by the microorganism Bacillus
    anthracis, not the bacterium itself or its spores.

    2Anthrax contamination had been found earlier in several Florida postal
    facilities that processed mail for the American Media Incorporated
    building there. However, no letter containing anthrax was ever found.

    incidents, several hoax letters indicating the presence of anthrax had
    been mailed to federal and state agencies, as well as to private sector
    organizations. In calendar year 2000, the Federal Bureau of Investigation
    (FBI) responded to about 250 cases potentially involving weapons of mass
    destruction. Of these, 200 were related to anthrax, although all turned
    out to be hoaxes. Nevertheless, these events raised the possibility that
    facilities could become contaminated and would therefore have to be
    evaluated for environmental contamination. However, federal agencies have
    not been fully prepared to deal with environmental contamination, that is,
    anthrax released through the mail, including the potential for multiple
    dispersals in indoor environments. 3

    In this report, we respond to your request that we

    o  	describe and assess federal agencies' activities to detect anthrax
    contamination in the postal facilities;

    o  	assess the results of the federal agencies' testing in the postal
    facilities; and

    o  	assess whether agencies' activities were validated and, if not,
    discuss any issues that arose from the lack of validation and any actions
    they took to address these issues.

    This report follows our May 19, 2003, testimony on anthrax testing at the
    Southern Connecticut processing and distribution center (P&DC), known as
    the Wallingford facility, and it completes our work in this area.4

    To respond to your request, we interviewed officials from federal agencies
    involved in sampling the postal facilities, including the Department of
    Defense (DOD) and the Centers for Disease Control and Prevention
    (CDC)-specifically, CDC's Agency for Toxic Substances and Disease

    3According to the head of the Postal Inspection Service, more than 7,000
    hoaxes, threats, and suspicious letters and packages-an average of almost
    600 a day-were reported to his agency in the weeks following the first
    anthrax incident. As a result, nearly 300 postal facilities had to be
    evacuated.

    4GAO, U.S. Postal Service: Issues Associated with Anthrax Testing at the
    Wallingford Facility, GAO-03-787T (Washington D.C.: May 19, 2003). See
    also GAO, U.S. Postal Service: Better Guidance Is Needed to Improve
    Communication Should Anthrax Contamination Occur in the Future, GAO-03-316
    (Washington D.C.: Apr. 7, 2003), and U.S. Postal Service: Better Guidance
    Is Needed to Ensure an Appropriate Response to Anthrax Contamination,
    GAO-04-239 (Washington D.C.: Sept. 9, 2004).

    Registry (ATSDR), National Center for Infectious Diseases (NCID), and
    National Institute for Occupational Safety and Health (NIOSH).5 We also
    interviewed officials from the Environmental Protection Agency (EPA), U.S.
    Army Corps of Engineers, U.S. Postal Service (USPS), Association of Public
    Health Laboratories (APHL), public health and private sector laboratories,
    and experts on microbial detection in indoor environments. In view of the
    ongoing criminal investigation, we did not review the FBI's sampling
    techniques. However, CDC, EPA, and USPS provided us with some data on the
    FBI's testing.

    We reviewed documentation provided or developed by ATSDR, CDC, DOD, EPA,
    the Occupational Safety and Health Administration (OSHA), and USPS, as
    well as sample collection strategies, guidance, environmental collection
    and analytical methods and protocols, and test results data, that is,
    sample collection and analytical data collected by federal agencies, their
    contractors, and public health laboratories. We did not independently
    verify these data.

    We conducted site visits to some postal facilities affected by anthrax and
    some public health and private sector laboratories that were involved in
    analyzing samples. We also carried out literature searches and reviewed
    studies on sampling methods for detecting biological substances, including
    anthrax, on surfaces and in the air. (See app. I for additional details on
    our scope and methodology.) We conducted our review from May 2003 through
    November 2004 in accordance with generally accepted government auditing
    standards.

    Although we did not assess anthrax testing done after the 2001 anthrax
    incidents, we believe that the issue we identified concerning the need for
    validated methods and sound sampling strategies would apply to such
    testing in future. This is particularly evident given the consequences
    arising from the March 2005 incident involving facility closures following
    preliminary anthrax testing in the Washington, D.C., area.

    Results in Brief 	CDC, EPA, and USPS, the federal agencies involved in
    sampling the postal facilities in 2001 to detect anthrax, undertook
    several activities: (1)

    5For the purposes of our study, we report on CDC, EPA, and USPS sampling.
    However, ATSDR staff, working in coordination with CDC's NIOSH staff, were
    involved in the anthrax responses in Connecticut, Florida, New York, and
    Washington, D.C.

    sampling strategy development, followed by (2) sample collection, (3)
    transportation, (4) extraction, and (5) analysis of the samples. As we
    discuss below, neither these activities nor the overall process have been
    validated for anthrax testing. Consequently, the agencies were challenged
    due to limited information available for reliably choosing one method over
    another and no information on the limits of detection to use when
    evaluating negative results. The sampling strategy used by the agencies
    could not provide any statistical confidence with regard to the basic
    question: Is this building contaminated? Therefore, in the future, in the
    absence of a positive result, a different strategy is needed that will
    provide statistical confidence, at a defined level, to answer this
    question.

    The first activity involved agencies' developing a sampling strategy,
    which included deciding how many samples to collect, where to collect them
    from, and what collection methods to use. The agencies primarily used a
    targeted strategy: They collected samples from specific areas considered
    more likely to be contaminated, based on agencies' technical judgments.
    This strategy was reflected in agencies' site-specific plans and guidance
    and included judgments about where anthrax was likely to be found, such as
    a mail processing area.6 Such judgments can be effective in some
    situations, for example, in determining (1) the source of contamination in
    a disease outbreak investigation or (2) whether a facility is contaminated
    when information on the source of potential contamination is definitive.
    However, in the case of a negative finding, when the source of potential
    contamination is not definitive, the basic question-Is this building
    contaminated?-will remain unanswered.

    The agencies did not use probability sampling in their initial sampling
    strategy. 7 Probability sampling would have allowed agencies to determine
    whether the building was contaminated with some defined level of
    confidence-in case of a negative result. Agency officials gave several
    reasons for choosing targeted sampling. For example, for CDC, targeted
    sampling was the most expeditious approach for quickly identifying
    contamination in facilities to support public health measures such as

    6These judgments, according to CDC, "relied primarily on using existing
    law enforcement, epidemiology, and event details to identify locations
    where anthrax was most likely to be found."

    7A probability sample is taken from a population by some random or
    stratification method, so that each item in the population has a known,
    nonzero probability of being selected. For negative results, a probability
    sample allows for conclusions at specific levels of confidence about the
    entire population sampled.

    decisions on the need to provide antibiotics. For USPS, the number of
    samples it could collect was limited due to insufficient laboratory
    analytic capacity. In the future, it would be reasonable for the agencies
    to develop a sampling strategy that would allow them to make statistical
    inferences about the negative results when the source of contamination is
    not definitive. This is important, considering that low levels of anthrax
    could cause disease and death in susceptible individuals.

    The second activity involved the agencies and their contractors using
    different methods to collect samples during sampling events.8 While USPS
    generally used dry swabs to collect samples (the least effective method),
    CDC and EPA used multiple methods-dry swabs, premoistened swabs, wet
    wipes, and a high-efficiency particulate air (HEPA) vacuum-in various
    combinations or alone.9

    The third activity involved the agencies, or their contractors,
    transporting the samples to laboratories for analysis according to federal
    regulations for transporting "infectious substances." The regulations were
    designed to prevent an inadvertent release of anthrax rather than maintain
    the samples' biological integrity for testing. While anthrax spores are
    robust, compared with other pathogenic microorganisms, the extent to which
    various transportation conditions might have affected their
    viability-their ability to germinate, divide, and multiply-was not
    specifically validated, although the effects of temperature and
    ultraviolet light on spore viability have been reported in the literature.

    The fourth activity involved laboratory personnel extracting the particles
    from the sample material, using extraction fluids and procedures specified
    by the laboratory. However, because no sample extraction efficiency data
    were available, interpreting anthrax analytic results was problematic.

    8We use "sampling event" to refer to initial sample collection by a
    specific agency on a specific day and at a specific time in a specific
    facility. Multiple agencies collected samples on the same day in some of
    the same facilities; therefore, each agency's sample collection is
    considered a separate sampling event. As a result, there were more
    sampling events than the total number of facilities sampled.

    9Earlier, according to USPS officials, they collected other types of
    samples, such as wet wipes, to be analyzed by a portable, field-based
    analytic method, as well as two "quick tests," or hand-held assays (HHA)
    in one facility in Washington, D.C. And in multiple facilities in the New
    York area, CDC used only dry swabs, following a requirement by New York
    public health laboratories.

    The final activity involved analyzing the extracted material with specific
    analytic methods for preliminary and confirmatory identification of
    anthrax. Some problems were experienced during preliminary analysis
    because (1) knowledge of the limits of detection for the field-based tests
    was lacking and (2) there were not enough trained personnel to use these
    methods.

    The results of the CDC, EPA, and USPS testing in 286 postal facilities
    were largely negative. But negative test results do not necessarily mean
    that a facility is free from contamination, a conclusion that, according
    to CDC, was stated at the time of the testing. Results can be negative if
    (1) samples were not collected from places where anthrax was present, (2)
    the detection limit of the sampling method was greater than the actual
    contamination level, (3) not enough samples were collected, (4) not enough
    spores were recovered from the sample material, (5) analysis of the sample
    extract did not detect anthrax spores, or (6) anthrax was not present in
    the facility. Of 286 facilities, 23 tested positive. For 2 of these 23
    facilities, test results were negative at first but positive on a
    subsequent testing. However, in 1 of these facilities-the Wallingford,
    Connecticut, facility-it was not until the fourth testing that positive
    results were obtained.

    The federal agencies' activities to detect anthrax contamination were not
    validated.10 The significance of the lack of validation of the agencies'
    various detection activities was highlighted in our discussions with
    scientists and researchers who have worked on microbial detection in
    indoor environments. Their opinions differed on sampling methods and
    sample material appropriate for environmental sampling and the processes
    necessary for validating methods. The opinions of public health and agency
    officials involved in making decisions on responding to anthrax
    contamination also differed. Validation, as it is generally understood, is
    a formal, empirical process in which the overall performance
    characteristics of a given method are determined and certified by a
    validating authority as (1) meeting the requirements for the intended
    application and (2) conforming with applicable standards. Because the
    agencies did not use an empirical process to validate their testing
    methods, the agencies had limited information available for reliably
    choosing one method over

    10In commenting on our draft report, CDC stated that the Laboratory
    Response Network (LRN) confirmatory test assays for detection of anthrax
    were validated. However, we were not provided with supportive
    documentation of methodologies used for such validation.

    another and no information on the detection limit to use when evaluating
    negative results.11

    Without validation, the sampling activities could have been based on false
    assumptions. For example, the lack of validated sample collection methods
    means that it is not known how many spores a particular method will
    collect from a surface and, thus, which method is appropriate for a given
    situation. Using an ineffective method or procedure could result in a
    finding of no contamination when in fact there is contamination-a false
    negative. Because the sampling methods are not validated, it is not known
    to what extent they will underestimate contamination. Thus, in the case of
    a negative result, agencies would have no sound basis for taking public
    health measures for the occupants of the contaminated facility.

    Validating the overall process is important because operational and
    healthrelated decisions are made on the basis of testing results generated
    by that process. In addition, validation would offer assurance that the
    results of using a particular method, which is part of that process, are
    robust enough to be reproduced, regardless of which agency, contractor, or
    laboratory is involved. Thus, agencies and the public could be reasonably
    confident that any test results generated by a process that includes that
    method would be reliable and, in particular, that any negative results
    would mean that a sample was free from contamination (within the method's
    limits of detection).

    In preparing for future incidents, the agencies have (1) made some changes
    based on what has been learned about some of the limitations of their
    sampling strategies, (2) made some revisions to their guidelines to
    reflect some of this knowledge and experience or developed new ones, (3)
    funded some new research, and (4) planned or conducted conferences
    addressing some of the issues we have identified. In addition, the
    Department of Homeland Security (DHS) has taken on the role of
    coordinating agencies' activities and has undertaken several new
    initiatives related to dealing with anthrax and other biothreat agents.

    However, while the actions DHS and other agencies have taken are
    important, they do not address the issue of validating all activities
    related

    11In commenting on our draft, CDC stated, "methods were selected based on
    factors such as comparable and available knowledge, studies on fungal
    spores, and in view of the immediate need for emergency response."

    to sampling. Since the fall of 2001, studies have been performed, or are
    under way, that may contribute to the validation of the individual
    activities. Nonetheless, these studies address only some aspects of an
    individual activity rather than the overall process. Finally, the agencies
    have not made appropriate and prioritized investments to develop and
    validate all activities related to other biothreat agents.

    Accordingly, we recommend that to improve the overall process for
    detecting anthrax and to increase confidence in negative test results
    generated by that process, the Secretary of Homeland Security develop a
    coordinated approach. This approach would include working with agencies to
    ensure that appropriate validation studies of the overall process of
    sampling activities, including the methods, are conducted. Specifically,
    the Secretary should (1) take a lead role in promoting and coordinating
    the activities of the various agencies with technical expertise related to
    environmental testing; (2) ensure that a definition of validation is
    developed and agreed on; (3) guarantee that the overall process of
    sampling activities, including methods, is validated so that performance
    characteristics, including limitations, are clearly understood and results
    can be correctly interpreted; (4) see that appropriate investments are
    made in empirical studies to develop probability-based sampling strategies
    that take into account the complexities of indoor environments; (5) ensure
    that appropriate, prioritized investments are made for all biothreat
    agents; and (6) make sure that agency policies, procedures, and guidelines
    reflect the results of such efforts.

    DHS stated that while it has the overall responsibility for coordination,
    EPA has the lead role in responding to biological attacks. However, DHS
    stated that it will coordinate with EPA to ensure that appropriate
    investments are made to explore improved sampling. But concerning our
    recommendation about probability-based sampling strategies, DHS said that
    it first wanted to develop sampling requirements and then evaluate both
    targeted and probability-based sampling against those requirements. We
    believe that DHS's evaluation of sampling will result in a conclusion that
    probability-based sampling strategies are necessary to (1) answer the
    question-Is this building contaminated?-and (2) achieve DHS's goal of
    having a "scientifically defensible sampling strategy and plan."

    While CDC and USPS, as well as APHL, agreed with the importance of using
    validated testing methods, they raised various concerns about our
    discussion of validation or targeted versus probability-based sampling.
    While we clarified our discussion to address these concerns where
    appropriate, we continue to believe that our findings on the need for

    Background

    validated testing methods and probability-based sampling strategies are
    well supported by the evidence, which we have presented in our report.

    Although anthrax can infect humans, it is most commonly found in
    planteating animals. Human anthrax infections are rare in the United
    States, and when infection does occur, it usually results from
    occupational exposure to infected animals or contaminated animal products,
    such as wool, hides, or hair. Anthrax infection can occur (1) cutaneously,
    usually from a cut or abrasion on the skin; (2) gastrointestinally, by
    ingesting undercooked, contaminated meat; and (3) through inhalation, by
    breathing aerosolized, or airborne, spores into the lungs.

    Anthrax is aerobic and facultative anaerobic; it can grow in aerobic (with
    oxygen) or anaerobic conditions. It is gram-positive-that is, when stained
    with a special solution (Gram's stain), the bacteria retain the color of
    the solution. Anthrax forms spores and is not capable of movement. The
    vegetative cell is 1 to 8 microns long and 1 to 1.5 microns wide; spore
    size is approximately 1 micron.12 Spores germinate, growing readily on
    most ordinary nutrient media in the laboratory.

    When the spores are germinating and are growing in a vegetative state,
    rather than dormant, and viewed through a microscope, they are said to
    look like "jointed bamboo rods." However, to the naked eye, anthrax
    vegetative cells growing on plates characteristically have the appearance
    of a "Medusa head" (with a curled edge) and "ground glass" (with a rough
    surface). Anthrax spores germinate and form vegetative cells after the
    spores enter a host. Vegetative cells multiply rapidly in an environment
    rich in nutrients, such as the blood or tissues of an animal or human
    host. Although vegetative cells have poor survival rates outside the host,
    anthrax spores are hardy and can survive for decades in the environment.

    The Environmental The response to the incident in the American Media
    Incorporated building

    Sampling Response 	in Florida in September 2001 led to the identification
    of mail as the potential source of contamination; eventually, it led to
    the sampling of the postal facilities. The agencies began sampling on
    October 12, 2001, in Florida and stopped on April 21, 2002, when the
    Wallingford, Connecticut,

    12A micron is 1 millionth of a meter, or about 1 thousandth of a
    millimeter. The period at the end of this sentence is approximately 500
    microns in diameter.

    facility was sampled for the last time. The following are key events
    related to the response in the postal facilities:

    On October 5, 2001, the death of an American Media employee from
    inhalation anthrax disease triggered an investigation by CDC, DOD, EPA,
    and the FBI. Since a contaminated envelope or package was not recovered in
    Florida, the agencies could not initially establish how the anthrax was
    delivered-whether by U.S. mail or some other means, such as courier.
    According to USPS, the combination of the Florida incident and the opening
    of the letter to Senator Daschle on October 15 established the link to the
    U.S. mail system. As early as October 10, CDC investigators had considered
    the possibility that USPS had delivered the letter containing anthrax. On
    October 12, USPS learned that it had delivered the contaminated letter,
    which was eventually recovered at the National Broadcasting Company.

    On or about October 9, 2001, at least two letters containing anthrax
    spores-those to Senators Daschle and Leahy-entered the U.S. mail system.
    Before the letters were sent to the Brentwood facility in Washington,
    D.C.,13 they were processed on high-speed mail sorting machines at a
    postal facility in Hamilton, New Jersey, known as the Trenton facility. In
    addition, two other recovered letters had been sent to a television news
    anchor at the National Broadcasting Company and to the editor of the New
    York Post in New York City; according to USPS, these letters were
    postmarked September 18, 2001. Discovering the contaminated letters
    resulted in a focus on the postal facilities involved in processing these
    letters.14 The agencies reacted to events as more information became
    available.

    On October 18, 2001, the USPS sampling effort began in the Brentwood
    facility. Its nationwide, or "precautionary," sampling, which was to rule
    out contamination in facilities considered less likely to be contaminated,
    began on or about October 28, 2001, according to USPS officials, and

    13The Brentwood facility was renamed the Joseph Curseen Jr. and Thomas
    Morris Jr. Processing and Distribution Center, in memory of the two
    Brentwood employees who died of inhalation anthrax.

    14The two recovered letters sent to the National Broadcasting Company and
    the New York Post were processed on high-speed mail sorting machines at
    the Trenton facility and the Morgan facility, the New York facility that
    also processed these letters, while the letters to the two senators were
    similarly processed at the Trenton facility in New Jersey and the
    Brentwood facility in Washington, D.C.

    ended on April 21, 2002, with the final sampling in the Wallingford
    facility.15 According to USPS, it followed the "mail trail" and sampled on
    the basis of the likelihood of finding additional contamination if it
    existed. Mail flow data were used as initial criteria to identify
    facilities that received 1 percent or more of their mail from either the
    Trenton or Brentwood P&DCs; other facilities were included on the basis of
    "plausible nonmail pathways," for example, a repair facility and stamp
    fulfillment center. The postal command center was responsible for facility
    testing and cleanup.

    Four contractors conducted USPS sampling. FBI sampling began on October
    12, 2001, in the Florida facilities and ended on November 16, 2001.16 CDC
    and EPA sampling began on October 15, 2001, and ended in the Florida
    facilities on November 3, 2001. In addition to CDC's sampling of the
    Florida facilities, CDC began sampling other postal facilities on October
    21, 2001, and ended on December 2, 2001. CDC's part in the sampling effort
    involved what it termed "outbreak investigation" sampling and included
    facilities associated with the primary facilities or with an employee's
    illness. According to CDC, the outbreak investigation included facility
    testing in part because postal employees at a specific facility had
    contracted anthrax (for example, Trenton and Brentwood P&DCs). In
    addition, CDC, as part of its epidemiologic investigations, 17 was looking
    for clues to the role that cross-contaminated mail might have played in
    nonpostal anthrax cases (for example, in the Morgan, Wallingford, and West
    Palm Beach facilities). Finally, based on mail flow patterns, CDC
    concluded that facilities may have been cross-contaminated, even if no
    anthrax cases were known (for example, all 50 post offices downstream from
    the Trenton facility).

    On October 30, 2001, according to USPS, the diagnosis of illness in a New
    York City woman raised the possibility of cross-contamination. According
    to CDC, a key finding, suggesting secondary contamination,

    15USPS officials told us that "nationwide" testing referred to the
    "downstream," or "precautionary," testing it performed. In this report, we
    refer to such testing as precautionary.

    16Additional testing may have taken place beyond November 16, 2001, due to
    the ongoing criminal investigation.

    17Epidemiology, a branch of medical science, investigates the incidence,
    distribution, and control of disease in a population. When CDC identifies
    the first confirmed case of an unusual illness, such as anthrax infection,
    it begins an investigation to identify new cases, unreported cases,
    contacts, and risk factors.

    was the cutaneous anthrax illness of a New Jersey mail carrier who did not
    work at the Trenton facility. CDC, EPA, USPS, and the FBI sampled 286
    postal facilities. According to USPS, to identify a good representation of
    facilities across the network for testing, it selected facilities based
    upon USPS knowledge of mail flows across the country. The intent was to
    show anthrax had not spread beyond the two sites that had been identified
    as being contaminated. Testing sites included facilities such as a mail
    recovery center, mail transport equipment center, and the Topeka repair
    center, as well as other P&DCs. The belief was that if anthrax
    contamination was found in these facilities-which USPS referred to as
    trading partners-then additional sampling would be required in downstream
    facilities connected to the trading partners.

                                    USPS Mail System

    The mission of USPS is to provide affordable, universal mail service. As
    of May 28, 2004, more than 800,000 workers processed more than 200 billion
    pieces of mail a year. The USPS headquarters office is in Washington, D.C.
    USPS has nine area offices; approximately 350 P&DCs; and about 38,000 post
    offices, stations, and branches; the P&DCs vary widely in size and
    capacity. The USPS mail system is involved in collecting, distributing,
    and delivering letters, flats (that is, catalogs and magazines), and
    parcels, as well as other items that vary in size and capacity.

    USPS provides for the security of the mail and for enforcing federal
    postal laws through its Postal Inspection Service. This service employs
    approximately 1,970 fact-finding and investigative postal inspectors and
    1,100 uniformed postal police officers.

    Mail processing facilities use several types of high-speed machines to
    process letters. At the facility that initially receives a letter for
    mailing, an advanced facer-canceller system cancels the postage stamp. For
    identification and sorting, other machines with optical character readers
    apply bar codes and markings (that is, identification tags) to the
    envelopes. The tags identify the time and date of processing, the machine
    and facility that processed the envelope, and the delivery destination.
    During fall 2001, USPS used this information to track the path of
    contaminated envelopes through the mail system.

    Delivery bar code sorter (DBCS) machines sort the mail. One machine alone
    processes about 37,000 letters an hour, using pinch belts that repeatedly
    squeeze the letters. During processing, paper dust accumulates,
    particularly near pinch rollers that move the mail through the machine.
    Since the rollers and optical readers are hard to access with vacuum

    nozzles, compressed air was typically used to blow debris out of the
    machine. The compressed air was, however, banned in October 2001 because
    of concern about the potential for spreading anthrax in mail processing
    facilities.

    Federal Agencies Involved in Anthrax Detection in Postal Facilities Had
    Differing Responsibilities

    The federal agencies involved in the response in the postal facilities had
    differing responsibilities. CDC and state and local health departments
    primarily provided public health advice and assistance to USPS. CDC has
    had primary responsibility for national surveillance of specific diseases,
    including anthrax; it has also conducted epidemiologic investigations to
    determine, among other things, the source of the disease. The FBI has been
    responsible for criminal investigations involving interstate commerce and
    the mail and crimes committed on federal property.18 EPA has been the
    nation's lead agency for responding to a release of hazardous substances
    into the environment.

    Responding to health emergencies, including bioterrorist attacks, is
    generally a local responsibility, but localities could and did request
    CDC's assistance in the fall of 2001. CDC performed the tests needed to
    confirm cases of anthrax and analyzed the substances in the two
    contaminated letters recovered in New York City. ATSDR and NIOSH within
    CDC helped USPS conduct environmental tests of some of its facilities and
    advised USPS on its facilities' decontamination. The U.S. Army Medical
    Research Institute for Infectious Diseases (USAMRIID) has conducted basic
    and applied research in the diagnosis, treatment, and prevention of
    hazardous infectious diseases for the military. It analyzed some
    environmental samples from postal facilities.19 It also performed detailed
    analyses, for the FBI, of anthrax spores in the letters addressed to
    Senators Daschle and Leahy. OSHA, responsible for employee health and
    safety issues, provided technical assistance and guidance to USPS on the
    decontamination of postal facilities.

    18The U.S. Postal Inspection Service-responsible for, among other things,
    protecting the mail system from criminal misuse-assists the FBI in
    criminal investigations. Numerous federal, state, and local agencies were
    also involved.

    19According to USAMRIDD officials, this work was only part of the
    comprehensive evaluation the FBI requested for analyzing the spores;
    additional laboratories participated, as requested, for specialized
    evaluations.

    On October 8, 2001, the President created the Office of Homeland Security
    to develop and coordinate a comprehensive national strategy for dealing
    with domestic terrorist threats or attacks. The office, which had limited
    involvement in the 2001 response, was superceded by the Homeland Security
    Act of 2002, which transferred many of its functions to DHS; it became
    operational in 2003. DHS was created by combining many previously separate
    agencies and is assigned a lead role in coordinating the efforts of
    federal agencies that respond to acts of terrorism in the United States.

    The Laboratory Response Network

    According to CDC, plans to mitigate anthrax outbreaks related to
    bioterrorism began in 1998, when CDC hosted a workshop on response to
    possible bioterrorism acts.20 The following year, HHS and CDC established
    the National Pharmaceutical Stockpile, and CDC collaborated with the FBI
    and APHL to develop the Laboratory Response Network (LRN) to coordinate
    detection and identification capabilities for threat agents associated
    with the testing of human specimens and environmental samples that could
    be related to bioterrorism.21 LRN was developed in 1999 to coordinate
    clinical diagnostic testing for bioterrorism. The primary purpose on the
    biological side was to detect the presence of biothreat agents in a number
    of specimen and sample types, especially since CDC was working closely
    with the FBI relative to its preparedness and response needs for law
    enforcement. Originally set up in four levels, A to D, LRN now consists of
    three levels of laboratory response: sentinel (level A), reference (levels
    B and C), and national laboratory (level D). Sentinel laboratories include
    clinical laboratories certified under the Clinical Laboratories
    Improvement Act (CLIA) of 1967, with biosafety level (BSL) 2 safety
    practices.22 These laboratories function as first responders that can
    perform standard initial tests to rule out, but not definitively confirm,
    anthrax.

    20B. T. Perkins, "Public Health in a Time of Bioterrorism," Emerging
    Infectious Diseases 8 (2002): 1015-17. See CDC, "Comprehensive Procedures
    for Collecting Environmental Samples for Culturing Bacillus anthracis"
    (Atlanta, Ga.: U.S. Department of Health and Human Services, rev. Apr.
    2002).
    http://www.bt.cdc.gov/agent/anthrax/environmental-sampling-apr2002.asp
    (Jan. 10, 2005).

    21Effective March 1, 2003, the National Pharmaceutical Stockpile became
    the Strategic National Stockpile.

    22Biosafety levels consist of different combinations of laboratory
    practices and techniques, the use of specific safety equipment, laboratory
    facilities suitable for the procedures performed, the hazard posed by the
    infectious agents, and the laboratory functions.

    Reference laboratories have core capacity for isolating agents and
    confirmatory testing. They include most state and local public health
    laboratories, with BSL 3 containment facilities that have been given
    access to nonpublic testing protocols and reagents. 23 These laboratories
    function to "rule in and refer" and thus have advanced capacity for the
    rapid identification of anthrax. The LRN national laboratories include
    only laboratories at CDC and USAMRIID. These laboratories, with the
    highest containment level (BSL 4), have expertise in diagnosing rare and
    dangerous biologic agents.

    CDC guidance, prepared and revised during the response, stated that
    lowrisk (nonpowder) environmental samples should be processed according to
    LRN level A protocols for rule-out testing in a CLIA-certified laboratory,
    using BSL 2 facilities and BSL 3 safety practices.24 In April 2002, CDC
    revised the guidelines, which stated that swab samples collected for
    ruleout testing should be analyzed at an LRN level A laboratory, using BSL
    2 facilities and BSL 3 safety practices.25 All other samples-including
    bulk (for example, a piece of carpeting), wipes, air samples, and vacuum
    samples-were to be analyzed for anthrax at an appropriate LRN level B or C
    laboratory, using BSL 3 facilities. Qualified laboratories report anthrax
    test results either qualitatively (for example, positive or negative) or
    quantitatively (for example, as a specific number of colony forming units
    [CFU] or living cells per gram or per square inch of material sampled),
    since CFUs do not equal one or a specific number of spores and the
    contamination may be either heterogeneously or homogeneously distributed.
    The results underestimate the total number of spores. It is important to
    note, however, that a negative result means only that no anthrax was
    detected in the sample analyzed and that it does not unequivocally
    determine the status of the facility or health risk to an individual.

    23A reagent is any substance used in detecting another substance by
    chemical, microscopic, or other means.

    24CDC, Procedures for Collecting Surface Environmental Samples for
    Culturing Bacillus anthracis (Atlanta, Ga.: U.S. Department of Health and
    Human Services, October 28, 2001). The guidelines included instructions
    for collecting bulk, premoistened swabs and HEPA vacuum samples as well as
    the level A testing protocol for premoistened swab samples. Presumptive
    positive swab samples were to be referred to an LRN state public health
    laboratory for confirmatory testing by LRN level B protocol for
    identification of anthrax.

    25CDC, "Comprehensive Procedures for Collecting Environmental Samples for
    Culturing Bacillus anthracis" (rev. Apr. 2002).

    Terms Associated with Sampling Methods and Procedures

    Sampling generally refers to the selection of a representative portion of
    a population, universe, or body. In this report, we refer only to the
    collection of environmental samples from the postal facilities and
    associated activities and procedures. Environmental sampling, in this
    context, refers to the collection of material from an environment, such as
    a surface or the air, by using a specific sample collection method and
    specific procedures or protocols. Environmental sampling is used to
    determine the extent and degree of contamination, assess the risk of
    exposure, support decisions related to medical treatment or cleanup, and
    determine when cleanup is sufficient to allow an area to be reoccupied.

    Objectives for sampling vary. For example, sampling to detect whether
    anthrax is present, or to rule its presence out, is referred to as initial
    sampling. Sampling to determine the extent of contamination-for example,
    how far it has spread in a facility-is referred to as characterization
    sampling. Sampling to determine whether decontamination of a facility has
    been effective is referred to as verification sampling.

    Air and surface sample collection methods are of several types. Swabs,
    premoistened or dry, have small surface areas (they are similar to
    Q-tips(R) cotton swabs) and are typically used to collect samples from
    small, nonporous surfaces that do not have a large accumulation of dust.
    Wet wipes-sterile gauze pads-are typically used to collect samples from
    larger, nonporous surfaces. A HEPA vacuum is a suction device with a
    nozzle that has a filter attached to it for collecting dust samples from a
    surface or the air. Microvacuuming techniques allow the collection of
    samples by air sampling pumps.

    Bulk samples can help detect the presence of contamination on building
    materials; office equipment; small articles such as letters or packages;
    carpeting; and heating, ventilating, and air conditioning (HVAC) filters.
    Air sampling that results in culturable samples can be done by a variety
    of methods, which include sampling pumps and filters (gelatin,
    polytetrafluoroethylene, and the like) placed inside sampling cassettes.
    Air sampling may be used to characterize the air concentration of anthrax
    spores.

    In this report, "sensitivity" refers to the minimum number of anthrax
    spores that a collection method can pick up and that an analytic method
    requires to generate a positive result. "Specificity" refers to a method's
    ability to accurately discriminate between anthrax and other bacterial
    species. "Repeatability" refers to a method's ability to produce the same

    results several times under the same conditions. "Reproducibility" refers
    to a method's ability to produce similar results under similar conditions,
    when performed by different persons independently in different locations.
    For example, for analytic methods, reproducibility would be a measure of
    the variations in test results across similar tests conducted in different
    laboratories or in the same laboratory on different occasions.

    "Collection efficiency" refers to the percentage of viable anthrax spores
    present in a sampled environmental area that are removed from the surface
    by various sample collection methods (e.g., a swab or wipe).

    "Recovery efficiency" refers to the number of viable spores- for example,
    the number of CFU in plate culture-that are detected as growing by various
    analytic methods.

    "Accuracy" in the context of analytical tests refers to the closeness of
    the test results obtained by a particular method to the true value.

    "Robustness" refers to a method's ability to yield a high number of
    correct responses when performed under a variety of different experimental
    conditions.

    "Limit of detection" refers to the lowest amount of analyte in a sample
    that can be detected, but not necessarily quantified, under stated
    experimental conditions. The detection limit is usually expressed as a
    concentration (e.g., percentage, or parts per billion) in a sample.

    CDC, EPA, and USPS, the federal agencies involved in sampling the postal
    facilities in 2001 to detect anthrax, undertook several activities: (1)
    sampling strategy development, followed by (2) sample collection, (3)
    transportation, (4) extraction, and (5) analysis of the samples (see fig.
    1). As we discuss below, neither these activities nor the overall process
    was validated for anthrax testing. Consequently, the agencies were
    challenged due to limited information available for reliably choosing one
    method over another and no information on the detection limit to use when
    evaluating negative results. The sampling strategy used by the agencies
    could not provide any statistical confidence with regard to the question:
    Is this building contaminated? Therefore, in the future, in the absence of
    a positive result, a different strategy is needed that will provide
    statistical confidence, at a defined level, to the answer to this
    question.

    Agencies Were Challenged by Problems Associated with Sampling Activities

    Figure 1: Agency Sampling Activities

    Source: GAO analysis of CDC, EPA, and USPS data.

                                           5

    Sample analysis

    Of living organisms in preliminary
    tests
    and
    confirmatory
    tests

    Activity 1: Sampling Strategy Development

    One challenge the agencies faced was to rapidly develop an effective
    sampling strategy to detect anthrax in facilities of varying sizes that
    had complicated machinery and complex surfaces. A sampling strategy
    includes such elements as how many samples to collect, where to collect
    them from, and what collection method to use. The targeted strategy the
    agencies used was reflected in their site-specific sampling activities.
    Sample sizes varied by facility and circumstances, increased over time,
    and excluded probability sampling.

    According to CDC, EPA, and USPS officials, they generally "followed the
    mail trail" in collecting samples. They determined which areas were
    involved in mail processing, from their knowledge of the potential path of
    the contaminated letters-that is, by analyzing the actual path of the mail
    from mail flow data-and from discussions with facility managers and from
    epidemiological data. We describe their site-specific approaches below.

    USPS's strategy was reflected in its Standard Sampling Plan, for use by
    USPS contractors in selected precautionary testing in postal facilities
    USPS considered less likely to be contaminated.26 According to USPS, the
    sampling strategy evolved in October and November 2001 as mail flows in
    the anthrax incident were identified and prescreening was assessed. The
    first version of the Standard Sampling Plan that USPS used was dated
    November 2, 2001; it was followed by numerous revisions.

    26According to USPS officials, before USPS developed its Standard Sampling
    Plan, it had been conducting testing in response to events at individual
    facilities. See USPS, Standard Sampling Plan, draft (Washington, D.C.:
    Nov. 9, 2001).

    The plan specified the number of samples to be collected, which increased
    as the investigation unfolded. In the beginning, in each facility, 23
    samples were to be collected from specific areas relating to mail
    processing and up to 20 additional "discretionary" samples were to be
    collected, depending on the type and size of the facility. Later, USPS
    increased the number of samples required to a minimum of 55, with up to 10
    additional discretionary samples for larger facilities. Consequently, the
    number of samples collected varied by facility, from a low of 4 to a high
    of 148.27

    CDC's and EPA's site-specific strategies were primarily discretionary.
    According to CDC, for example, decisions as to the number and location of
    samples to be collected were based on discussions with facility managers
    and others, reviews of facility floor plans, and observations to identify
    possible contamination pathways and locations, which were then targeted
    for sample collection. The numbers of samples CDC collected varied by
    facility, ranging from a low of 4 to a high of 202.28

    EPA's site-specific strategy for the postal facilities it sampled in
    Florida, in coordination with CDC, on October 31, 2001, was to
    "characterize" the extent of anthrax spores in a post office suspected of
    having handled contaminated mail; it also focused on decontamination
    issues. Like USPS, EPA's plan involved a targeted sampling strategy but
    did not specify the number of samples to be collected. However, it did
    state that "characterizing a PO [post office] where there is no evidence
    that suspect contaminated mail has been handled is problematic." It also
    stated that from discussions with postal service officials, employees, and
    unions, as well as a review of suspected locations, the "sampling
    locations will be identified that are most likely to contain residue from
    suspect postage." 29

    27These numbers represent samples collected for culture analysis: the 4
    excluded samples USPS collected for analysis by the portable polymerase
    chain reaction (PCR) instrument that USPS used in some facilities or the 2
    "quick tests" performed at Brentwood on October 18, 2001.

    28According to CDC, the facility in which the 4 were collected did not
    include mail-sorting equipment.

    29Post Office Sampling Plan-Florida, Revision 1 (Oct. 31, 2001).

    The numbers of samples EPA collected ranged from a low of 4 to a high of

    71. EPA did not develop a guidance document.30

    USPS and CDC developed guidance. USPS, responding to the finding of
    contamination in a number of its facilities, developed draft interim
    guidance, revised several times, intended solely for USPS facilities.31
    The November 5, 2001, guidance addressed issues such as sampling,
    decontamination, communication, employee notification, and interim
    cleaning procedures.32

    Shortly after CDC became involved in sampling activities in postal
    facilities, CDC developed "Comprehensive Procedures for Collecting
    Environmental Samples for Culturing Bacillus anthracis" for industrial
    hygienists and environmental health professionals who might be called on
    to collect samples. 33 The document addressed issues to consider when
    collecting anthrax samples, such as general locations and collection
    methods and procedures. The guidance stated that the number of samples
    collected was to be "influenced by the circumstances of the potential
    contamination." It also stated "a sufficient number of samples must be
    taken to increase the probability that the sampling is representative of
    the extent of contamination." However, it did not define "representative"
    or specify what methodology to use to determine sample size.

    30In September 2002, the National Response Team, chaired by EPA, developed
    a draft "reference tool" to reflect agencies' experience responding to the
    2001 anthrax contamination; see National Response Team, Technical
    Assistance for Anthrax Response, Interim-Final Draft Phase I Update
    (Washington, D.C.: Nov. 2003).
    http://www.nrt.org/Production/NRT/NRTWeb.nsf/AllAttachmentsByTitle/A-47AnthraxTAD/
    $File/Anthrax.pdf?OpenElement (Jan. 9, 2005).

    31USPS, Draft Interim Guidelines for Sampling, Analysis, Decontamination,
    and Disposal of Anthrax for U.S. Postal Service Facilities (Washington,
    D.C.: Nov. 8, 2001, rev. Dec. 4, 2001).

    32The guidance evolved. In the beginning, for smaller facilities, it
    provided for collecting 23 samples from specific areas involved in mail
    processing, with up to 10 additional discretionary samples; for larger
    facilities, the guidance eventually provided for collecting 55 samples,
    with up to 10 discretionary samples.

    33NIOSH said it had posted the guidance on CDC's Web site and that the
    guidance was updated in April 2002. See CDC, "Comprehensive Procedures for
    Collecting Environmental Samples for Culturing Bacillus anthracis"
    (Atlanta, Ga.: U.S. Department of Health and Human Services, rev. Apr.
    2002).
    http://www.bt.cdc.gov/agent/anthrax/environmental-sampling-apr2002.asp
    (Jan. 10, 2005).

    Agencies Primarily Used a Targeted Strategy

    CDC and USPS officials said that they used targeted sampling-they
    collected samples from specific areas considered, based on agencies'
    technical judgments, more likely to be contaminated. This strategy was
    reflected in agencies' site-specific plans and guidance and included
    judgments about where anthrax was likely to be found, such as a mail
    processing area.34 Such judgments can be effective in some situations, for
    example, in determining the source of contamination in a disease outbreak
    investigation, provided results are positive. However, if the results are
    negative, the basic question-Is this building contaminated?-cannot be
    answered with statistical confidence.

    According to CDC, in a situation where there is confidence that the source
    and path of contamination are known, a targeted sampling strategy (that
    is, a judgmental approach) can result in a successful and efficient use of
    resources.35 In addition, CDC stated that it used empirical methods to
    gather information from various sources in order to identify plausible
    contamination pathways and to classify locations by contamination
    potential. CDC used "judgmental" worst-case approaches to collect from the
    "most likely contaminated" locations. CDC further stated that these
    approaches were derived from "maximum risk employee" approaches,
    traditionally used for initial sampling strategies in occupational health
    to identify employees believed to have the greatest exposure in a
    workplace.36

    CDC believes that targeted sampling "allows scientific inferences about
    contamination." In commenting on the draft of this report, CDC stated that
    in a well-developed targeted sampling strategy, "if you cannot detect the
    agent in a high-probability area, it is improbable that a low-probability
    area

    34These judgments, according to CDC, "relied primarily on using existing
    law enforcement, epidemiology, and event details to identify locations
    where anthrax was most likely to be found."

    35CDC provided the following example of a targeted strategy: The use of
    postal code information, from the recovered Senator Daschle letter, to
    identify that Brentwood DBCS machine # 17 was the one that processed the
    letter. Machine # 17 was then targeted for sampling with specific
    attention to those locations closest to the mail path through the machine.
    Judgmental approaches not only produced the highest probability of
    identifying a positive sample during the 2001 response but they helped to
    establish those locations that posed the greatest risk of exposure.

    36In 2001, preliminary information was used to identify, for sampling,
    those surfaces (e.g., sorting machines and mail sorting bins) considered
    "most likely to have been contaminated."

    would produce a positive result." This assertion appears to be reasonable
    at face value. However, finding a positive result depends on several
    factors, such as the accuracy of identifying the source of contamination
    and the detection limit of the sample collection and analytical methods.

    According to CDC, it conducts outbreak investigations in a manner that in
    its judgment optimizes the chance of locating the source of contamination.
    CDC's preference for targeted sampling is based on the need to rapidly
    identify the source of contamination in order to institute early public
    health interventions. CDC, however, agrees: "targeted sampling does not
    support statistical inferences."

    CDC and USPS officials said that they used a targeted strategy for several
    reasons, including limitations on how many samples could be collected and
    analyzed. They also said that in 2001 they lacked the data necessary to
    develop an initial sampling strategy that incorporated probability
    sampling.37 We disagree with this interpretation. Probability sampling is
    statistically based and does not depend solely on empirical criteria
    regarding the details of possible contamination.

    CDC officials said that the numbers of samples that could be collected
    were limited by laboratory capacity for analyzing samples. For example,
    according to the CDC official in the Morgan facility in New York, CDC
    sampling was restricted to "56 dry swab samples because the New York City
    health department was `overwhelmed' with samples from so many other
    places." CDC also had a number of different location-specific sampling
    goals.38

    CDC, EPA, and USPS officials provided various comments on factors involved
    in developing sampling strategies. According to a CDC official, CDC "does
    not use statistical sampling in its initial assessment sampling"; the
    official was not aware of any literature demonstrating that "statistical

    37According to CDC, the missing data included "size of the hot spot, limit
    of detection, surface contamination risk criteria."

    38According to CDC, epidemiologic sampling was needed in some locations to
    examine potential exposure pathways for nonpostal anthrax cases. Some
    local public health authorities requested that locations less likely to be
    contaminated (such as public areas at P&DCs) be sampled in order to help
    decide the need to provide personal protective equipment to members of the
    public.

    sampling" is any more effective than CDC's approaches.39 However, we did
    not find any empirical support that for initial sampling, targeted
    sampling is better than probability sampling.

    According to an EPA official, "if there is some knowledge available about
    the release, then sampling in targeted locations is the best approach;
    otherwise, a grid sampling approach could be used, but a sufficient number
    of samples then would need to be collected."40 The official said that
    developing a statistically based sample size requires knowing the
    substance (for example, anthrax) and the performance efficiency of the
    sample collection methods. EPA did not have this knowledge for anthrax.
    Finally, the official stated that statistical sampling could be done for
    other substances, such as polychlorinated biphenyl, a chemical that can
    cause cancer in humans.

    USPS officials said that they had learned lessons from the sampling
    conducted in the Wallingford facility in 2001. Consequently, in April
    2002, for both initial and verification sampling of the Wallingford
    facility, according to USPS officials, they used grid-based sampling. This
    resulted in their finding three positive samples containing only a few
    spores out of the total samples collected in the facility's high-bay area,
    that is, elevated areas including pipes, ducts, lights, joists, beams, and
    overhead conveyors.

    Incorporating Probability According to CDC, a targeted sampling strategy
    may be effective in Sampling Would Allow Greater detecting contamination
    in a facility when sufficient site-specific Confidence in Negative Results
    information exists to narrow down the locations in which the release and

    contamination are most likely to have occurred.41 CDC's assumptions for
    this strategy are that at the outset, (1) a scenario where all locations
    have

    39Citing a paper by Carlson and colleagues, CDC stated, "Statistical
    sampling approaches for surface contamination most commonly address
    verification (clearance sampling) approaches." The paper also includes
    other information requirements, such as specifying the (1) maximum
    acceptable level (that is, the level above which a surface is not
    considered clean enough), (2) largest acceptable hot spot, and (3) desired
    probability with which a single hot spot must be discovered. See T. M.
    Carlson and others, Sampling Requirements for Chemical and Biological
    Agent Decontamination Efficacy Verification (Washington, D.C.: U.S.
    Department of Energy, Mar. 29, 2001).

    40Grid sampling is a probability sampling method used to systematically
    sample twodimensional areas.

    41Examples of such information range from law enforcement findings about
    the location of an event to engineering information about machines that
    might aerosolize spores, to medical epidemiology findings about where
    affected individuals worked.

    an equal chance of being contaminated is generally the exception rather
    than the rule; (2) information collected about the event, combined with
    technical judgment about exposure pathways, can be used to identify
    locations where contamination is most likely to be found; (3)
    contamination levels of highest public health concern can usually be
    detected using a variety of available methods, despite their limitations;
    and (4) there is important public health value in quickly identifying
    contaminated locations. However, these assumptions may not always apply.
    For example, there may be limitations in the available information that
    restrict the ability to reliably identify target locations. The method of
    contamination spread could conceivably be via a mechanism where there is
    an equal chance of any area being contaminated. Lastly, all results may be
    negative, which will lead to a requirement for additional testing, as was
    the case in Wallingford. This, in turn, will result in the loss of the
    critical time needed for public health intervention. Therefore, there is a
    need for probability sampling at the outset to provide statistical
    confidence in the interpretation of negative results.

    We consider probability sampling to be a viable approach that would
    address not only the immediate public health needs but also the wider
    public health protection, infrastructure cleanup, and general
    environmental contamination issues. In any particular facility,
    probability sampling could operate in the following ways: At the outset of
    a response, a statistically based probability sampling plan would be
    drawn, based on the facility dimensions, complexities, and other
    characteristics. We recognize that in a major incident, the number of
    samples that may need to be collected and analyzed may challenge available
    laboratory resources. Accordingly, there is a need to develop innovative
    approaches to use sampling methods that can achieve wide-area coverage
    with a minimal number of individual samples to be analyzed. For example,
    HEPA vacuum techniques, in combination with other methods, appear to be
    one such approach that could achieve this. In addition, because of limited
    laboratory capacity, samples may need to be stored after collection for
    subsequent analysis, on a prioritized basis.

    Initial outbreak sampling, within the dictates of the probability-sampling
    plan, could be targeted to those areas that, based on technical judgments
    (if available), would be considered-when the information on the source of
    contamination is definitive-most likely to be contaminated. Thus, targeted
    sampling, from a public health perspective, is recognized as an important
    component of the wider probability sampling plan. If these early
    (targeted) samples yield positive results, then appropriate public health
    measures could be instituted. Further sampling of the facility could take

    place over a longer period in order to address characterization and
    cleanup issues. Conversely, if initial targeted samples were negative,
    completing the probability sampling plan would then permit an assessment,
    with appropriate confidence limits, of the likelihood of contamination,
    even if all of the samples collected under the plan were negative.

    We recognize that the use of probability sampling could have strained
    laboratory capacity in 2001 (for example, limited analytical capacity may
    not permit a laboratory to analyze all samples on a given day). However,
    there are several potential solutions, including (1) not all samples, once
    collected, have to be analyzed on the same day (given the fact that
    anthrax spores do not deteriorate while in acceptable storage conditions
    in the laboratory) and (2) laboratory capacity can be increased by hiring
    more staff for the existing laboratories, transporting the samples for
    analyses to more than one laboratory, and establishing additional
    laboratories. Probability sampling would offer a defined degree of
    statistical confidence in the negative results. A known level of
    confidence is needed because evidence suggests that even a few anthrax
    spores could cause disease in susceptible individuals.

    The situation in 2001 was unique, and the agencies were not fully prepared
    to deal with environmental contamination. Therefore, we are describing and
    assessing agencies' activities not to fault the agencies but to learn
    lessons. In the future, if the agencies decide to use a targeted rather
    than a probability sampling strategy, they must recognize that they could
    lose a number of days if their targeted sampling produces negative test
    results. In this case, additional samples would need to be collected and
    analyzed, resulting in critical time, for public health interventions,
    being lost. This was so at the Wallingford postal facility in the fall of
    2001, when about 3 weeks elapsed between the time the first sampling took
    place and the results of the fourth testing, which revealed positive
    results. Furthermore, about 5 months elapsed between the time of the first
    sampling event and the time anthrax was found in the Wallingford
    facility's high-bay area.42

    Therefore, in the future, strategies that include probability sampling
    need to be developed in order to provide statistical confidence in
    negative

    42According to CDC, earlier identification of anthrax in the high-bay area
    would most likely not have altered public health intervention
    recommendations. The decisions about postexposure prophylaxis were based
    on the available epidemiology.

    results. Further, even if information on all the performance
    characteristics of methods is not yet available, a probability sampling
    strategy could be developed from assumptions about the efficiency of some
    of the methods. And even if precise data are not available, a
    conservative, approximate number could be used for developing a sampling
    strategy. This would enable agencies and the public to have greater
    confidence in negative test results than was associated with the sampling
    strategy used in 2001.

    Activity 2: Collecting Samples

    The agencies used a variety of sample collection methods, alone or in
    combination. USPS primarily used the dry swab method. CDC and EPA used
    premoistened and dry sterile, synthetic (noncotton) swabs, wet synthetic
    wipes, and HEPA vacuums for sampling. To determine whether anthrax was
    airborne, CDC performed air sampling in the Brentwood facility 12 days
    after the contaminated letters were processed.43 Airborne anthrax spores
    pose a health risk because they can cause inhalational anthrax, the most
    serious form of the disease. Agency officials stated that laboratory
    requirements had influenced the choice of methods. For example, in the New
    York area, CDC used only dry swabs, following a requirement by New York
    public health laboratories.

    The majority of the samples were collected by the dry swab method, which
    experts and others we interviewed considered the least effective. As shown
    in table 1, 304 sampling events involved single methods-that is, CDC and
    USPS collecting dry swab samples (185) and CDC and others collecting
    premoistened swabs (119). However, for some sampling events, CDC used wet
    wipes, HEPA vacuum, and air samples at Brentwood and swabs, wet wipes, and
    HEPA vacuum samples at Wallingford.

    43CDC said that it did not do more air sampling because the air samples in
    Brentwood were all negative, despite known surface contamination.
    Therefore, air sampling was not seen as useful if (1) the samples were
    collected days after buildings were closed, (2) the ventilation systems
    were turned off, and (3) anthrax spores had settled on surfaces. CDC
    stated that air sampling is more appropriate for clearance (verification
    sampling) purposes. CDC also did research on air sampling at Trenton in
    February 2002, which we discuss later in the report; according to USPS, a
    Canadian Defense Ministry unit conducted additional air sampling under
    CDC's guidance.

    Table 1: Agencies' Sample Collection Methods for Initial Sampling, October 2001
                                   through April 2002

         Methods at different   Sampling                Agency
           sampling events        eventa CDC  CDC and   USPSb  FBI NJDHSS Unknown
                                                EPA
              Dry swabs              185  X               X
          Dry swabs and HEPA
                vacuum                 2  X
              Dry wipes                1                  X
          Premoistened swabs         119  X      X              X    X
        Premoistened swabs and
                 bulk                  1                        X
        Premoistened swabs and
                 wet
                wipes                  1         X
        Premoistened swabs and
             HEPA vacuum               5  X                     X
    Wet wipes, HEPA vacuum,
           and air sampling            1  X
          Wet wipes and HEPA
                vacuum                 1  X
                Wipes                  1                  X
             HEPA vacuum               2                  X
        Swabs, wet wipes, and
             HEPA vacuum               1  X
    Other (hand-held assay)         1                  X
          Method and agency
               sampling
               unknown                 3                                     X
                Total                324

    Source: CDC, EPA, and USPS.

    aSampling event refers to sample collection by one agency on a specific
    day, at a specific time, in a specific facility. Multiple agencies could
    have collected samples on the same day; we consider these separate
    sampling events.

    bEarly, in some facilities, USPS also collected samples for analysis by a
    portable, polymerase chain reaction (PCR)-based instrument that
    contractors operated for analyzing these particular samples.

    USPS officials said that the choice of dry swabs for the USPS Standard
    Sampling Plan was based on advice from CDC and an APHL working group,
    which had coordinated with the head of LRN. According to APHL, the working
    group was made up of "members from various state public health
    laboratories, APHL, and CDC, as well as other federal agencies." USPS said
    that the goal was to develop a consistent approach that all states could
    use and that would ensure that all laboratories analyzing the

    samples would have the same capabilities.44 USPS also said that use of
    this method would avoid overwhelming the laboratories' capacities.45

    According to APHL officials, the working group was to select a collection
    method. This group consulted with CDC's NCID in November 2001. APHL said
    that an NCID official, who was a member of the group, agreed that the dry
    synthetic swab method could be used but that premoistened swabs would pick
    up more spores. (See fig. 2 for examples of swab methods.) NCID assisted
    in developing the analytic procedures for the dry swab samples USPS
    collected.

    44USPS was referring to the approach that was developed for the USPS's
    contractors to use under USPS's November 2001 agreement with APHL for
    public health laboratories to analyze the samples collected by the
    contractors. In commenting on this statement, APHL officials stated, the
    goal of the LRN is to standardize test procedures and reagents nationwide,
    and the USPS was mirroring that approach.

    45APHL disagreed with USPS, stating that the capacity of the LRN was not a
    topic of concern and that more testing of a variety of environmental
    samples could have taken place if validated methods for those samples had
    been available through the LRN. However, in discussing major issues that
    arose during the anthrax attacks of 2001, including all testing, APHL
    referred to the fact that many LRN laboratories were "overwhelmed and near
    their breaking point" and "laboratories were required to test specimens
    that, in most cases, did not pose a threat."

                       Figure 2: Three Sample Collection Methods

    During our fieldwork, we tried to determine what specific advice CDC gave
    APHL on using dry swabs. In responding to our inquiry, CDC did not
    specifically deny APHL's statement that an official from CDC's NCID told
    APHL that dry swabs could be used. However, an official from CDC's NIOSH,
    which was not a member of the working group, said that CDC has always
    recommended using premoistened swabs. Nevertheless, according to APHL,
    "the NIOSH recommendation was not known by the NCID working group members,
    nor did they advocate on its behalf." We noted that all versions of CDC's
    guidelines included instructions for using premoistened swabs.

    The choices for sampling methodology are apparent in the USPS interim
    guidelines, earlier versions of which included instructions for
    premoistened swabs, later versions omitting them; USPS initially developed
    the guidelines following the finding of contamination in a number of its
    facilities. However, the USPS Standard Sampling Plan always provided for
    dry swabs, as part of the November 7, 2001, USPS agreement with APHL. This
    agreement designated public health laboratories to analyze samples
    collected by USPS contractors.46 In particular, this plan was used by the
    contractors and formed the basis for the development of a site-specific
    plan for each facility sampled.

    When the contractors were sampling the facilities, the interim guidelines
    were still in draft form. In commenting on a November 7, 2001, version of
    the guidelines, which included instructions for using premoistened swabs,
    CDC suggested, on November 9, 2001, that USPS use other methods, such as
    bulk and vacuum samples. 47 CDC stated that the reason for the use of
    swabs was an accommodation USPS had reached with APHL. CDC also said that
    state laboratories might be less familiar with analyzing bulk and vacuum
    samples. A USPS official said that there were issues related to which
    laboratories could handle the other types of samples and that laboratories
    tended to be conservative, preferring to accept only the types of samples
    they were used to handling, such as swabs. According to APHL,

    46Several versions of the USPS guidelines were developed while the
    contractors sampled
    the postal facilities, but they followed only the Standard Sampling Plan.
    The most recent
    guidelines are USPS, "Interim Guidelines for Sampling, Analysis,
    Decontamination, and
    Disposal of B. anthracis Spores in USPS Facilities," revision 1.0
    (Washington, D.C.:
    Dec. 2003).

    47NIOSH comments were on USPS interim guidelines, not the Standard
    Sampling Plan,
    which USPS contractors used under USPS's November 7, 2001, agreement with
    APHL for
    analyzing samples.

    the decision to recommend the use of dry swabs was based on a variety of
    concerns, including

    the use of an untrained and poorly equipped workforce collecting the
    environmental samples; maximized isolation of viable Bacillus anthracis
    through preservation of spores during transport when temperature and
    exposure to light was least controlled; the ability to eliminate other
    environmental bacteria and fungi that would inhibit isolation of anthrax;
    and the lack of standardized validated methods for laboratory testing of
    HEPA socks.

    The decision to use dry rather than premoistened swabs stemmed partly from
    the concern of some public health officials, including APHL officials we
    interviewed, that moistened swabs would allow anthrax spores to germinate,
    growing into vegetative cells instead of remaining as spores.48 Other
    public health officials we interviewed said it was highly unlikely that
    anthrax spores would germinate into vegetative cells in a premoistened
    swab. APHL officials said that it was feared that such vegetative cells
    would be destroyed during certain analytic procedures. However, none of
    the agencies' collection methods were evaluated for anthrax detection in
    environmental samples. The published literature provided some information
    on the efficiency of a few sample collection methods. In all the methods
    studied, swabs were always premoistened before samples were collected.
    However, according to one study, the most efficient method caused problems
    when used with certain analytic methods.49 In the absence of empirical
    research, agencies had no information available for reliably choosing one
    method over another and no information on the limits of detection to use
    when evaluating negative results.

    Activity 3: Transporting Agencies transported samples by land or air to
    laboratories for extraction

    Samples 	and analysis (activities 4 and 5). The USPS sample collection
    plan included shipping instructions that were based on regulations for
    shipping infectious substances and designed to prevent their inadvertent
    release.50

    48According to an NCID official, this belief about anthrax spore
    germination was held for only a short time. However, APHL disagreed,
    stating that the concern about spore germination is still a significant
    concern today.

    49Mark P. Buttner, Patricia Cruz-Perez, and Linda D. Stetzenbach,
    "Enhanced Detection of Surface-Associated Bacteria in Indoor Environments
    by Quantitative PCR," Applied and Environmental Microbiology 67 (June
    2001): 2564-70. The study compared the performance of swab kit, sponge
    swipe, cotton swab, and bulk sampling methods.

    50This is not intended to imply that the environmental samples were to be
    mailed through the postal system.

    EPA's sample collection plan did not refer to transportation requirements.
    According to CDC's guidelines, anthrax samples were to be considered
    infectious substances and packaged according to applicable federal
    regulations enforced by the Department of Transportation. These
    regulations were aimed at "ensuring that the public and the workers in the
    transportation chain are protected from exposure to any agent that might
    be in the package." 51 Among other potential requirements, infectious
    material must be contained in a securely sealed, pressure resistant,
    watertight, primary receptacle surrounded by an absorbent and cushioning
    material. This material must, in turn, be enclosed in a securely sealed,
    watertight, and durable secondary packaging, which has to be enclosed in
    an outer packaging constructed of fiberboard or equivalent material, as
    well as shock absorbent material if more than 50 milliliters are shipped
    in one package.

    However, these regulations did not address one of the most important
    issues-maintaining the biological integrity of samples while being
    transported. Failure to do so could result in false negative test results.
    For example, analysis by culture requires that spores can germinate,
    divide and multiply, so that tests can determine whether a sample contains
    anthrax. Temperature and exposure to certain kinds of light, such as
    ultraviolet light, can be deleterious to some microorganisms. Therefore,
    it is important that every sample collected retain its original physical
    form before and during transportation.52

    We recognize that it may not be possible to maintain the original form of
    samples collected by various methods. A 2002 study recognized some of the
    factors that must be considered when determining conditions for

    51Department of Transportation, 49 C.F.R. subchapter C-Hazardous Materials
    Regulation. The USPS regulations mirror the Department of Transportation
    regulations. However, to be transported as mail, material must be
    classified as mailable. By statute, infectious materials, such as anthrax
    spores, that are "disease germs or scabs, [or] other natural or artificial
    articles, compositions, or material which may kill or injure another"
    cannot be mailed. Such materials are termed "nonmailable matter."
    Knowingly mailing such material is a criminal offense, and doing so with
    the intent to kill or injure is a felony. When an etiologic material is
    not "outwardly or of [its] own force dangerous or injurious to life,
    health, or property," USPS may allow it to be mailed, subject to
    appropriate rules and regulations governing its preparation and packing.
    As a result, USPS allows the mailing of small quantities of appropriately
    packaged infectious material, but only if it is intended for medical or
    veterinary use, research, or laboratory certification related to public
    health.

    52Environmental Protection Agency, General Field Sampling Guidelines
    (Washington, D.C.: Aug. 11, 1994).

    transporting biological samples, such as temperature, exposure to light,
    and time before processing.53 CDC's guidance stated that samples,
    including premoistened swabs, wet wipes, HEPA vacuum, air, and bulk (for
    example, a piece of carpet), should be transported to the appropriate
    laboratory at ambient temperature.54 The USPS Standard Sampling Plan
    required that dry swab samples be transported at ambient temperatures.
    However, both CDC and USPS said that laboratories were also consulted for
    specific transportation requirements.

    Before the 2001 incidents, LRN analytical protocols were designed to
    address sample integrity for human clinical specimens, which differ from
    environmental samples.55 According to a DOD expert, human samples may or
    may not be expected to contain spores. In addition, all human samples,
    including nasal swabs, contain substances that could lead to germination.
    Vegetative cells require some form of stabilization. According to a public
    health official, ideally all samples, whether clinical or environmental,
    should be refrigerated while being transported. However, he also stated
    that the temperature conditions for some environmental samples could vary.
    For example, because HEPA vacuum samples are usually dry, they can be
    shipped at ambient temperatures. Premoistened swabs and wet wipes should
    be kept cold or shipped with ice packs to prevent fungal growth,
    particularly if their transportation will be delayed. Nevertheless, he
    said, both clinical and environmental samples should be analyzed within 24
    hours.

    According to APHL officials, transportation delays could result in swabs'
    degradation from overgrowth of molds or fungus. A DOD expert we

    53The study stated: "A major concern in transporting biological samples is
    ensuring that the microorganisms remain viable and active with
    multiplication until the testing procedures have been performed. Samples
    should be protected from ultraviolet light, heating or freezing. When
    transit times are less than 6 hours, the sample may be maintained at the
    original ambient temperature. If 6 hours is insufficient time for transit
    of samples, the general consensus is to lower the temperature to less than
    10 degrees centigrade to restrict the amount of growth and deleterious
    interactions between the intrinsic species present in the sample. These
    samples should be processed within 24 hours of retrieval." E. Raber and
    others, "Chemical and Biological Agent Incident Response and Decision
    Process for Civilian and Public Sector Facilities," Risk Analysis 22 (Apr.
    2002): 195-202.

    54EPA's plan, which applied only to the Florida postal facilities, did not
    address transportation requirements. However, CDC and EPA worked together
    to sample the Florida postal facilities.

    55LRN protocols state that if the transport time of moistened clinical
    swab samples will be greater than 1 hour, they should be transported at 2
    to 8 degrees centigrade.

    interviewed said that spores would have no trouble surviving at room
    temperature for much longer than 6 hours, and background growth at ambient
    temperatures is negligible. Nevertheless, according to another public
    health official, while laboratories were familiar with transporting
    clinical specimens, they were unsure about transporting environmental
    samples. Therefore, experiments under controlled situations are needed to
    resolve this issue.

    We did not attempt to ascertain (1) the specific transit times for
    delivering all the samples to laboratories, (2) whether sample
    transportation was delayed, and (3) if it was, how long it was delayed. We
    also did not attempt to ascertain the environmental conditions the samples
    were shipped under or when they were received at the laboratories.
    Finally, we did not attempt to ascertain the degree to which spores could
    have been exposed to varying environmental conditions from the time of
    release to the time of sample collection, which could have affected sample
    integrity. Anthrax spores are robust, compared with other pathogenic
    microorganisms, but whether transportation affected their viability cannot
    be known because the conditions of their transportation were not
    validated. Transport conditions, once validated, would have to be
    standardized to ensure reproducibility.

    Activity 4: Extracting Samples

    LRN protocols required that sample material be extracted with specific
    extraction procedures and fluids (such as sterile saline or water) and
    that the extracted fluid be subjected to specific analytic methods. For
    the samples USPS collected under the APHL agreement, the extraction
    methods included adding a sample processing solution to the conical tubes
    containing the dry swabs before "plating." This process was adapted from
    LRN protocols for extracting swabs.56 However, the private laboratory (not
    part of LRN) that originally analyzed the samples for USPS did not use an
    extraction fluid; it inoculated the noncotton, rayon-tipped dry swab
    directly onto a culture plate.

    Several factors could have affected extraction efficiency. For example,
    according to public health officials and other experts, the degree to
    which swabs or wipes can retain spores depends on the material they are
    made

    56LRN protocols for environmental swabs required placing each swab in a
    3-milliliter sample processing solution (0.3 percent Tween 20(TM), a
    surfactant) in phosphate-buffered saline before plating.

    of. Cotton is more retentive than some artificial fibers like rayon and
    may be more difficult for extraction of spores for analysis. In addition,
    according to a public health official, cotton swabs are characterized by a
    "lipid matrix, which gives poor results for culture." However, a CDC
    official also commented that cotton is a natural material and that some
    laboratories using polymerase chain reaction (PCR) believed that cotton
    swabs would interfere with PCR analysis.57 CDC also found, after
    collecting additional samples in Brentwood to determine the extent of
    contamination, that cotton wipe material (that is, moistened sterile
    cotton gauze) decreased spore recoveries.

    Other factors affecting spore extraction are the physical nature of the
    collection device and surface properties. For example, swabs are easier to
    manipulate and immerse in extract fluid than more bulky wipes are.
    Further, extraction fluids, whether water or a saline solution, with or
    without detergents, differ in their efficiency for extracting spores from
    a swab or wipe. The extraction fluid is also important in that it may
    affect subsequent analyses, by affecting spore germination or by
    interfering with analytic methods, such as PCR. CDC has acknowledged that
    "the recovery efficiency of the analytical methods has not been adequately
    evaluated." The possibility of interference by sponges, used as a sample
    collection method, with PCR, an analytic method, was a factor in CDC's
    decision to use synthetic swabs rather than sponge sampling kits.58

    The reproducibility of the results when an extraction fluid is used can
    also be an issue. For example, a USAMRIID official we interviewed told us
    of an unpublished USAMRIID study conducted to determine the efficiency of
    extracting anthrax from swabs; the study showed that even if the same
    procedure was followed, the results were not always the same.59 Although
    the importance of reproducibility has been recognized, definitive
    scientific information regarding extraction efficiency is lacking. In its
    absence, it is not clear whether sampling results were affected,
    particularly with respect

    57PCR is a process in which a deoxyribonucleic acid (DNA) molecule is
    extracted from a sample and then analyzed with a specific procedure to
    detect the genetic code of known pathogens, such as anthrax.

    58Buttner, Cruz-Perez, and Stetzenbach, "Enhanced Detection of
    Surface-Assisted Bacteria."

    59Using synthetic swabs and a particular type of buffer could lead to 70
    to 75 percent extraction. However, repeating the test with the same type
    of buffer made by different companies yielded different results. The
    official said that this test showed that there were too many variables.
    Even when analysts followed the same procedure, the results were not
    always reproducible, casting doubt on the reliability of the test results.

    to samples that may have contained few spores. Without knowing the
    extraction efficiency, a false negative result may potentially be seen as
    a true negative.

    Activity 5: Analyzing Samples

    Analyzing the samples involved a variety of methods and required two
    steps-preliminary and confirmatory-to generate a final result. The
    laboratory analytic methods that were used for detecting anthrax in
    clinical samples already existed, but they had not been used for
    environmental samples. As a result, different analytic approaches were
    taken at the preliminary step, involving adaptations of such protocols.
    Samples deemed positive at the preliminary step were not always confirmed
    as positive, as was to be expected. However, this could cause problems for
    the agencies. In addition, some agencies considered preliminary analyses
    by field-based instruments unreliable, while others maintained that they
    were reliable but had been used inappropriately. However, once sample
    extracts were subjected to the required confirmatory tests, a positive
    result was indeed a positive.

    In analyzing the postal samples, laboratories used a variety of methods
    for preliminary and confirmatory testing (see fig. 3). Preliminary tests
    included colony morphology, Gram's stain, hemolysis, and motility tests.60
    Any culture isolates that could not be ruled out in the preliminary step
    of testing were considered presumptively positive and referred for
    confirmatory testing. Confirmatory tests included culture analyses
    (traditional microbiological and biochemical analyses), gamma phage lysis
    (a test that identifies the susceptibility of the organism to
    anthrax-specific viruses that create a kill zone in anthrax cultures), and
    direct fluorescent antibody assay, or antibody analyses employing a
    two-component test that detects the cell wall and capsule, or outer
    covering, produced by vegetative cells of anthrax.61

    60When bacteria stained with Gram's stain retained the color of the
    primary stain (crystal violet), they were considered gram-positive, a
    characteristic of anthrax. Hemolysis, a procedure involving culturing,
    identified whether the colonies gave no evidence of red blood cell lysis,
    a characteristic of anthrax. Motility refers to whether the colonies
    showed no movement in microscopic observation, another characteristic of
    anthrax.

    61CDC noted that performance of all the analytical tests did not always
    take place in practice. For example, according to some public health
    laboratory officials we interviewed, different combinations of tests were
    used in some instances or only one component of the direct fluorescent
    antibody was used for confirmatory testing; real-time PCR was used in
    early and later stages of testing for some sampling events.

    Other specialized tests, such as molecular subtyping, were also conducted
    to determine what strain of anthrax was involved. The test results were
    reported as positive-anthrax was found-or negative-anthrax was not found.
    Traditional microbiological analyses require 18 to 24 hours before a
    result can be generated, depending on the laboratory protocols and
    procedures.62 In a few instances, results were also reported as number of
    CFUs per gram of sample material.

    Figure 3: Laboratory Analysis of Samples in Preliminary and Confirmatory
    Tests

                                      Preliminary

                                      Confirmatory

    Do preliminary tests (e.g., Gram's stain and motility) on suspect
    organisms. If they resemble anthrax, report the sample as positive
    (presumptive) and proceed to confirmatory testing. If they do not resemble
    anthrax, report the sample as negative; no further tests are needed.

    Do confirmatory tests (e.g., capsule, gamma phage, and direct fluorescent
    antibody) on preliminary positive sample extract to confirm that it
    contains anthrax. If it does, report the sample as positive (confirmed).
    If it does not, report as negative.

               Source: GAO analysis of CDC, DOD, LRN, and USPS documents.

    Laboratory Protocols Were Adapted for Analyzing Samples

    According to CDC guidelines, LRN laboratories were to analyze samples by
    appropriate LRN protocols.63 According to CDC, all LRN laboratories were
    qualified to perform the preliminary tests, and most could perform
    confirmatory and other specialized tests. While a lower level of LRN
    laboratory could analyze swab samples for preliminary testing, all other
    samples-such as bulk, wipes, air samples, or vacuum samples-were to be
    analyzed at a higher level of LRN laboratory. Samples could also be
    analyzed at CDC laboratories. Presumptive positives found at a lower-level

    62According to USPS, presumptive positive results may be determined on the
    basis of culture-appropriate growth at the first plate reading (18 to 24
    hours of growth) by APHL and LRN procedures. In commenting on our draft
    report, USPS also said that while LRN and APHL procedures call for first
    reading at 18 to 24 hours, during 2001, USPS received calls that indicated
    presumptive growth based upon 12-hour plate culture readings.

    63Earlier CDC guidance, prepared in October 2001, stated that low-risk
    (nonpowder) environmental samples should be processed according to LRN
    level A protocols for ruleout testing in a CLIA-certified laboratory,
    using biosafety level (BSL) 2 facilities and BSL 3 safety practices. The
    guidance was revised in April 2002.

    LRN laboratory had to be referred to an appropriately qualified laboratory
    for confirmatory testing.64

    The LRN analytic protocols already existed for detecting anthrax in
    clinical swab samples, but they had not been used for detecting anthrax in
    environmental samples. Consequently, several revisions were made to the
    LRN protocols. LRN confirmatory protocols provided for processing
    environmental swabs but not other types of samples, such as wet wipes or
    HEPA vacuum. However, according to public health officials, once the
    sample material was extracted, the same procedures used for analyzing
    clinical samples would apply. According to agency and public health
    laboratory officials, LRN protocols were generally used for the samples
    they analyzed, with some exceptions. For example, according to a
    laboratory official, not all components of a particular confirmatory test
    were always performed because of time constraints. CDC also acknowledged
    that, in practice, procedures sometimes differed from approved protocols.

    According to data we reviewed, CDC-approved laboratories, such as public
    health laboratories, and a private laboratory analyzed the samples. The
    dry swab samples, which were the majority, were analyzed by public health
    laboratories or the private laboratory but with different preliminary
    protocols. Before its agreement with APHL in November 2001, USPS used a
    private laboratory to analyze the dry swab samples; this laboratory
    developed its own method of analysis, which it described as "direct
    plating," after which it performed the standard preliminary tests (LRN
    level A protocols).65 CDC officials told us that level A laboratories did
    not have access to the specialized test reagents for gamma phage lysis and
    the two direct fluorescent antibody tests.

    The method involved inoculating a dry swab directly onto a culture plate,
    without first extracting the sampling material and immersing it in a
    solution. According to laboratory officials, once the sample material was

    64Initial tests of unknown clinical and environmental samples under LRN
    level A protocols included colony morphology, Gram's stain, and hemolytic
    and motility tests designed to efficiently rule out Bacillus anthracis.
    Suspect isolates were then subjected to confirmatory testing. LRN level B
    protocols have two methods for confirmatory identification of Bacillus
    anthracis. The first is a demonstration of a capsule combined with
    susceptibility to lysis by gamma phage. The second is a direct fluorescent
    antibody assay for cell wall polysaccharide and capsule antigens.

    65The laboratory was not a member of LRN.

    extracted, the sample extracts were subjected to LRN protocols for
    preliminary testing. Any positives were sent to CDC for confirmatory
    testing. Unlike the testing under the APHL agreement, the laboratory did
    not subject the dry swabs to vortex (that is, rapid rotation of the
    fluid), or "heat shock," procedures. According to an official from the
    private laboratory, direct plating is a good technique when there are low
    levels of spores, and, for the samples, it was more efficient to do
    culture plates than PCR.

    A public health official we interviewed disagreed with the private
    laboratory's approach. His laboratory had a similar method, called "touch
    plate," for environmental monitoring, he said, but direct plating using
    dry swab samples was not the best approach for growth because of the lack
    of efficiency in transferring the spore from the swab to the plate.66
    According to CDC, it also used direct plating for some of the premoistened
    swabs it collected from some facilities. CDC also stated that regardless
    of the method, 100 percent of a contaminant would not be recovered from a
    surface.67

    In some instances, CDC stated, touch plates, commonly called replicate
    organism detection and counting (RODAC) plates, provide better recovery.
    Touch plates are commonly used for sampling smooth, hard surfaces.68 CDC's
    description of a touch plate method differs from the one used by the
    private laboratory. We did not find any comparative studies that assessed
    the relative efficiency of the two methods.

    Under the APHL agreement, a working group with input from CDC developed a
    method that public health laboratories were to use in analyzing the dry
    swab samples collected by USPS contractors. The method was adapted from
    LRN protocols for analyzing premoistened

    66This laboratory was a member of LRN. The laboratory's touch plate method
    consisted of taking a contact plate with agar (nutrient medium) and
    touching surface areas to detect the presence of anthrax or other
    biothreat agents, followed by incubation for growth of colonies.

    67In commenting on our draft, USPS said that it contended that the dry
    swab method, coupled with the analytical methods used by the private
    laboratory, was as effective as any methods initially used to detect the
    presence of viable anthrax spores.

    68One technique using RODAC plates is to press the agar surface of the
    plate onto the sample surface.

    environmental swabs.69 According to APHL officials, the adopted method was
    not validated. Consequently, it had to be revised during the testing. For
    example, the APHL officials found that the heat shock procedure was
    unnecessary, and therefore it was discontinued.

    In addition to traditional analytic tests, such as culture for preliminary
    tests, other laboratory tests were to be used on the postal samples. For
    example, for the USPS dry swab samples, some public health laboratories
    performed real-time PCR. Similarly, for some samples CDC and EPA
    collected, real-time PCR was performed. According to CDC, the confirmatory
    tests were generally reliable, provided that multiple tests were performed
    to confirm the results. However, in testimony in May 2003, a scientist
    from Johns Hopkins University questioned certain aspects of the protocols
    for the analytical procedures USPS and CDC used on premoistened and dry
    swab samples.70

    For example, the scientist stated that (1) USPS procedures did not
    incorporate detergent in sample extraction to aid spore release, (2) the
    volume of fluid used to extract the swab differed between CDC and USPS
    (CDC required 3 milliliters, USPS 1.5 milliliters), (3) the fraction of
    the total extract volume inoculated onto the culture plates differed
    between CDC and USPS, and (4) the number of culture plates that were
    inoculated per sample also differed (CDC 3, USPS 1). Further, according to
    the scientist, both methods cultured too little of the total extract
    volume for use as a rule-out test.71

    69Adaptations of the laboratory protocols included (1) heat shock the
    environmental sample for 30 minutes rather than 10 minutes to clean up the
    sample and allow for an easier read of the microbiological plates for
    growth of Bacillus anthracis spores; (2) the possible use of a sterile,
    disposable serological transfer pipette to inoculate sheep's blood agar
    plates, the 100 microliters being approximated; and (3) because the 100
    microliter inoculum is large for the plates, the use of dry plates for the
    inoculum to soak in sufficiently.

    70The procedures were included in USPS, Draft Interim Guidelines for
    Sampling, Analysis, Decontamination, and Disposal of Anthrax, and in CDC,
    "Comprehensive Procedures for Collecting Environmental Samples."

    71Robert G. Hamilton, testimony, Subcommittee on National Security,
    Emerging Threats, and International Relations, Committee on Government
    Reform, House of Representatives, United States Congress, Washington,
    D.C., May 19, 2003.

    Agencies Encountered Problems with Preliminary Analytic Methods

    The problems agencies encountered in preliminary testing included issues
    related to training and quality control, as well as problems with using
    fieldbased analytic methods with limitations that were not well
    understood. In preliminary testing, a suspect organism must first be
    selected; at this point, human error or quality control issues can affect
    the results. For example, we identified a problem involving culture in the
    preliminary tests-that is, a reliance on the naked human eye to identify
    and select the growth of anthrax on the petri dish. Many different types
    of organisms could be growing that looked like, but were not, anthrax.
    This is significant because when negative results were obtained during
    preliminary testing, no further testing was to be done.

    According to some public health officials, because environmental samples
    often contain Bacillus species commonly found in the environment, any
    organisms isolated by culture would have to be examined carefully to rule
    out the presence of the anthrax organism. Therefore, adequate training was
    essential. According to one public health official, a bacterium called B.
    cereus "looks very similar to anthrax in Gram's stain." As we showed in
    figure 3, Gram's stain is one of the preliminary tests used to rule out
    anthrax. A DOD anthrax expert explained, "Both B. cereus and B. anthracis
    are members of the genus Bacillus but are different species. Gram's stains
    are useful as an initial step in determining whether an unknown
    microorganism could be Bacillus anthracis. If it is gramnegative, it is
    not." The problem with differentiation of various Bacillus species might
    lead to false positive results. Therefore, confirmation is needed.

    Other problems can also affect the reliability of laboratory results.
    According to a public health official, false negatives can result from not
    using positive controls in performing a specific test. For example, a
    defective reagent can cause a test to malfunction and not reveal
    anthrax.72 According to an expert microbiologist, this was a problem with
    laboratories inexperienced in working with anthrax. Although this type of
    microbiology is standard in clinical laboratories, he said, staff
    experience is key. According to another expert, speaking generally,
    determining who is qualified during a crisis is not easy, because standard
    operating procedures may have to be adapted to the situation. Likewise,
    regulatory

    72Laboratories are required to have quality control and quality assurance
    procedures and to use controls to ensure that such an event does not
    occur. In this case, a positive control would involve analyzing a sample
    known to contain a contaminant and performing the required tests, using
    the reagent in question, which should produce a positive result.

    and certification agencies would not have the capacity to set standards or
    proficiency examinations criteria in a crisis.73

    In addition, for about 10 days, in some of the postal facilities,
    beginning on October 31, 2001, USPS contractors collected swab and wipe
    samples for analysis by a portable, PCR-based instrument.74 According to a
    USPS official, USPS collected side-by-side samples, a certain percentage
    of which were to be analyzed by the portable instrument and a certain
    percentage by culture. According to data we received, all the results
    obtained by the portable instrument and culture analysis were negative.
    However, USPS officials told us that the results from the portable
    instrument were inconclusive because of problems associated with false
    positive and false negative response rates. According to USPS, it
    discontinued using the portable instrument, based on advice from DOD. USPS
    officials said there were concerns about the limitations of the instrument
    and level of user experience required, among other things.75

    Preliminary analyses were also performed with field-based analytic tests
    designed to produce more rapid results than laboratory testing. For
    example, on October 18, 2001, USPS arranged for a local hazardous
    materials response team to perform two "quick tests," using hand-held
    assays (HHA) in the Brentwood facility. The results were reported as
    negative the same day.76 According to a USPS official, USPS wanted to get

    73According to the expert, "on-the-fly" proficiency can be evaluated by
    including blind samples. These should include both negative and positive
    samples for qualitative analysis. When the analysis returns a quantitative
    result, the proficiency examination should include different samples
    spanning the range of what the analysis personnel will encounter. Such
    proficiency testing would help determine the value of the test results
    generated during a crisis. Action decisions based on on-the-fly
    proficiency test results could factor in potential analysis problems.

    74A portable device that detects various microbes associated with
    infectious diseases and biowarfare agents; such devices are used by first
    responders, such as hazardous materials teams, to detect hazardous
    substances.

    75USPS provided the following details about the instrument: (1) the
    minimum level of detection is greater than 100 CFUs; (2) inhibitors can
    affect PCR methodology; and (3) although USMARIID data show reasonable
    performance in a laboratory setting with greater quantities of testing
    solution, a high degree of user experience is required to obtain the
    lowest possible false positive (20 percent) and false negative (10
    percent) rates and, in the field, the false positive rate may approach 100
    percent and the false negative, 40 percent.

    76HHAs are small biological test strips, similar to that used in a
    pregnancy test, with colored bands to detect the presence of a live agent.
    The two HHAs were collected from the ventilation air filters above mail
    processing machines in Brentwood.

    a rapid preliminary result while waiting for the results of dry swab
    sampling that USPS contractors performed the same day, which were to be
    analyzed by culture methods. The culture results were not available until
    about 4 days later, when some were reported as positive.

    Concerns about field-based methods created confusion about their
    reliability and the circumstances under which they should be used. For
    example, HHS did not recommend field-based methods, nor did the Office of
    Science and Technology Policy (OSTP), because of concerns about their low
    sensitivity and potential for false positive results caused by nonanthrax
    bacteria, chemicals, or inadequately trained personnel. However, DOD
    stated that the field-based methods were effective when employed
    appropriately.

    In an October 2001 advisory, CDC stated that the utility and validity of
    the HHAs were not known and that it did not have enough scientific data to
    recommend their use. CDC stated further that the analytic sensitivity of
    these methods was limited by the technology; data that manufacturers
    provided indicated that a minimum of 10,000 spores was required to obtain
    a positive result. In a July 2002 memorandum to federal mail managers and
    first responders, OSTP recommended against using commercially available
    detection and identification technologies such as HHAs for detecting
    suspect biological samples, noting that no federal agency had certified or
    approved them. The memorandum also cited the limited sensitivity and
    specificity of HHAs and their risk of false positives and false
    negatives.77

    Nevertheless, according to DOD, it had used HHAs successfully in military
    situations and was continuing to develop the technology. In responding to
    OSTP's memorandum, DOD stated that HHAs were effective when employed as
    part of a "systematic, layered detection approach supported by additional
    levels of confirmation." DOD further stated that HHAs had provided the
    first indication that the letter sent to Senator Daschle contained
    anthrax. According to a DOD expert we interviewed, the instruments were
    reliable. The expert explained that the reference to their unreliability
    stemmed from a combination of factors, including the types of assays that
    were used in the instruments, interpretation of the results,

    77According to OSTP, an HHA is easy to use but should not be used under
    conditions such as (1) sampling porous surfaces, which contain grooves
    that may contain dirt or other substances that could hinder the device's
    effectiveness, or (2) sampling where there is an excessive concentration
    of a suspect agent, which may cause clogging and lead to an inconclusive
    result.

    and how those results were used. He stated that both instruments-that is,
    HHAs and PCR-based instruments-were extremely reliable when their
    constraints were understood, they were used properly, and the results were
    interpreted correctly. He added that the results obtained by HHAs should
    never have been used to make health care recommendations- DOD had never
    recommended this to the user community. Finally, according to an EPA
    official we interviewed, HHAs can be useful when used appropriately by
    first responders who understand their limitations.

    The agencies were also faced with problems when deciding how to respond to
    preliminary positive results that might eventually turn out to be
    confirmed otherwise. For example, agencies did not have clear criteria for
    when to close facilities. In addition, as we recently reported, although
    HHAs were considered preliminary tests, concerns were raised that the
    negative results might lead to a false sense of security.78 During the
    2001 incidents, USPS kept the Brentwood facility open, following CDC's
    advice that closing it was not warranted. According to USPS officials, the
    correctness of this advice appeared to be confirmed by the HHA results
    obtained on October 18, 2001. When CDC confirmed a case of inhalation
    anthrax in a Brentwood employee on October 21, 2001, the facility was
    closed that day. According to USPS, it was not until October 22, 2001,
    that the laboratory's culture tests of the other samples, collected on
    October 18, revealed positive results.

    In a more recent instance, on November 6, 2003, USPS shut down 11 postal
    facilities in and around Washington, D.C., after a preliminary test-not a
    confirmed result-from a routine air sample taken on November 5 indicated
    that a naval mail processing facility might be contaminated with
    anthrax.79 USPS tracked the flow of mail through its own facilities and
    closed 11 postal facilities that delivered mail to the naval facility. The
    subsequent confirmatory tests were negative, and the facilities were
    reopened about 3 days later.

    78See GAO, U.S. Postal Service, GAO-04-239.

    79According to USPS, Navy personnel performed the original sampling and
    analysis, and the preliminary positive result was found at a non-USPS mail
    processing facility. However, USPS also performed testing at the
    designated facilities and other associated facilities; thus, the
    facilities were reopened following confirmatory results obtained by both
    agencies.

    The Five Activities Involved Many Variables That Can Affect Results

    All the activities discussed above are interdependent, and many variables
    for each one can affect the results. Further, problems associated with any
    one of these activities could affect the validity of the results generated
    by the overall process. Given that there are so many variables, the use of
    different sample collection strategies, reflected in site-specific plans,
    could yield different results. For example, three potential sample
    collection plans could be used in one facility-plan A, using one
    collection method (for example, a swab); plan B, using two methods (for
    example, a swab and wipe); and plan C, using three methods (for example,
    swab, wipe, and HEPA vacuum). How these collection methods are to be
    applied-that is, how they are physically used and how much area each
    sample covers-is a variable.80 Within each plan, sample transportation
    protocols could differ, involving variables such as temperature-plans A
    and B might require transporting at ambient temperature, while plan C
    might require freezing temperature-the sample collection method's
    moistness during transport, and the size and construction of the
    packaging.

    In addition, within each plan, laboratory extraction and analysis
    protocols could differ, involving variables such as (1) different
    manufacturers' different formulations of extraction fluids, (2) different
    ways to physically release spores from a particular collection method
    (such as a swab) into the liquid extract (such as by shaking or
    vortexing), and (3) a combination of analytic methods, such as culture or
    PCR for DNA amplification to identify anthrax.81 Any problems experienced
    with any of these variables across any of these plans could affect the
    final result.

    80Material variables could include premoistened or dry swabs or wipes, as
    well as swabs made of cotton or synthetic fiber, wipes made of
    multilayered gauze or cloth, or devices of similar description made by
    different manufacturers. Variables of technique could include coverage
    area per sample and mechanical details of manual collection, such as
    surfacesweep pattern; manner of moistening, holding, and rotating swabs
    and of folding wipes; and firmness of applied pressure related to HEPA
    vacuum hose-nozzle handling or filled sample "nozzle sock" handling.

    81Other variables include different treatments (typically, heat shock) to
    achieve both resistant spore enrichment (relative to more susceptible
    vegetative bacteria and molds) and spore activation in extracts (to
    physiologically induce rapid germination), different techniques for
    concentrating or splitting (or aliquoting) extracts to inoculate culture
    plates, and a combination of qualitative or quantitative analytical
    methods, such as culture for plate count assay or PCR for DNA replicative
    amplification in preparation for strainspecific DNA sequencing analyses
    for anthrax species.

    The Sampling Results

    Were Largely Negative

    The majority of the samples collected from the postal facilities tested
    negative.82 In all, federal agencies collected about 10,000 samples during
    initial testing. USPS contractors collected the majority of the samples
    (7,040), followed by CDC (2,313), in a total of 301 separate sampling
    events. Samples were also collected by EPA, the FBI, and the New Jersey
    Department of Health and Senior Services (NJDHSS), a state public health
    laboratory, for a total of 23 additional sampling events (see table 2).

                  Table 2: Total Samples Collected and Sampling Events

                                   Agency    Samples collected    Sampling events
                                     USPS                7,040
                                      CDC                2,313
                    CDC and EPA (Florida)                  183
                                      FBI                  225
                  State health department                   20
                                   Othera                   26
                                    Total                9,807

    Source: GAO analysis of CDC, EPA, and USPS data.

    aThe sampling agency was not indicated in the data we received.

    82After anthrax was discovered in the Florida postal facilities, CDC and
    USPS began sampling the balance of the facilities. CDC began outbreak
    testing, which was associated with, for example, cases of illness or links
    to the facilities that processed the letters-that is, the primary
    facilities. USPS began precautionary testing of facilities.

    We recognize that the sampling activities of CDC, USPS, and other agencies
    may have been conducted for different reasons and goals.83 Nevertheless,
    it is interesting that of the 9,807 samples that the agencies collected,
    more than 98 percent, or 9,648, were negative; a little more than 1
    percent, or 159, were positive. In all, 286 facilities were tested for
    anthrax contamination. Of these, Brentwood, Trenton, and Morgan were
    primary facilities; that is, these 3 facilities processed the original
    letters containing the anthrax. Although one of the Florida facilities was
    suspected of having processed a contaminated letter, none was found. The
    remaining facilities (283) were mostly tested as part of the FBI
    investigation, USPS precautionary, or CDC outbreak testing between October
    2001 and December 2001.84

    Testing in the Three Primary Facilities Revealed More Positive Results
    Than in the Wallingford Facility

    Testing results differed between the primary facilities and Wallingford
    (as shown in fig. 4). First, in the three primary facilities, results were
    positive each time a facility was tested, with the important exception of
    the two quick tests in Brentwood. In Wallingford, considered less likely
    to be contaminated, results were positive only on the fourth sampling.
    Second, in the primary facilities, sampling with a single method produced
    some positive results, regardless of the sample collection method. In
    Wallingford, neither dry nor premoistened swabs produced any positive
    results. Third, in the primary facilities, both single and multiple
    methods produced positive results; in Wallingford, only multiple methods
    produced positive results.

    83According to CDC, it conducted sampling as part of the outbreak
    investigation. Given that these activities were usually conducted in
    highly suspect contaminated buildings, response actions had to be
    initiated rapidly for the good of public health and all analyses were
    coordinated with level B (or higher) LRN laboratories. In contrast, most
    of USPS's activities were conducted as precautionary testing. USPS
    activities were conducted in selected P&DCs (those USPS facilities that
    exchanged mail with Trenton, Brentwood, or Morgan); therefore, because
    there existed a potential for cross-contamination, response actions were
    conducted in the long term, when compared with CDC activities, and
    analyses were conducted with APHL laboratories that may not have been LRN
    (or, at the least, not level B).

    84Two facilities were also retested in 2002.

    Figure 4: Primary and Wallingford, Connecticut, Facilities: Sampling Methods and
                                        Results

    Source: GAO analysis of CDC and USPS data.

    Note: Numbers in parentheses are total samples collected and total
    positive results for a particular sampling effort (e.g., 29/14).

    aIn April 2002, USPS also sampled elevated areas of the facility, using
    HEPA vacuum.

    bUSPS also had a hazardous materials unit collect two HHAs samples in
    Brentwood on October 18, 2001, which were negative.

    cThe NJDHSS laboratory also collected 20 premoistened swabs, which were
    negative, in the Trenton facility on October 18.

    When comparing the positive results, obtained with dry swabs, across the
    primary facilities, the proportions differed. For example, in one sampling
    event in Brentwood, out of 29 samples collected using dry swabs, 14 were
    positive (48 percent), whereas in Morgan, out of 56, only 7 were positive
    (13 percent). In addition, for the West Palm Beach, Florida, facility,
    sampled several times during one sampling event, out of 38 dry swab
    samples collected, only 1 was positive (about 3 percent). While we did not
    define this facility as primary, it was suspected of processing a
    contaminated letter, although none was found. The use of both wet and

    dry swabs produced positive results in this facility, however (see app. II
    for details).

    Such results cannot be used for drawing specific conclusions, because
    other variables may have been involved. For example, according to CDC, the
    anthrax powder in the letters that these facilities processed differed,
    and the Morgan facility did not use compressed air, which may have
    contributed to increased aerosolization and the spread of spores
    throughout other facilities. The level of contamination in the facilities
    differed. Brentwood ambient air was sufficiently contaminated so that USPS
    employees acquired inhalation anthrax.

    The Majority of the USPS and CDC sampled facilities that processed mail
    from the primary

    Facilities' Test Results facilities to determine whether any other
    facilities had become

    Were Negative 	contaminated. USPS tested 177 of the facilities in
    precautionary testing; CDC tested 113 facilities as part of its outbreak
    investigation testing.85 The majority of test results from these
    facilities were negative: Of 286 facilities sampled, 23 tested positive,
    including the 3 primary facilities, and 263 tested negative (see fig. 5).

    85The sum of facilities sampled by USPS and CDC is greater than the total
    number of facilities sampled because USPS and CDC sampled in some, but not
    all, of the same facilities.

                      Figure 5: Test Results Were Largely Negative

                              Facilities testing positive

                              Facilities testing negative

    The following discusses results for some of the positive facilities,
    excluding the primary ones:

    o  	Generally, only 1 or 2 of the total samples collected for each
    facility were positive, such as several post offices that received mail
    from Brentwood, including Dulles (11 samples collected, 1 positive),
    Friendship Station (32, 1 positive), Pentagon Station (17, 2 positive),
    and Raleigh, North Carolina (42, 1 positive). These facilities were
    considered cross-contaminated.

    o  	West Palm Beach and Wallingford tested positive only on retesting,
    whereas initially they had tested negative. The West Palm Beach facility
    tested positive on the second testing. According to CDC, the sampling
    strategy used in this facility was found to have limitations and was not
    used again.86 However, Wallingford did not test positive until

    86Technically, West Palm Beach tested positive on the second sampling
    event. According to CDC, the strategy for the first sampling event was to
    clean the facility and then collect samples, which may have caused the
    negative result. Additional samples were collected in this facility in
    subsequent sampling events.

    the fourth testing.87 These results underscore the importance of retesting
    and cast doubt on the efficiency of the testing process.

    Of the 263 facilities that tested negative, only 9 were sampled more than
    once.88 A facility in West Trenton tested negative, even though an
    employee had contracted cutaneous anthrax. The facility in West Trenton
    was tested twice by the FBI and once by CDC, during which a total of 57
    samples were collected, with negative results.

    Obviously, final, or confirmed, results will be negative if contamination
    is not present in a facility. However, a result can be negative for
    several other reasons, such as (1) the sampling method was not efficient
    enough, (2) samples were not collected from places where contamination was
    present, (3) not enough samples were collected, (4) not enough spores were
    recovered from the sample material, or (5) analysis of the sample extract
    was not sensitive enough to detect anthrax spores that were present (that
    is, the result was a false negative).

    The sampling at the Wallingford facility is a good illustration of the
    complexities of sampling. According to postal officials, they did not
    expect Wallingford to be contaminated. USPS sampled it twice-on November
    11, in its precautionary sampling, and on November 21, 2001, after a case
    of inhalation anthrax in a postal customer was confirmed. All results were
    negative. USPS contractors, using dry swabs, collected 53 samples on
    November 11 and 64 samples on November 21. However, on November 25, CDC
    collected 60 samples, using premoistened swabs. Still, all results were
    negative.

    Finally, CDC performed what it called extensive and directed sampling on
    November 28, with multiple methods-swabs, wet wipes, and HEPA vacuum (see
    table 3)-and this time, testing produced positive results. Of 202 samples,
    4 wet wipe and 2 HEPA vacuum samples were positive. Some of the samples
    from the mail sorting machines were positive, including a

    87USPS retested Wallingford in April 2002, before cleaning its high-bay
    areas, using a different method. On retesting, positive results were
    obtained for 3 of the 64 samples collected.

    88The facilities were tested more than once for various reasons. For
    example, the Seymour facility in Connecticut was retested because of a
    death in the community related to anthrax.

    sample collected from a machine that primarily processed letter mail. The
    sample was found to contain about 3 million CFUs.

    But it took several sampling events to identify the anthrax spores in the
    mail processing equipment. While the sample from the machine containing 3
    million CFUs was collected on November 28, 2001, another machine (# 6) was
    sampled 5 times, and a total of 77 samples were collected, before anthrax
    was eventually found in an area that held mail for the postal customer who
    had contracted inhalation anthrax. 89 This particular machine would have
    sorted mail by the customer's carrier route and address. In addition, not
    until April 2002 was anthrax identified in Wallingford's high-bay area.
    This further highlights the importance of developing an appropriate
    sampling strategy.

    Table 3: Delivery Bar Code Sorting Machines Sampled, Wallingford Facility, 2001

               USPS precautionary                      CDC outbreak
               testing                                investigation
                                                             Dec. 2, 2001
                                                        (characterization   Total
    Machine Nov. 11, 2001 Nov. 21,   Nov. 25, Nov. 28, 2001 sampling)a samples
               2001                     2001
                                               1            8
                                               1            8

    8

    11 (1) 48 (1)

    2 12

    1 2 3 23 48 (1)

    2 12

    8

    18

    b

    8 (4) 52 (30)

    1 8 (1) 52 (3)

    8

    89Letters addressed to the customer could have been processed on any of
    the 13 DBCSs at the facility during the preliminary sort. The final sort,
    on a designated machine sorting letters to 9-digit or 11-digit Zip
    Codes(TM), involved several runs through the machine. However, this final
    sort of the customer's letter would have been processed on DBCS # 6
    because this machine had specific bins designated for the carrier route to
    the customer's address. According to CDC, samples were collected by
    compositing all bins in a column. Of the 48 columns, only the column
    containing the bins for the Zip Codes(TM) that included the customer's
    town was found positive.

               USPS precautionary                      CDC outbreak
               testing                                investigation
                                                             Dec. 2, 2001
                                                        (characterization   Total
    Machine Nov. 11, 2001 Nov. 21,   Nov. 25, Nov. 28, 2001 sampling)a samples
               2001                     2001
         13                                    1            8                   9
        Total                       1 6        8         130 (6) 200 (35)

    Source: GAO analysis of CDC data.
    Note: Numbers in parentheses indicate positive results.

    aThe first time anthrax spores were found in sample collected from
    delivery bar code sorter # 6.

    bAbout 3 million anthrax spores were found in one sample.

    Interpreting the Test Results

    Since the minimum infectious dose of anthrax for inhalation exposure is
    not known, interpreting environmental sampling data with respect to health
    risks is speculative. Added to this, the lack of performance data on
    sampling efficiency means that the level of confidence that a negative
    result is truly negative is not known (that is, contamination, if present,
    is below a certain level when all samples are negative). Consequently,
    health risk is not known. Therefore, in our May 2003 testimony, we
    recommended that USPS (1) reassess the risk level, based solely on a
    negative sampling result, for postal workers at those facilities deemed to
    be free of spores and the general public served by those facilities and
    (2) reconsider the advisability of retesting those facilities-should the
    decision be made to retest any of these facilities-and use the most
    effective sampling methods and procedures.

    We also recommended that the results of reassessment be communicated to
    the postal workers and the general public.90

    When communicating testing results to, and interpreting data for, a lay
    audience, it is important to include appropriate caveats. If a confirmed
    positive result is obtained in facility testing, saying that a facility is
    contaminated is clearly not a problem. However, it is not possible to
    assess the real degree of contamination, except in a general way, because
    of the lack of empirical data that would allow extrapolation of the
    results from various parts of a facility to the entire facility.

    Problems arise mainly because facilities are not uniform in their complex
    geometry, as well as surface types-rough, smooth, porous, oily, and so

    90GAO, U.S. Postal Service, GAO-03-787T, p. 24.

    on. Even surfaces of identical materials may differ qualitatively,
    depending on their geometrical or spatial arrangement-vertical or
    horizontal, fully exposed or shielded. Although collecting large numbers
    of samples can help in obtaining an accurate assessment, the problem
    remains fundamental. In 2001, difficulty in understanding the true degree
    of contamination was compounded by a lack of knowledge about the
    efficiency of the available sampling and analytical methods.

    When all samples from a facility are negative, interpretation is even more
    difficult. The facility may not be truly contaminated; the negative result
    may stem from the investigators having missed a contaminated area during
    sampling; or the sampling and analytical methods may not be sufficiently
    sensitive. Properly validating the process, including sampling and
    analytical methods, can increase the level of confidence that
    contamination, if present, would be detected in the areas sampled.

    Nevertheless, empirical studies-involving model facilities that can be
    experimentally contaminated to different degrees and then sampled-may
    yield practical information about the number of samples needed to detect
    contamination. This may, in turn, suggest a degree of confidence in
    interpreting negative test results that could be equated with the absence
    of contamination.

    When investigators consider the lack of knowledge about the efficiency of
    sampling and analytic methods, as well as the absence of risk-based data,
    it is important to reassess risk in the light of the challenges the
    agencies faced. In August 2003, USPS stated that to respond to the
    recommendation in our May 2003 testimony, it had formed a working group,
    including representatives from CDC, EPA, OSHA, and postal unions, to
    conduct the recommended review and analysis.91 According to USPS, a
    February 2002 CDC assessment had determined that such retesting was not
    required.

    On August 27, 2004, USPS stated that the working group had concluded, "No
    further testing is warranted for postal facilities that were deemed to be
    free of anthrax spores following the attacks of 2001."92 The group also

    91USPS stated that in its assessment, the group would study the entire
    sampling chronology and the process USPS and others had used, including
    the initial sampling strategy while events unfolded and sampling
    guidelines were enacted, as well as the engineering controls and work
    practices that were implemented.

    92USPS, USPS Response to GAO Recommendation on the Anthrax Attack of 2001
    (Washington, DC.: Aug. 2004).

    concluded that the anthrax risk level, for postal employees in the
    facilities tested and the general public they served, was negligible and
    additional testing would not increase the safety of postal premises for
    employees and customers. According to USPS, factors contributing to this
    conclusion were that (1) no facility was deemed free of anthrax solely on
    the basis of a single sampling result; (2) illness tracking by USPS and
    federal, state, and local health agencies had shown no epidemiological
    evidence of inhalational or cutaneous anthrax among postal employees or
    customers since November 2001; and (3) USPS continued to use engineering
    controls related to anthrax and work practices that reduced the potential
    for another incident in which anthrax could become airborne.

    With respect to USPS conclusions about risk level, we agree that the risk
    is now probably low and that other actions, such as changes to operational
    procedures, have probably decreased the risk even further. But because the
    least efficient method was used to sample a large proportion of the postal
    facilities and since neither the methods nor the process as a whole was
    validated, we concluded that a reassessment of risk was necessary. The
    working group is confident that risk is negligible and it has so informed
    employees and others. We understand that the rationale for not retesting
    is primarily based on the fact that (1) no other postal employees or
    customers has contracted inhalation anthrax disease since November 2001
    and (2) operational changes have lessened the potential for remaining
    spores to become airborne. According to CDC, most of the sampling done at
    facilities with a higher probability of contamination included methods
    other than dry swabs. Therefore, this use of less effective methods where
    contamination might have been lower is counterintuitive and supports the
    concern that incidents of contamination in some facilities may have been
    missed.

    Our recommendation was aimed at facilities deemed free of anthrax from a
    single negative sampling event. The data we received from CDC, EPA, and
    USPS indicated that 254 such facilities were sampled only once. A large
    proportion, 164, were sampled with just dry swabs. Only 9 facilities that
    tested negative were retested; the others were deemed negative from a
    single negative sampling result.93 A large proportion of these facilities
    were considered less likely to be contaminated. However, information

    93In its August 2004 report, USPS apparently interpreted our use of
    "single negative sampling result" to mean only one sample, whereas we used
    the term to refer to a single sampling event.

    obtained during our review suggests that the use of the least sensitive
    method is problematic in such facilities.

    It also appears that the number of samples collected in precautionary
    sampling may not have been enough in such facilities. For example, after
    the extensive sampling at Wallingford, according to CDC, it became
    apparent that considerably more samples were needed and that wet wipes and
    vacuums, which are used to sample larger areas than are swabs, should be
    used in large facilities.94 Therefore, while no more instances of anthrax
    disease related to the release of anthrax in 2001 have occurred, our focus
    remains on the efficacy of the sampling approaches, with a view to
    improvements in the future.

    Finally, it appears that in 2001, on the basis of a single sampling event,
    spores could have been present in some of the facilities that tested
    negative with the least effective method. USPS reported that the risk is
    now low in the facilities tested in 2001, but a sampling strategy that
    addresses a range of situations that may be encountered, including
    facilities with both high and low probabilities for contamination, is
    needed. This strategy should include methods and sample sizes appropriate
    for sampling objectives. This should increase the chances of finding
    contamination in such facilities and confidence that a negative is indeed
    a negative, at least with some level of statistical confidence. In our
    view, the Wallingford experience-in which (1) several initial sampling
    efforts did not identify anthrax in contaminated machinery in November
    2001 and (2) a different sampling strategy identified less contamination
    in other areas of the facility several months later-further indicates the
    importance of a sampling strategy that includes validated methods and
    incorporates probability sampling.

    The agencies took some public health-related actions to respond to
    incidents related to bioterrorism, but they were not fully prepared for
    the nature of the 2001 anthrax incidents. No agency activity to detect
    anthrax contamination in the postal facilities had been validated prior to
    the event. Because validation for select agents is complex and
    time-consuming, it was not possible to perform validation studies during
    the emergency response itself. Therefore, agencies had limited information
    available for

    94E. H. Teshale and others, "Environmental Sampling for Spores of Bacillus
    anthracis," Emerging Infectious Diseases 8 (Oct. 2002): 1083-87.

    Lack of Validation Raises Questions about the Reliability of Negative
    Results

    reliably choosing one method over another and limits of detection for
    interpreting negative results. Our discussions with agency officials and
    other scientists who have worked on microbial detection in indoor
    environments highlighted the significance of the lack of validation of the
    agencies' methods.

    In addition, the opinions of the officials from different agencies
    differed with respect to which methods were appropriate for environmental
    sampling. Public health officials and agency officials involved in making
    decisions in response to anthrax contamination also differed in their
    opinions. For example, the officials differed on whether a swab should be
    moistened or dry before it is used for sample collection. Although
    agencies have made some progress, significant limitations and
    uncertainties continue with respect to the testing process. In particular,
    serious concerns have been raised about the reliability of negative test
    results.

    No Activity or Process Was Validated

    None of the agencies' activities to detect anthrax contamination in the
    postal facilities were validated.95 Validation is a formal and
    independently administered empirical process. For validation, the overall
    performance characteristics of a given method must be certified as meeting
    the specified requirements for intended use and as conforming with
    applicable standards. Because the agencies did not use an empirical
    process to validate their methods of sampling and analysis, the agencies
    had limited information available for reliably choosing one method over
    another and no information on the limits of detection to use when
    evaluating negative results. Consequently, the methods were selected based
    on factors such as available knowledge and sampling associated with fungal
    spores and asbestos particles, as well as the context of an emergency
    response.

    As we noted above, CDC had made some preparation to respond to incidents
    related to bioterrorism in 1998, leading to LRN's establishment in 1999.
    CDC stated that LRN focused first on developing procedures for clinical
    samples. However, since CDC was working with the FBI, environmental
    testing was also a priority for LRN. According to CDC, for example,
    standard laboratory manuals were developed for LRN, with written protocols
    for identifying anthrax and other category A biologic agents. Procedures
    for identifying anthrax were validated and, in some

    95In commenting on the draft report, CDC stated that LRN confirmatory test
    assays for detection of anthrax were validated. However, we were not
    provided with supportive documentation of methodologies used for such
    validation.

    instances, developed or redeveloped from older methods.96 CDC also
    standardized, produced, and distributed to participating laboratories
    reagents for testing.97 According to CDC, in the fall and winter of 2000,
    scientists at state public health laboratories were trained to use these
    analytic methods for identifying anthrax and other biologic agents.

    In October 2001, however, environmental samples far outnumbered clinical
    samples, so that environmental sampling had a much larger role than CDC's
    preparedness efforts had anticipated.98 In October 2001, CDC stated,

    Currently, no occupational or environmental exposure standards exist for
    B. 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. anthracis

    spores from the sample collection media) has not been adequately evaluated
    and limits of detection have not been established.99

    Lacking validation, therefore, it was not known what (1) the overall
    performance characteristics of the various sample collection methods that
    the agencies used were and (2) the recovery efficiency and limits of
    detection of the agencies' methods.

    Performance characteristics include, for example, determining how many
    spores must be present on a tested surface to give a positive result-the

    96See B. Perkins and others, "Public Health in the Time of Bioterrorism,"
    Emerging Infectious Diseases 8:10 (Oct. 2002): 1015--618. Since the
    document did not specify details, what constituted validation was not
    discussed.

    97Capacity for specialized or more developmental diagnostic and other
    tests for anthrax- for example, real time PCR; direct fluorescent
    antibody, immunohistochemical, molecular subtyping; and antimicrobial
    testing-were established at CDC and, in some instances, other advanced
    laboratories such as USAMRIID.

    98According to CDC, LRN laboratories processed more than 121,700 samples
    for anthrax, the majority being environmental samples from areas of
    suspected or confirmed contamination.

    99CDC, "Comprehensive Procedures for Collecting Environmental Samples," p.
    1.

    Validation Is Independently Administered

    lowest limit of detection-and whether the same process, if repeated under
    similar conditions, will produce similar results. Because all the methods
    the agencies used to collect samples and analyze them had inherent
    limitations, these methods produced results that were accurate and
    specific only within certain limits. To interpret and apply the resultant
    data meaningfully, it is essential to know what these limitations were.
    Otherwise, a negative result may tell us only that the amount was less
    than whatever the detection limit was, rather than that there was no
    contamination.

    As validation is generally understood, it is independently administered by
    an accredited third party, the validating authority, as (1) meeting the
    specified requirements for its intended application and (2) conforming to
    applicable standards.100 Formal validation can lead to regulatory
    acceptance and approval listing. Third-party validation is commonly
    performed on a wide range of certifiable methods, employed in such
    wellregulated areas as pharmaceuticals, clinical laboratory analysis, and
    environmental monitoring. However, before formal validation procedures can
    be applied, the specified goals for overall performance, including peer
    review of the method's operating principles, must be thoroughly developed.

    This process involves well-controlled performance testing, including both
    positive and negative controls, as well as calibrated standards. If a
    method is to become widely accepted, its principles of operation should
    pass the test of expert peer review.101 When recognized performance
    standards and validation criteria do not exist, a set of validation
    criteria for the method, acceptable to prospective end users for some
    intended purpose, must be specified. Besides being validated, a reliable
    method-as part of a wellmanaged quality assurance process-must be well
    controlled during the proficiency training of its prospective
    practitioners and in actual field

    100Validation generally requires accredited third-party administration of
    concurrent empirical trials at multiple independent laboratories.
    Generally, methods are validated by a process of statistical conformity
    assessment; by a series of trials with reference to a set of applicable
    performance standards, including appropriate positive controls (procedures
    employing calibrated physical standards); and by overall method
    performance criteria.

    101This generally involves the coordinated use of a method on calibrated,
    unknown test samples by multiple practitioners (often independent testing
    laboratories), under some accredited third party's administration, and
    statistical analysis of the results to assess multiple performance
    parameters and suitability for use under some specified conditions.

    Nonvalidated Sampling Strategies Could Be Based on False Assumptions

    practice.102 Successfully completing validation offers some assurance that
    a method's final results are sufficiently robust that the method can be
    relied on for reproducible results, regardless of the practitioner,
    agency, contractor, or laboratory.

    Without validated techniques for sampling, transporting, extracting, and
    analyzing, agencies' sampling strategies could be based on false
    assumptions (see fig. 6). Use of nonvalidated sampling methods means that
    the collection efficiency of these methods was not known. Not validating
    transportation requirements means that spore viability could have been
    reduced, leading to a false interpretation of a negative test result-that
    is, that an original sample contained no anthrax spores when in fact it
    did, producing a false negative. Transportation conditions were largely
    determined by regulations for transporting etiologic agents. There is no
    reason, however, why both safety and sample integrity cannot be achieved
    by appropriate packaging and shipping conditions. CDC stated that the
    evidence in the literature suggests that the integrity of the samples was
    never jeopardized. While we agree with this statement, it fails to
    recognize that in establishing sampling and associated activities, to
    demonstrate that procedures are appropriate and effective, there is a
    burden of proof that can only be effectively addressed by validation.

    102Controls consist of standardized materials and associated procedures
    for using them effectively. They are generally of two types: Negative
    controls (typically, sterile specimen blanks) are expected to result in
    negative tests and are used to guard against false positives, such as
    those stemming from cross-contamination. Positive controls are expected to
    yield well-calibrated positive results and are used to guard against
    variable inaccuracies and false negative testing results.

    Figure 6: Lack of Validation Can Affect Individual Activities and the
    Overall Process

    Source: GAO analysis of CDC, EPA, and USPS data.

    Without validated extraction methods, the basic capabilities and technical
    skills that the laboratory staff performing this task need cannot be
    understood. For example, if it is expected that a high percentage of
    spores will be lost during extraction, the staff must be highly trained so
    that the maximum number of spores can be extracted. Similarly, the staff
    should be sufficiently trained to analyze the sample extract to reflect
    whether the sample was originally contaminated (positive) rather than
    containing no spores (false negative). Nonvalidated analytic methods
    cannot be demonstrated to be uniform (or reproducible) or trusted to yield
    correct results. Consequently, any negative results involving nonvalidated
    activities raise questions about their reliability.

    Agencies' Advice Cannot Be Definitive without Validation

    The agencies' advice on issues related to sample collection and analytic
    methods could not be definitive, given the lack of validation and how
    information was evolving from agencies' experiences during the
    investigation. According to APHL, for example, on November 5, 2001, in a
    dialogue between CDC and APHL working group members, an NCID
    representative stated that dry DacronTM swabs could be used to sample
    environmental surfaces but that wet wipes, although not necessary, would
    probably pick up more material. The working group was in the process of
    selecting a collection method for USPS contractors to use while doing
    precautionary sampling and developing an analytical protocol. The NCID
    official had been assisting in adapting the LRN analytic protocol for the
    collection method selected-the working group's final decision was to use
    dry swabs.

    In commenting on USPS draft guidelines-being developed when USPS
    contractors were already sampling the facilities under the APHL agreement
    (using the USPS Standard Sampling Plan)-CDC and EPA suggested that USPS,
    for several reasons, include other methods besides premoistened and dry
    swabs. On November 9, for example, CDC advised USPS that using bulk and
    vacuum samples, in addition to swabs, could be useful to investigators,
    but CDC acknowledged that some state public health laboratories might be
    less familiar with the methods needed to analyze such samples.103 CDC
    stated that it recognized that USPS's decision to use only swabs was
    related to an accommodation reached with APHL's laboratories to more
    effectively use state health laboratories for analysis.

    In a December 4, 2001, letter to USPS. EPA stated that using methods other
    than dry swabs would benefit USPS. EPA said that its experience in
    sampling on Capitol Hill indicated that USPS should incorporate other
    collection methods into its interim guidelines-such as wet wipes,
    premoistened swabs, HEPA vacuum, bulk, and air sampling-for its
    environmental sampling from initial steps through decontamination. EPA
    said, "All [would] likely benefit from increased sensitivity of these
    methods over the current dry swab technique used by USPS."

    By this time, USPS had completed the majority of its sampling. However, in
    sampling Wallingford's high-bay areas in April 2002 before annual

    103CDC's NIOSH officials were commenting on USPS guidelines, Guidance for
    the Sampling, Analysis, and Decontamination and Disposal of Anthrax for
    Postal Facilities, interim draft 1.2 (Washington, D.C.: Nov. 7, 2001).

    cleaning, USPS used not dry swabs but HEPA vacuum, to ensure that no
    anthrax was present.104

    The scientific community did have some knowledge about the performance of
    some of the agencies' methods. For example, evaluations had been carried
    out in laboratory settings with swabs, wipes, and vacuuming to detect
    anthrax or similar bacteria but with varying results in sample extraction.
    In addition, swabs, wipes, HEPA vacuum, and air had been used to collect
    samples for hazardous substances other than anthrax. Culture, an analytic
    method, was well established for detecting anthrax in clinical samples,
    but it was not validated for detecting anthrax in environmental samples.
    According to CDC, the analytic methods that the agencies used were
    generally considered reliable when multiple tests were performed to
    confirm a result.

    Nevertheless, the relative efficiency and accuracy of traditional methods
    were not known. CDC reported in December 2001 that little data on the
    accuracy of the analytic methods for detecting spores existed. According
    to a public health official, real-time PCR was the most accurate of the
    methods (culture, direct fluorescent antibody, gamma phage lysis) for
    analyzing samples collected by different methods (swab, wipe, HEPA vacuum,
    and air). However, PCR does not provide information on the viability of
    the organism.105 Knowing whether the organism is viable is important in
    considering health risks and what antibiotic will be effective against
    disease.

    To better understand the efficiencies of some of the methods of collection
    and analysis, agencies performed limited studies in some of the primary
    facilities during the incidents. The studies provided additional
    information about the methods' efficiency but did not validate them. One
    of the studies-the December 2001 CDC side-by-side study of the performance
    of

    104CDC completed sampling about December 2, 2001. Its peak sampling events
    were October 22 to November 3. USPS sampled between October 18 and
    November 28, 2001, except for the South Jersey facility, sampled in
    February 2002, and Wallingford's high-bay areas, sampled in April 2002.

    105According to DOD, PCR tests were traditionally used in laboratories to
    presumptively identify most biological agents, but were time-consuming,
    taking approximately 6 hours to produce a result, whereas real-time PCR
    (excluding sample preparation time) could take 2 hours or less. However,
    according to a DOD official, sample extraction cannot be separated from
    the PCR process. In the anthrax incidents, real-time PCR was used for
    preliminary testing.

    dry and premoistened swabs, wet wipes, and HEPA vacuum sample collection
    in the heavily contaminated Brentwood facility-confirmed the views of the
    experts and researchers, particularly about the relatively poor
    performance of dry swabs. The study found that premoistened swabs detected
    spores more than 33 percent of the time when spores were detected by wipe
    and HEPA vacuum sock samples. Dry swabs failed to detect spores more than
    66 percent of the time when they were detected by wipe and HEPA vacuum
    samples. This study concluded that dry swabs should not be used to sample
    the environment for anthrax.

    However, according to the study, it was not adequate to evaluate the
    sampling efficiencies of the wipe and HEPA vacuum samples. The study also
    concluded that there was a need to "quantify sampling efficiency to
    develop the type of limit-of-detection data normally created for other
    types of sampling and analytic methods." As to analysis, two CDC studies
    in 2001 found that real-time PCR and direct fluorescent antibody were
    sensitive, specific methods for detecting anthrax in environmental samples
    and isolates.106 Some of the agencies' studies are described in table 4.

    106A. F. Hoffmaster and others, "Evaluation and Validation of a Real-Time
    Polymerase Chain Reaction Assay for Rapid Identification of Bacillus
    anthracis," Emerging Infectious Diseases 8 (Oct. 2002): 1178-82, and B. K.
    De and others, "A Two-Component Direct Fluorescent-Antibody Assay for
    Rapid Identification of Bacillus anthracis," Emerging Infectious Diseases
    8 (2002): 1060-65.

    Table 4: Agency Evaluations, October 2001 through February 2002

    Date Agency Objective and method

    Conclusion

    October CDC      To learn more about the potential for a
                                                   contaminated
    2001a       mail processing machine as a continual
                   source of
                   aerosolized anthrax spores and whether
                   particle
                   concentration in the air could be estimated.

    Collected surface samples (using 10 RODAC plates and 10 premoistened
    swabs) from a Brentwood mail sorting machine (after the facility closed)
    to see if it was still contaminated; air samples (10 banks of slit
    samplers) were placed about 5 feet above the machine.

    October EPA To learn the extent of indoor secondary aerosolization 2001b
    of anthrax spores under active and inactive office RODAC plates and swabs
    showed anthrax growth. Two plates showed low-level contamination and 3
    CFUs; they were negative by the swab method. Air sampling detected anthrax
    before and after the machine was activated. Even after processing more
    than 1.2 million letters following initial contamination and surface
    cleaning, aerosolized particles containing anthrax can still be detected
    around a contaminated machine.

    Defining the risk of inhalational anthrax in primary and secondary
    aerosolization of anthrax spores needs more study.

    Anthrax spores released in a U.S. Senate office building were
    re-aerosolized in common office activities. The potential for secondary
    aerosolization of viable anthrax spores originating from contaminated
    surfaces in indoor environments has implications for appropriate
    respiratory protection, remediation, and reoccupancy of contaminated
    office environments.

    conditions.

    Collected surface samples (swabs and microvacuums), air samples (Anderson
    6-stage sampler and 2-stage sampler), and open agar plates.

    December CDC, To compare the relative efficiency of sampling 2001c ATSDR,
    methods used to collect spores from contaminated and surfaces.

    USPS 	December 17-20, 2001, side-by-side performance comparisons of
    surface samples collected at Brentwood with dry and premoistened swabs,
    wet wipes, and HEPA vacuum.

    February CDC To compare air sampling methods side by side.

    2002d	and Evaluated air sampling methods (mixed cellulose,USPS
    polytetrafluoroethylene, gelatin-coated, dry filters, and Swabs performed
    poorly. Dry swabs were least effective, failing to detect spores more than
    75% of the time that wipes and HEPA vacuums detected them from nonporous
    surfaces. Dry swabs should not be used to sample the environment for
    anthrax; premoistened swabs could be used in certain circumstances. Wipe
    and HEPA vacuum sampling yielded similar results on nonporous surfaces.
    Developing numerical criteria for surface contamination and potential
    human exposure requires understanding sampling efficiency.

    All methods detected spores to some degree; walking and light work may
    re-aerosolize spores; failure to plate entire sample in analysis may
    result in false negative; the Anderson method seemed the most sensitive
    for spore collection; and the dry filter unit may have reduced the number
    of spores available for collection because of high flow rate and may be
    least sensitive, given the air volume passing through a sampler.

    Anderson single-stage cascade impactors) for anthrax spores before and
    after the operation of mail sorting equipment in the Trenton P&DC.

    Source: GAO analysis of CDC, EPA, and USPS data.

    aP. M. Dull and others, "Bacillus anthracis Aerosolization Associated with
    a Contaminated Mail Sorting Machine," Emerging Infectious Diseases 8
    (2002): 1044-47.

    bC. P. Weis and others, "Secondary Aerosolization of Viable Bacillus
    anthracis Spores in a Contaminated U.S. Senate Office," Journal of the
    American Medical Association 288 (Dec. 11, 2002): 2853-58.

    cW. T. Sanderson and others, "Surface Sampling Methods for Bacillus
    anthracis Spore Contamination," Journal of Emerging Infectious Diseases 8
    (2002): 1145-50.

    d CDC, "Hazard Evaluation and Technical Assistance Report: NIOSH
    Evaluation of Air Sampling Methodologies for Bacillus anthracis in a
    United States Postal Service Processing and Distribution Center, Trenton,
    New Jersey," report HETA 2002-0109-2927 (Cincinnati, Ohio: Department of
    Health and Human Services, CDC, National Institute for Occupational Safety
    and Health, 2004).

    Lack of Validation Highlighted Experts' and Agencies' Different Opinions

    The significance of the lack of validation of the agencies' various
    detection activities was highlighted in our discussions with scientists
    and researchers who have worked on microbial detection in indoor
    environments. Their opinions differed on sampling methods and sample
    material appropriate for environmental sampling and processes necessary
    for validating methods. Public health and agency officials involved in
    making decisions on responding to anthrax contamination also differed.

    Experts at USAMRIID indicated that they knew before October 2001 that dry
    swabs were ineffective at collecting spores and that the swabs should be
    moistened before being used. According to a scientist at Johns Hopkins
    University School of Medicine, using dry swabs in environmental testing
    has not been justified since the swab-rinse assay was introduced in
    1917.107 A NASA study indicated that premoistened swabs were useful in
    attempting to detect biological substances on smooth, nonporous surfaces
    in a spacecraft.108 As to air sampling methods, according to an expert,
    highvolume air samplers would detect spores, even in minimally
    contaminated facilities, whether or not the spores had settled onto
    surfaces. While not discounting this opinion, another scientist stated
    that in air sampling, spores might deteriorate and thus cause negative
    results (through culturing), since they would not germinate or grow on a
    plate because of stresses encountered during air sampling. This scientist
    also noted that air sampling was an expensive approach and would require
    isolation of an area. According to a DOD expert, "Endospores are highly
    resistant to desiccation (for example, drying process or very low humidity
    environment), which is the most likely stress they would encounter in air
    sampling and then only if they were sampled onto a dry filter."

    As for extraction, experts have stated that spores could not be recovered
    efficiently-that is, extracted during laboratory processing-from dry swabs
    and that swabs made of synthetic material were more efficient than cotton
    swabs for picking up particles, including bacterial spores. As we

    107Robert G. Hamilton, testimony, May 19, 2003, pp. 3 and 7.

    108National Aeronautics and Space Administration, Office of Space Science,
    " NASA Standard Procedures for the Microbial Examination of Space
    Hardware," in NASA Procedures and Guidelines, NPG: 5340.1D, final draft
    (Washington, D.C.: no date).

    discussed earlier, concerned that microbial growth would affect analysis
    of the sample material, agency and public health officials differed on
    whether swabs should be moistened.

    Although most agency officials and scientists agreed that the agencies'
    methods were not validated, they held different opinions and took
    different approaches with regard to the procedures that are necessary
    before any detection method can be considered validated. In addition, we
    found that agency officials were defining the term "validation"
    differently. According to the NIOSH Manual of Analytical Methods, a
    validated method is one that "meets or exceeds certain sampling and
    measurement performance criteria."109 However, according to a public
    health official and some agency officials, a less formal approach-such as
    the established use of a method with demonstrated, consistent outcomes
    over time by different users-could also establish a method's validity.

    In this regard, a public health official we interviewed said that because
    the results from the use of real-time PCR during the 2001 incidents agreed
    with the results achieved by culture, real-time PCR was essentially
    validated. CDC stated that it had validated the use of real-time PCR for
    environmental samples but not direct fluorescent antibody. In addition,
    agencies may differ as to what constitutes validation. For example, a 1997
    report looking at the validation and regulatory acceptance of
    toxicological methods found that the agencies it reviewed did not have a
    definition of test validation, although, with variations, they followed
    certain procedures to accomplish validation.110

    109P. C. Schlecht and P. F. O'Connor, eds., "Glossary of Abbreviations,
    Definitions, and Symbols," in NIOSH Manual of Analytical Methods
    (NMAM(R)), 4th ed., HHS (NIOSH) publication 94-113 (Washington, D.C.: Aug.
    1994), p. A-10.

    110National Institute of Environmental Health Sciences, Validation and
    Regulatory Acceptance of Toxicological Test Methods: A Report of the Ad
    Hoc Interagency Coordinating Committee on the Validation of Alternative
    Methods, NIH publication 973981 (Research Triangle Park, N.C.: Mar. 1997).

    Agencies Have Taken Some Steps to Prepare for Future Incidents

    To prepare for future incidents, the agencies have taken some steps,
    basing them on what has been learned about some of the limitations of
    their sampling strategies and associated methods. For example, they have
    revised their guidelines or developed new ones to reflect some of this
    knowledge and experience. However, the information in these guidelines
    related to environmental testing is not based on empirical validation
    studies.

    After the 2001 incidents, the National Response Team, chaired by EPA,
    developed a technical assistance document, which is still in draft, as a
    specific resource for responding to an actual or a suspected release of
    anthrax outside an agricultural environment.111 It was designed for a wide
    audience to use, including first responders who discover a potential
    release, government agencies responding to a release on their own property
    or as part of a federal effort, and facility managers and owners who may
    discover a release. The document does not prescribe specific actions for
    every case. It provides scientific background and viable options for users
    to consider in facing specific circumstances.112

    The draft Technical Assistance for Anthrax Response discusses sample plan
    development, objectives, approaches, and methods. It also reflects several
    statements in CDC's guidance. For example, it states that there are
    currently no validated methods of sampling and analysis specifically for
    anthrax in environmental samples. In addition, with respect to field-based
    methods-that is, HHAs and PCR-based instruments-it states that until
    further validation testing is completed and guidelines have been developed
    for these methods, they should not be used alone and that any results
    should be confirmed with samples analyzed by laboratory culture
    methods.113 The draft does not list dry swabs as a collection method.
    However, unlike its references on the appropriate use of field-based
    methods, it excludes references to the dry swab method's limitations and
    whether it should be used for sampling.

    111National Response Team, Technical Assistance for Anthrax Response,
    Interim-Final Draft Phase I Update (Washington, D.C.: November 2003),
    sect. 1.1. The Interim-Final Draft was issued in September 2002.
    http://www.nrt.org/Production/NRT/NRTWeb.nsf/AllAttachmentsByTitle/A-47AnthraxTAD/
    $File/Anthrax.pdf?OpenElement (Jan. 9, 2005).

    112The General Services Administration and OSHA, among others, also
    developed guidance on anthrax.

    113Section 1.1 of Technical Assistance for Anthrax Response states that
    new information related to detection and decontamination will be added as
    soon as it is available.

    With respect to sampling strategies, the draft suggests that targeted
    sampling may be appropriate to determine whether anthrax is present when
    the source of the contamination is known and quickly isolated. However,
    the draft also states that if the source of the contamination is not known
    or quickly isolated, the sampling approach should include statistically
    based sampling. This draft document seems to recognize the risks
    associated with the use of targeted sampling when certainty does not exist
    with respect to the presence or location of the anthrax. Another risk
    associated with the use of a targeted approach, identified during our
    review, relates to the level of contamination present in the facility.
    Since at the outset one may not know for sure the level of contamination,
    and if the level of contamination is below some detection limit, negative
    results from targeted sampling may give a false sense of security.
    However, through using probability sampling, negative results can be
    interpreted to provide an evaluation, at a certain level of confidence, of
    the maximum level of contamination that may be present. This is the key
    advantage of probability versus targeted sampling.

    With respect to sampling and analysis, USPS's December 2003 revision of
    its "Interim Guidelines for Sampling, Analysis, Decontamination, and
    Disposal of B. anthracis Spores" generally reflected related sections in
    Technical Assistance for Anthrax Response. For example, for sampling
    objectives, sampling approach, and analytic methods, USPS refers to the
    relevant chapters in Technical Assistance for Anthrax Response, stating
    that USPS "may use any of the sampling methods prescribed in the TAD
    [Technical Assistance Document] depending on the nature of the sampling
    and site-specific conditions."114 According to USPS officials, USPS is in
    the process of updating its guidance and intends to replace the December
    2003 guidelines for anthrax with a more comprehensive "all hazards"
    emergency response plan for addressing future natural and artificially
    created emergencies.

    USPS has also begun implementing a biodetection system, having deployed
    303 units at 44 sites, as of October 2004. The system collects and does a
    preliminary analysis of samples from the environment, triggering an alarm
    if anthrax is detected. The technology will detect only anthrax and not
    other threat agents. Guidelines for implementing the new detection system
    call for taking immediate emergency action, including evacuation,

    114USPS, "Interim Guidelines for Sampling, Analysis, Decontamination, and
    Disposal of B. anthracis Spores in USPS Facilities," p. 20.

    as soon as it is activated. Facilities are to reopen only if a follow-up
    analysis of a sample is negative for anthrax-a process that can take
    several days.115 According to USPS, OSTP has evaluated the technology.
    However, we have not reviewed OSTP's evaluation because it was beyond the
    scope of this report.

    CDC testified in May 2003 that it was planning to update its November 9,
    2001, Interim Anthrax Response Plans and Guidelines.116 An April 2, 2002,
    CDC document, "Comprehensive Procedures for Collecting Environmental
    Samples for Culturing Bacillus anthracis," stated that preliminary
    analytic methods should not be used alone and that any results should be
    confirmed, with samples analyzed by laboratory culture methods. In May
    2004, CDC officials said that CDC had learned a great deal in fall 2001
    about the potential for aerosolization but that its knowledge and approach
    were not yet fully reflected in its guidance. According to these
    officials, CDC had expected to publish updated guidance on its approach by
    the end of 2004, with recommendations for responding to positive
    environmental samples in postal facilities.

    CDC has made public some of its views and conclusions about the testing
    methods and approaches in the postal facilities. For example, it testified
    in May 2003 that none of the premoistened or dry swab samples collected in
    Wallingford were positive. As a result, CDC recommended that wet wipe and
    HEPA vacuum sampling be used in collecting environmental samples for
    investigating large facilities. Further, CDC stated that its investigation
    in Wallingford showed that extensive sampling was required and that
    epidemiologic investigation was essential in identifying sites for
    sampling.117

    USPS said that by the time it sampled the high-bay areas of Wallingford,
    it had applied what had been learned about the collection methods. This

    115Follow-up analysis involves culturing the sample that triggered the
    alarm. Spores 	collected on a filter in the detection system are cultured
    so that the resulting bacteria can 	be positively identified.

    116Kenneth F. Martinez, "CDC and ATSDR Activities at the Southern
    Connecticut Processing 	and Distribution Center in Wallingford, CT,"
    statement before the Subcommittee on 	National Security, Emerging Threats,
    and International Relations, Committee on 	Government Reform, House of
    Representatives, U.S. Congress, Washington, D.C.,	May 19, 2003.

    117W.T. Sanderson and others, "Surface Sampling Methods for Bacillus
    anthracic Spore 	Contamination," Journal of Emerging Infectious Diseases 8
    (2002): 1145-50.

    time, USPS used not dry swabs but HEPA vacuums. CDC is also participating
    with other agencies, including EPA and DHS, in related research on various
    methods, which we discuss later in this report.

    In contrast to the situation in 2001, DHS now has a significant role in
    responding to acts of terrorism by coordinating the homeland security
    functions of many federal agencies, including research. 118 It is not
    clear, however, which agency will be specifically responsible for
    validation studies. DHS has authority to support research in bioterrorism
    and is undertaking or sponsoring some studies, including studies on HHAs,
    as are other agencies.119 DHS states that it regularly attends interagency
    meetings with representatives from ATSDR, CDC, EPA, and the Defense
    Advanced Research Projects Agency. It says that it plans to sponsor
    workshops with DOD on biothreat agents and the appropriate method for
    sampling in a given scenario. Category A biothreat agents and resultant
    diseases include Variola major (smallpox), Clostridium botulinum
    (botulism), Yersinia pestis (plague), and Francisella tularensis
    (tularemia), as well as anthrax. To the officials' knowledge, agencies
    without DHS support are not required to inform DHS of such projects.

    DHS is planning several projects related to anthrax sampling. For example,
    it is involved in a domestic demonstration and application, a
    collaborative project with EPA and CDC's NIOSH. The goals are to identify
    "how clean is clean"; improve sample collection efficiencies; identify
    rapid viability determination, statistical sampling methods, and a
    sampling database; and develop a rapid viability determination method for
    spore strips. DHS has also established a working group to develop
    standards for surface and air sampling. In January 2005, DHS and DOD's
    Technical Support Working Group (TSWG) convened the First Annual National
    Conference on Environmental Sampling for Bio-Threat Agents, a forum for
    government, industry, academia, and first responders to address critical
    issues in environmental sampling.

    118In 2001, no federal response plans were triggered, DHS did not exist,
    and no agency was designated the overall lead.

    119DHS said that it is also developing a Knowledge Management Center at
    the National Biodefense Analysis and Countermeasures Center. The center is
    to collect and reference all data, such as sampling methodology, that the
    federal community can use for response decisions. According to DHS, having
    a single repository for information would allow a more timely analysis of
    current methodology and capability during an event.

    According to a DHS official, Homeland Security Presidential Directive 5
    (HSPD-5) identifies DHS as being in charge of managing a federal
    response.120 HSPD-5 states that

    the Secretary of Homeland Security is the principal federal official for
    domestic incident management. Pursuant to the Homeland Security Act of
    2002, the Secretary is responsible for coordinating federal operations
    within the United States to prepare for, respond to, and recover from
    terrorist attacks, major disasters, and other emergencies.

    According to the official, DHS would coordinate all involved agencies so
    there is one response objective. However, DHS would not override the
    existing authority of involved agencies. During the 2001 anthrax
    incidents, USPS officials told us that although they received input on
    sampling or public health matters from various other agencies, including
    DOD, they deferred to the agency of jurisdiction, which they considered
    CDC to be. During that period, there was some confusion over which methods
    should be used, primarily because of the lack of validated methods and
    difficulty arranging for laboratory analysis of all types of samples, as
    well as large numbers of samples.

    In addition, each of the different agencies had a different focus. For
    example, according to CDC,

    The lines of authority for managing this line of crisis were very
    confusing, and there was a

    lack of assigned responsibility government-wide for taking the lead role
    in coordinating a

    response to these attacks, a situation which has been rectified with the
    creation of the DHS.121

    It is still not clear which agency would have the lead responsibility for
    conducting validation studies and whether DHS would fund them.

    120HSPD-5 also states, "To prevent, prepare for, respond to, and recover
    from terrorist attacks, major disasters, and other emergencies, the United
    States Government shall establish a single, comprehensive approach to
    domestic incident management. The objective of the United States
    Government is to ensure that all levels of government across the Nation
    have the capability to work efficiently and effectively together, using a
    national approach to domestic incident management. In these efforts, with
    regard to domestic incidents, the United States Government treats crisis
    management and consequence management as a single, integrated function,
    rather than as two separate functions." George W. Bush, Homeland Security
    Presidential Directive 5/HSPD-5, the White House (Washington, D.C.:
    February 28, 2003), sect. 3.

    121See GAO, U.S. Postal Service, GAO-04-239.

    According to APHL surveys since 2001, intended to provide information on
    the status of laboratory capability and capacity, there have been some
    improvements in this area, following supplemental funding received in
    2003. 122 For example, when surveyed in 2002, respondents in 13 states
    reported having only one staff member trained to perform confirmatory
    testing for one or more category A biothreat agents, including anthrax.
    Most reported needing physical facility upgrades, including biosafety
    capabilities, and seven did not have real-time PCR capability. The 2003
    follow-up survey showed some improvement; for example, all respondents had
    at least one staff member trained to perform confirmatory tests for
    anthrax and only one had no real-time PCR capability. However, respondents
    continued to report the need for facility upgrades, including biosafety
    capability, and some had only one real-time PCR instrument and, thus, no
    surge capacity. Concern about staffing continued: The "issue most
    frequently cited was recruiting new staff." APHL has stated that it
    intends to continue to conduct such surveys annually.

    While the actions by agencies and others will help increase knowledge,
    make available some general guidelines, and provide a forum for
    discussion-all important aspects in emergency preparedness-it is not clear
    how (1) a well-planned and coordinated effort-that will ensure the
    problems and limitations we and others identified will be addressed-is to
    be achieved or (2) the limitations in laboratory capacity will be
    addressed.123 For example, it is not clear how the entire process for
    anthrax detection will be validated. It is also not clear how an
    appropriate approach-that will increase confidence that anthrax will be
    detected in facilities with both high and low probabilities for
    contamination-is to be developed, managed, and funded. Finally, agency
    officials also did not

    122According to APHL, in May 2002, it surveyed 53 state and territorial
    public health laboratories (receiving 48 responses) to establish a
    baseline status of laboratory capability and capacity, as of December 31,
    2001, which was before the allocation of federal emergency supplemental
    funds for terrorism preparedness, released in June 2002. This allocation
    provided $146 million for state and local public health laboratory
    improvements. APHL conducted a follow-up survey in February 2003 of active
    members in 50 states, the District of Columbia, and three territories (51
    responded).

    123In commenting on our draft report, EPA stated, "Increasing lab capacity
    takes considerable time and involves locating appropriate and available
    lab space, hiring or locating qualified microbiologists, obtaining
    security clearances (6 months to one year), rebuilding laboratory space
    for Safety Level 3, immunizing staff to work with anthrax, arranging for
    safe sample transport, and obtaining standards and reagents from CDC's
    limited repository. Increasing lab capacity is a long term goal, with
    extensive efforts underway to do so."

    Ongoing Studies May Help Validate Individual Activities but Are Not Aimed
    at the Process as a Whole

    disagree that the individual activities and the process need to be
    validated for other biothreat agents, although officials expressed
    concerns about the resources needed for such an undertaking.

    Since the fall of 2001, limited comparative studies have been performed or
    are under way. However, these studies are limited in addressing only some
    aspects of an individual activity rather than the overall process. CDC has
    performed some studies of surface sampling methods and analytic methods.
    For example, with the FBI, it conducted a study of selected HHAs and
    concluded that the 2002 devices were not reliable.124 CDC stated that it
    has further studies under way, in conjunction with DOD and EPA, to
    determine the collection efficiency and limits of detection for surface
    and air sampling methods. In addition, CDC stated that studies involving
    researchers from CDC, EPA's National Homeland Security Research Center,
    and DOD were to begin to validate sampling methods. According to EPA, its
    Homeland Security Research Center is participating in a research project
    with CDC and others; a project with a national research laboratory; and
    the planning of a third project with the Department of the Navy to
    standardize and validate sampling and analytical techniques for surface
    sampling methods for anthrax. We describe the findings of some recent and
    ongoing studies below:

    o
    A study of some of the collection methods concluded that macrofoam swabs,
    when processed using certain methods, were superior to other swabs
    studied, such as those made of cotton, polyester, or rayon.

    125

    o
    A study of some of the analytical procedures for ruling out anthrax in the
    preliminary phase found that (1) using a particular nutrient growth
    medium, rather than the one used during the response, was more efficient
    and (2) occasional false positive results had been obtained

    124FBI and CDC, "Preliminary Findings on the Evaluation of Hand-Held
    Immunoassays for 	Bacillus anthracis and Yersinia pestis," Forensic
    Science Communications 5:1 	(Jan. 2003).

    125The study included minimal agitation, sonification, and vortexing
    processing methods 	and premoistened and dry swab preparations to evaluate
    four swab materials-cotton, 	macrofoam, polyester, and rayon-used to
    sample stainless steel surfaces inoculated with 	a known concentration of
    anthrax spores. Results showed that (1) premoistened swabs	were more
    efficient at recovering spores than dry swabs, (2) premoistened macrofoam
    and 	cotton swabs that were vortexed during processing recovered the
    greatest proportions of	spores, and (3) premoistened and vortexed
    polyester and rayon swabs were less efficient. 	See L. Rose and others,
    "Swab Materials and Bacillus anthracis Spore Recovery from 	Nonporous
    Surfaces," Emerging Infectious Diseases 10:6 (June 2004): 1023-29.

    with PCR protocols previously evaluated with reference strains of anthrax
    rather than field isolates.126 It is important to note that the
    investigators did not have access to the LRN's PCR and were not using the
    tests used by U.S. laboratories. CDC has stated that these findings are
    irrelevant to the U.S. ability to accurately identify environmental and
    clinical Bacillus anthracis isolates.

    o
    A study on detection systems and diagnostic support systems for
    bioterrorism response, including HHAs and PCR-based instruments,
    concluded, "Many of the evaluations performed to date are critically
    deficient."127 The study stated that if testing is performed at a
    relatively

    high pretest probability (as in a heavily contaminated building), a
    negative test result will be convincing only if the sensitivity of the
    system is very high. This brings to mind the situation at Brentwood, where
    the two preliminary HHAs, or "quick tests," were negative in a facility
    that was considered likely to be contaminated, based on the fact that it
    had processed the contaminated letters. However, the study also stated
    that some of the systems reviewed were the subject of ongoing evaluations
    that would provide additional information and help users interpret the
    results provided by such systems.

    o
    Several studies, funded by TSWG, focused on the sensitivity and
    specificity of sample collection methods for detecting anthrax and other
    biological substances.128

    o
    A March 2004 study TSWG funded showed that no one method or procedure
    could by itself be relied on, because of the many variables involved.
    Variations in humidity, temperature, air pressure and movement, uniformity
    (or more likely nonuniformity) of particle

    126See J. Papaparaskevas and others, "Ruling Out Bacillus anthracis,"
    Emerging Infectious Diseases 10:4 (Apr. 2004): 732-35. The study included
    72 environmental nonbacillus anthracis bacilli to study methods for ruling
    out anthrax. The study concluded that the most effective methods were (1)
    the use of horse blood agar, (2) motility testing after isolates had a
    2-hour incubation in trypticase soy broth, and (3) screening isolates with
    a Bacillus anthracis-selective agar.

    127See D. M. Bravata and others, "Evaluating Detection and Diagnostic
    Decision Support Systems for Bioterrorism Response," Emerging Infectious
    Diseases 10:1 (Jan. 2004): 100-

    08.

    128TSWG operates and is overseen by a number of agencies, including DOD
    and the Department of State. A national forum that includes several
    multiagency subgroups, it identifies, prioritizes, and coordinates
    interagency and international research and development requirements for
    combating terrorism.

    distribution, and sampling technique (not just the type of sampler but the
    actual sampling technique and movements) can affect results.129 In
    addition, the study showed that sampling efficiencies varied widely (from
    1 percent to 85 percent), depending on the surface sampled (glass,
    concrete, nylon, and computer screens) and the sampling method (cotton
    swab, sponge swipe kit). For one study task, a comparison of the analytic
    methods used showed that quantitative PCR was more sensitive than culture
    when detecting the microorganism used in the study (that is, Bacillus
    globigii spores).130 In addition, through air sampling, it became evident
    that the spores had become reaerosolized while surface samples were
    collected.

    o
    With TSWG funding, the Pacific Northwest National Laboratory is studying
    how to enhance the capabilities of the Visual Sample Plan, a software
    package. The software enables an agency to quickly create a site-specific
    sampling plan that allows it to define, with high confidence, the
    magnitude and extent of contamination; guide decontamination efforts; and
    verify the effectiveness of decontamination.

    The completed and ongoing studies, as well as the evaluations performed in
    the facilities, have contributed to, and are likely to continue to
    contribute to, knowledge about how to sample for anthrax. However,
    questions still remain about the performance characteristics of the
    methods. Since the overall process has not been validated, several
    unresolved issues remain:

    o
    It is still not possible to know (1) with what level of confidence each
    sample collection method will reveal the true extent of contamination and
    (2) how efficient the methods are, compared with one another when sampling
    different types of surfaces, such as those that are (a) porous or
    nonporous, (b) simple or complex, or (c) made of different materials, such
    as plastic, glass, or carpet.

    129Mark P. Buttner, Patricia Cruz-Perez, and Linda D. Stetzenbach,
    Development of Biocontaminant Detection/Identification Strategies for CBrN
    Countermeasures, draft final technical report (Las Vegas, Nev.: Harry Reid
    Center for Environmental Studies, University of Nevada, March 1, 2004).

    130Quantitative PCR is a fast, accurate, and reliable way to measure the
    distribution and expression of target DNA or RNA.

    o
    It is not known how each sample collection method compares when processed
    by each analytic method (culture, direct fluorescent antibody, or PCR).
    For example, will a HEPA vacuum sample extract analyzed by culture produce
    the same result if analyzed by PCR, and how do these results compare with
    results of similar analysis of, for example, a wet wipe.

    o
    Quantitative results based on standardized sample collection and analytic
    methods do not yet give information that relates to health risk to
    individuals.

    o
    It is not clear (1) how effective emerging technologies will be (such as
    those that provide rapid preliminary results in the field), without
    improved sensitivity, specificity, and reproducibility, and (2) under what
    conditions these technologies can be used in sampling postal facilities or
    other indoor environments.

    In addition, the five activities are interdependent, and the problems of
    one activity can affect the results of a subsequent activity. Therefore,
    according to an academic expert we consulted, validation and performance
    evaluation of the end result must span all five activities. It is
    possible, and even likely in some cases, that different individuals,
    agencies, and organizations will perform the activities individually, at
    different locations. Standard operating procedures for each activity must
    be validated, but an additional validation method that embraces all the
    activities-the overall process-is also needed. Further, the analysis
    process could be tested in real time in crisis situations to determine
    whether overall processing has been effective and accurate. Such testing
    could be accomplished by using negative and positive control samples that
    pass through all five activities.131

    With respect to interpreting sampling results, according to this expert,
    it is important to know the limits of detection and the error rate. In
    addition, when the error rate is low, ironically, validation exercises to
    estimate the

    131According to the expert, negative controls might include swabs that are
    not used for sample collection but that are passed along with the real
    sampling swabs. Crosscontamination from the samples or other controls
    would be apparent if the mock swabs generated a positive result. Likewise,
    to ensure that sampling and subsequent processing can detect a real
    result, positive swabs could be placed in the sample stream at the
    collection site. Passage through the five activities should generate a
    positive result at the end. The lack of a positive result would indicate
    that the sampling scheme and processing could not detect a positive result
    reliably, even if there was one.

    rate become even larger. For example, at a 1 percent error rate, he said,
    one would have to process 100 control samples to detect a single erroneous
    result. To estimate a 0.1 percent error rate, the exercise becomes tenfold
    larger. In such cases, the actual error rate might not be determined, but
    an upper limit can be estimated. For example, the rate might be estimated
    at no greater than 1 percent. Understanding the error rate for the
    individual activities and the overall process becomes invaluable for
    interpreting sampling results.

    Without a validated process, the issues and problems we have highlighted
    will remain unresolved. Therefore, in detecting anthrax, there can be
    limited confidence about the reliability of any of the surface collection
    methods or the whole process. As a result, negative results cannot be
    viewed with a great degree of confidence, in particular because the
    minimum human infectious dose for anthrax is still not known. Therefore,
    methods that are sensitive, specific, reproducible, robust, and with a
    greater chance of identifying low levels of anthrax are needed. Commenting
    on the overall anthrax investigation in the United States, CDC stated in
    October 2002 that the investigation had several limitations:

    Environmental sampling of potentially contaminated facilities used
    different testing methods; because less sensitive testing methods were
    used, certain sites may have

                                          132

    underrepresented the degree of contamination.

    Test Reliability Remains Uncertain Despite Agencies' Progress

    In summary, CDC and other agencies have taken a number of actions since
    2001 to address testing issues, including research on the comparative
    efficiency of certain collection and analytic methods. DHS was not
    established until 2003, and the Office of Homeland Security played only a
    limited role during the 2001 incidents. Since then, however, DHS has taken
    steps to manage subsequent events involving anthrax. Despite these
    efforts, the significant limitations and uncertainties with respect to
    various aspects of the testing process raise serious concerns about the
    reliability of test results. They include federal agencies' lack of
    assurance that anthrax will be detected in facilities that are not heavily
    contaminated or that are considered to have a low probability of
    contamination.

    132Daniel B. Jernigan and others, "Investigation of Bioterrorism-Related
    Anthrax, United States, 2001: Epidemiologic Findings," Emerging Infectious
    Diseases 8 (Oct. 2002): 1019-

    28.

    DHS has met with other federal agencies doing studies on testing methods,
    and it is doing its own studies, but no comprehensive overall plan exists
    to identify needed research, priorities, agency responsibilities, or time
    schedules. DHS could work with relevant agencies to see that a systematic
    validation plan is developed. It could also make a proactive effort to
    ensure that testing methods are validated; necessary research is carried
    out; and agency guidance, policies, and procedures reflect lessons learned
    as a result of the incidents that have occurred and research that has been
    performed since 2001.

    Key questions we believe need answers include the following:

    o
    What sampling strategies can provide a known level of confidence that
    anthrax will be detected in facilities, especially those that are not
    heavily contaminated?

    o
    How efficient are the various testing methods, and what minimum amounts of
    anthrax spores have to be present if anthrax is to be detected by these
    methods?

    o  Do transportation conditions affect the integrity of samples?

    o
    How effective are the various methods for extracting material from samples
    for analysis?

    o
    Do laboratories have the capability and capacity to analyze different
    types of samples?

    o  How should validation be defined?

    o
    What changes to agency policies, procedures, and guidance should be made
    to reflect lessons learned and the results of the validation process?

    Conclusions
    Federal agencies responsible for responding to the 2001 anthrax incidents
    have not been fully prepared. They adopted a targeted sampling strategy
    that they based on their best judgment at the time. We agree that the
    situation in 2001 was unique and that the agencies faced many challenges.
    Therefore, we are not faulting agencies for their actions in 2001.
    However, an approach for the future that incorporates probability sampling
    is needed. Without probability sampling, samples will not be
    representative of the total environment. Therefore, testing will not be
    able to provide

    reasonable assurance, with any defined level of confidence, to workers,
    customers, and the public that negative test results mean that a tested
    facility is free of contamination, within the limits of detection for the
    methods of sample collection and analysis.

    Site-specific sampling strategies in 2001 were essentially targeted to
    detect anthrax in locations believed to have the highest likelihood of
    contamination. While this was effective in facilities with a high level of
    contamination, or where information could be obtained on the likely
    contamination pathways, such as those facilities that processed the
    contaminated letters, it may not have been as effective in facilities
    found to have low or localized levels of contamination and when such
    information was not available.

    When the level of contamination is extremely high and dispersed in a
    facility, the method of sampling (for example, wipes versus swabs) may not
    be as critical, if the purpose is to find some contaminant. However, at
    lower levels, a way of interpreting negative results is needed, and this
    requirement emphasizes the importance of validation of methods and
    statistically based sampling strategies.

    Therefore, it is necessary to invest in empirical studies so as to develop
    a probability-based sampling strategy that will account for the complex
    geometry and surface types of many facilities. Using a probability-based
    sampling strategy, together with validated methods for detecting
    contamination, would provide a known level of confidence with which to
    interpret any negative results and would thus enable agencies to be more
    definitive in determining necessary actions.

    The lack of validated methods for assessing contamination in postal
    facilities impeded the agencies in responding to the incidents. The
    significance of the lack of validated methods was exemplified in the case
    of the Brentwood facility, where negative preliminary results were
    obtained by field-based methods of analysis, with limitations that appear
    to have been not well understood by some agencies. In commenting on our
    draft report, CDC stated that "it is important to note that CDC issued a
    Health Advisory on October 18, 2001, the very same day that these samples
    were collected, which explicitly addressed the severe limitations in

    handheld immuno assays for detection of anthrax spores."133 In contrast,
    USPS comments on our draft report stated:

    The Brentwood facility was kept open based on the advice of the CDC and
    not as a result of the two quick field assay tests. Additional swab-based
    sampling had been completed on October 18, 2001, but results were not
    available until October 22, 2001, the day after closure of the Brentwood
    facility (October 21, 2001) due to diagnosis of a confirmed case of
    inhalation anthrax disease in a worker. The swab-based sample results
    showed that the facility was indeed contaminated. Thus, either the
    confirmed anthrax disease case or the positive analytical results for the
    presence of viable anthrax spores would have resulted in facility closure.

    Whatever the reasons, the facility remained in operation with the
    potential of continuing exposure of workers. CDC confirmed a case of
    inhalation anthrax in a worker, and the facility was immediately closed on
    October 21, 2001. Similarly, the Wallingford experience shows that had a
    mail recipient not had a case of inhalation anthrax, the facility might
    have been regarded as clean, given the initial negative testing results.

    The Brentwood and Wallingford examples demonstrate the need to ensure that
    all the methods that are used are validated, so that their performance
    characteristics, including their limitations, are clearly known and their
    results can be correctly interpreted. "Validation," interpreted in
    different ways, should be clearly defined in one way that is agreed on
    among the relevant agencies. Validation should be defined so that, at the
    very least, it provides information about a method's performance
    characteristics and covers specificity, reproducibility, and limits of
    detection.

    The need that all methods, from sampling to final analysis, be validated,
    so that their performance characteristics can be clearly understood, is
    not in doubt. But any combination of methods that makes up the overall
    process should also be validated because the effect of different
    permutations of methods may not be predictable. However, the number of
    ways methods may be combined is large, and which particular set of methods
    may be used in particular circumstances is not known. Therefore, it may
    not be possible to always validate the entire process, especially if
    agencies are to

    133http://www.bt.cdc.gov/agent/anthrax/environment/handheldassays.asp.
    This advisory, which was distributed via Health Alert Network, provided
    agencies, including USPS, with clear warnings that (1) these methods were
    not sufficiently sensitive; (2) a negative result does not rule out a
    level of contamination below 10,000 spores; and (3) the utility and
    validity of these assays was unknown.

    have flexibility in dealing with particular circumstances. It must be
    recognized, however, that an inability to validate the entire process
    reduces, to some degree, the level of confidence in the results. To assess
    the impact of relying on the validation of individual activities,
    experiments could be performed with a limited number of processes,
    combining different methods.

    We collected data only on initial sampling done in connection with the
    2001 anthrax incident, but the issues we have raised clearly apply to all
    aspects of sample collection, from initial sampling to verification
    sampling. The issues we have raised in this report, however, also apply to
    any anthrax incident, including the March 2005 incident involving DOD
    facilities in the Washington, D.C., area.

    In addition, while the 2001 events involved anthrax, many other biothreat
    agents exist. Differences in their characteristics mean different
    solutions. Accordingly, efforts to develop sampling strategies and to
    validate methods should address requirements specific to those threat
    agents as well. However, since addressing other agents would consume
    resources and time, all these efforts should be prioritized in a long-term
    strategy.

    The several agencies that dealt with the anthrax attacks generally worked
    well together, but we have identified areas that would have benefited from
    one agency's taking the lead in coordinating the response. Given the
    mission of DHS and its responsibilities, it appears that DHS is now well
    positioned to take a lead role in promoting and coordinating the
    activities of the various agencies that have technical expertise related
    to environmental testing. In addition, it is important that all
    participating agencies recognize and support DHS in that role and that
    they have an effective structure for participating in identifying and
    addressing the appropriate issues.

    Recommendations for Executive Action

    Given the lack of validated methods for detecting anthrax contamination in
    facilities, we recommend that the Secretary of Homeland Security develop a
    coordinated approach to (1) improve the overall process for detecting
    anthrax and (2) increase confidence in negative test results generated by
    that process. This approach would include working with agencies to ensure
    that appropriate validation studies of the overall process of sampling
    activities, including the methods, are conducted. Specifically, the
    Secretary should

    Agency Comments 	and Our Evaluation

    1.
    take a lead role in promoting and coordinating the activities of the
    various agencies that have the technical expertise related to
    environmental testing;

    2. ensure that a definition of validation is developed and agreed on;

    3.
    guarantee that the overall process of sampling activities, including
    methods, is validated so that performance characteristics, including
    limitations, are clearly understood and results can be correctly
    interpreted;

    4.
    see that appropriate investments are made in empirical studies to develop
    probability-based sampling strategies that take into account the
    complexities of indoor environments;

    5.
    ensure that appropriate, prioritized investments are made for all
    biothreat agents; and

    6.
    ensure that agency policies, procedures, and guidelines reflect the
    results of such efforts.

    We obtained written comments on a draft of this report from CDC, DHS, and
    USPS. We also obtained written comments from APHL on excerpts from the
    draft that pertained to its role in anthrax testing. Although we requested
    comments from DOD and EPA, DOD said it had no comments and EPA provided
    only technical comments. The written comments we received from CDC, DHS,
    and USPS, as well as APHL, are reprinted in appendixes III (CDC), IV
    (DHS), V (USPS), VI (APHL), and VII (DOD). Their key concerns are
    discussed below. In addition, most of these agencies, as well as APHL,
    provided technical comments, which we addressed in the body of our report,
    as appropriate.

    CDC, DHS, and USPS, as well as APHL, agreed with our conclusion- methods
    for detecting anthrax contamination in facilities were not validated-and
    with the thrust of our recommendations-calling for a coordinated,
    systematic effort to validate the methods to be used for such testing.
    CDC, DHS, and USPS (1) disagreed with or expressed concern about our
    conclusions or the recommendation dealing with targeted versus probability
    sampling, (2) emphasized that validated testing methods for anthrax were
    not available in 2001 and that federal and state organizations did the
    best they could under the circumstances, and (3) identified factors or
    issues that need to be considered in validating testing methods. DHS
    indicated that some validation efforts are now under

    way and other federal agencies, including EPA and HHS, have important
    roles to play in addressing the sampling and validation issues we raised.
    DHS also said that it would work with these agencies to define and address
    validation and "develop a scientifically defensible sampling strategy." In
    addition, USPS disagreed with our conclusion about negative results-that
    there can be little confidence in their reliability due to the sampling
    strategy used and the lack of validated testing methods.

    CDC generally agreed with our recommendations addressing the need to
    coordinate across various federal agencies and to improve and validate
    environmental sampling methods for anthrax and other biothreat agents.
    However, CDC did not believe that our draft report sufficiently recognized
    the importance of targeted sampling. CDC, in its technical comments,
    indicated that "the report could be more useful and informative" if it
    provided additional information and clarification on the following four
    issues: (1) public health context for environmental sampling, (2)
    validation, (3) sampling and analytical methods, and (4) probability
    versus targeted sampling.

    In particular, first, CDC stated that the report's focus on environmental
    sampling contributed to "a narrower view" than is needed to "understand
    the full picture." According to CDC, when evaluating potential risks at a
    given facility, it "typically combines environmental results from initial
    assessment sampling information with other information, such as
    outbreak-specific epidemiology findings and facility engineering and work
    practice factors." In addition, CDC stated, "Environmental samples most
    often identify surface contamination which is not the same as exposure.
    Surface contamination is not directly translatable to risk of inhalation
    anthrax and more research is needed on this correlation." We agree with
    CDC that surface contamination is not directly translatable to risk of
    infection. However, it is important to recognize that in our report, we
    addressed fundamental and broad issues concerning sampling and analysis,
    with implications far beyond public health, including key environmental
    contamination issues, and that our report did not suggest that surface
    contamination is directly translatable to risk. Nevertheless, a clear
    understanding of the extent and degree of surface contamination is the
    most basic requirement for understanding the risk to humans. Therefore, we
    agree with CDC that more research should be done to improve sampling and
    analytical methods, as well as the evaluation of risk.

    Second, CDC stated that "it would not have been technically possible for
    CDC or the other agencies to arrange for validation in a few days time
    while in the midst of a national emergency." Further, CDC stated that

    proper validation would require significant time and personnel. Therefore,
    "full validation of every possible scenario variation would be impractical
    and [that it] could not take the place of scientific judgment and
    evaluation of the specific event." In our report, we identified the
    absence of validation and some of the consequences; we did not state that
    validation could have been carried out during the response phase. However,
    we clarified our report in response to CDC's concern, as well as similar
    concerns expressed by DHS and USPS. Furthermore, our report clearly stated
    that empirical validation is a rigorous process, which requires time and
    resources. In addition, we recommended that DHS conduct appropriate
    validation studies, on a prioritized basis. These studies would not
    attempt to address every scenario, but as an adjunct to scientific
    evaluation of the specific event, they would enhance the reliability of
    sampling and analytical methods.

    Third, according to CDC, although validation was not performed, there was
    an objective basis for choosing one sampling method over another during
    the 2001 incidents. CDC cited its 20-year history of sampling and
    analytical method development, collaborative work with LRN level B
    laboratories, and a study conducted after the incident. We agree with CDC
    that these factors may be useful in assisting agencies in choosing
    methods. However, it is also important to note that these factors cannot
    adequately substitute for empirical validation studies. We were pleased
    that CDC agrees that more information is needed to fully establish the
    validity of testing methods and that CDC has indicated that it is now
    collecting data in support of validation.

    Finally, CDC stated that "probabilistic sampling by itself [emphasis in
    original statement] is unlikely to be as effective or expeditious as
    targeted sampling" for initially identifying contamination for those cases
    where there is some knowledge about the source. According to CDC, a
    targeted sampling strategy can provide a rapid determination of whether
    contamination is present. However, we believe a major weakness of the
    approach is that if all samples are negative, it is not possible to
    conclude, with a defined level of confidence, that a facility is free of
    contamination. CDC does agree that targeted sampling does not support
    statistical inference. In contrast to targeted sampling, probability
    sampling allows more detailed interpretation of negative results. Such
    sampling allows extrapolation, in a statistically valid way, as to the
    level of contamination. In interpreting negative results, a known level of
    confidence is needed because evidence suggests that even a few anthrax
    spores could cause disease in susceptible individuals.

    According to CDC, the report's focus on negative results might obscure a
    number of larger issues. For example, CDC pointed out that "A key goal of
    initial assessment is rapid [emphasis in original statement] determination
    of whether contamination is present so public health decisions can be
    quickly made." CDC then suggests that timeliness is important because
    public health interventions such as "provision of post-exposure
    prophylaxis is most effective if administered within a short time window
    after exposure." Nevertheless, if results were falsely negative, agencies
    would have no basis for taking public health measures for the occupants of
    the contaminated building. Thus, relying solely on targeted sampling could
    actually result in delays in implementing time-critical public health
    interventions. On the other hand, we recognize that implementation of
    probability sampling would generate a larger sample size than initial
    targeted sampling, which may not be possible for a laboratory to analyze
    at one time. However, we believe that steps can be taken to address this
    issue, as we discussed in this report; that is, samples can be analyzed in
    a prioritized way. Furthermore, we agree that targeted sampling can play a
    useful role in prioritizing sample collection or in selecting areas or
    surfaces considered most likely to be contaminated, when the source of
    contamination is definitive.

    We consider probability sampling to be a viable approach that would
    address not only the immediate public health needs-provision of
    antibiotics-but also the wider general environmental contamination issues,
    such as infrastructure cleanup. In any particular facility, probability
    sampling could operate in the following ways: At the outset of a response,
    a statistically based probability-sampling plan would be drawn, based on
    the facility dimensions, complexities, and other characteristics. Initial
    outbreak sampling, within the dictates of the probability-sampling plan,
    could be targeted to those areas that, based on scientific and technical
    judgments (if available), are considered most likely to be contaminated.
    We believe that targeted sampling can be an important component of the
    wider probability-sampling plan, provided information on the source of
    contamination is definitive. If these early (targeted) samples yield
    positive results, then appropriate public health measures could be
    instituted. Further sampling of the facility, to address characterization
    and cleanup issues, could take place subsequently. But if initial targeted
    samples were negative when the likely contamination source is not
    definitive, completing the probability-sampling plan would then permit an
    assessment, with appropriate confidence limits, of the likelihood of
    contamination, even if all of the samples taken were negative.

    DHS stated that while it has the overall responsibility for coordination
    for future biological attacks, EPA has "the primary responsibility of
    establishing the strategies, guidelines, and plans for the recovery from a
    biological attack while HHS has the lead role for any related public
    health response and guidelines." DHS further stated that EPA "is
    developing specific standards, protocols, and capabilities to address the
    risks of contamination following a biological weapons attack and
    developing strategies, guidelines, and plans for decontamination of
    persons, equipment, and facilities [emphasis in original statement]." DHS
    pointed out that in the Conference Report on H.R. 4818, the conferees
    expressed their expectation that EPA will

    enter into a comprehensive MOU [memorandum of understanding] with DHS no
    later than	August 1, 2005 that will define the relationship and
    responsibilities of these entities with 	regard to the protection and
    security of our Nation. The Conferees expect the MOU to 	specifically
    identify areas of responsibilities and the potential costs (including
    which entity 	pays, in whole or part) for fully meeting such
    responsibilities. EPA shall [is to] submit to	the House and Senate
    Committees on Appropriations a plan no later than	September 15, 2005 that
    details how the agency will meet its responsibilities under the	MOU,
    including a staffing plan and budget.

    Finally, DHS stated, "Even though DHS is in charge during a biological
    attack, EPA is primarily responsible for the coordination of the recovery
    process. So, DHS will coordinate with EPA to ensure appropriate
    investments are made to explore improved sampling."

    With respect to our recommendation that DHS develop probability-based
    sampling strategies, DHS said that it must first define the necessary
    requirements for the sampling process and then evaluate targeted and
    probability-based sampling strategies against those requirements. DHS said
    that targeted sampling may be beneficial for some applications. We agree
    with DHS on the need to define the requirements for the sampling process
    and to evaluate sampling approaches against those requirements. On the
    basis of the work we have done on this review, we believe that (1) DHS
    will find that targeted sampling will not always meet all the requirements
    to answer the question of whether a facility is contaminated and (2)
    probability-based sampling will be necessary when information on the
    source and path of potential contamination is not definitive. In our view,
    this will be the case in order for DHS to achieve its goal of having a
    "scientifically defensible sampling strategy and plan."

    Although USPS disagreed with some aspects of our findings and conclusions,
    it generally agreed with our recommendations to DHS and

    noted in particular its belief that validated detection methods should be
    developed for all biothreat agents. USPS disagreed with our finding about
    the reliability of negative results, that is, that there can be little
    confidence in them because the agencies involved did not use probability
    sampling and there was no validation of agencies' sample collection and
    analytical methods. It pointed out that it followed the advice it received
    from the best available experts at the time. USPS further pointed out that
    the efficacy of its testing approach was confirmed by the August 2004
    report of a working group that was convened to respond to a recommendation
    we had previously made; this group's findings were discussed earlier in
    this report. On the other hand, USPS acknowledged that (1) disputes about
    its testing protocols arose among experts after it had substantially
    completed its testing and (2) even today, there is still no consensus
    among experts on the best sampling method to use.

    We are not challenging the conclusions reported in the August 2004 report
    by the working group that more that 3 years after the anthrax incident
    postal workers in the postal facilities tested in the fall of 2001 faced
    little risk from anthrax. However, for the reasons discussed in our
    report, the working group's conclusions do not mean that the negative test
    results--produced in 2001 using targeted sampling and nonvalidated testing
    methods-were reliable. As the working group points out in its August 2004
    report, the fact that no one at these facilities has become sick from
    anthrax since 2001 provides strong evidence that the number of residual
    spores is low or that conditions no longer exist for spores to become
    aerosolized and create harmful exposures. But this rationale cannot be
    used to conclude that the sampling approach and testing methods used in
    2001 produced reliable results. As we learned in 2001, a few spores can
    cause death (as was the case with the elderly Connecticut woman); given
    that the potential for more people to have been affected by anthrax
    existed in 2001, it was fortunate that no one else has become sick. We
    cannot rely on the argument that no one has become sick to answer the
    question of whether facilities are contaminated. In the future, we need to
    base such conclusions on appropriate sampling methods and validated
    testing methods. USPS, however, agreed with our recommendations and on the
    importance of validation.

    APHL agreed with the need for validated testing methods and indicated that
    the lack of such methods hindered laboratories in 2001 and remains a
    critical gap in preparedness and response today. APHL also said that with
    the advent of the Biohazard Detection System in postal facilities, there
    is an urgent need for standard sampling and testing methods to avoid
    complication and lack of confidence in resolving any initial positive

    screening results. In addition, APHL expressed some concerns with the
    excerpts from our draft report that it reviewed. APHL said that it was
    concerned that the sections of the report it reviewed could be interpreted
    as an indictment of its decisions without adequately considering (1) the
    urgency of the situation, (2) the context in which decisions had to be
    made, and (3) the basis of the decisions. In particular, APHL said that
    the report implied decisions were made based on convenience and hearsay
    instead of scientific knowledge of conventional microbiology and bacterial
    spore characteristics. APHL also said that it found numerous scientific
    inaccuracies, misinterpretations, and undocumented statements.

    It was not our intent to suggest that APHL's decisions were based on
    convenience and hearsay. We stated in our report that limitations existed
    in the amount of information available in 2001 about anthrax and testing
    methods to detect it. We also pointed out that since the fall of 2001,
    agencies have not been fully prepared. In addition, we stated that experts
    have differing views on the definition of validation and how validation
    should be done. We were not in a position to resolve these disagreements
    and based our recommendation for more research partly on these
    disagreements. Furthermore, we do not believe that the report contained
    inaccuracies and misinterpretations. We reported the information provided
    to us by various agencies and experts and obtained documentation or
    corroboration whenever possible. As we said in the report, however, there
    were instances in which people expressed differing views; in the case of
    the use of dry versus premoistened swabs, conflicts existed between what
    was sometimes said orally and-based on documents we were provided-what was
    written. As we also state in the report, agencies were not in a position
    to give definitive advice on issues related to sample collection and
    analytic methods due to the lack of validated methods. We believe this
    situation contributed to APHL's concerns.

    APHL also identified what it considered an example of a technical error,
    that is, our statement that

    the decision to use dry rather than wet swabs stemmed partly from the
    concern of some public health officials, including APHL officials, that
    moistened swabs would allow anthrax spores to germinate and grow into
    vegetative cells instead of remaining as spores. APHL officials said that
    it was feared that vegetative cells would be destroyed during certain
    analytical procedures. Other public health officials we interviewed said
    this was highly unlikely.

    Several experts, as well as other public health officials we consulted,
    said it was unlikely that anthrax spores would germinate on premoistened
    swabs into vegetative cells. Consequently, spores (as opposed to
    vegetative cells) would not be killed during the heat shock procedure. We
    clarified the text to reflect this. In addition, APHL stated that concern
    that some of the unknown substances (such as paper debris, dust, and
    harmless environmental bacteria and fungi) combined with moisture, would
    promote germination. APHL was concerned that during transport, the
    resulting vegetative cells would be more susceptible to dying off in the
    complex milieu than the spores. However, we stated in this report that
    anthrax spores are robust compared with other pathogenic microorganisms.
    Nevertheless, we also stated that whether transportation conditions could
    have affected the postal samples' viability is not known because the
    conditions of their transportation were not validated. We addressed APHL's
    other technical comments in the report as appropriate.

    As we agreed with your office, unless you publicly announce the contents
    of this report earlier, we plan no further distribution until 30 days from
    its issue date. We will then send copies of this report to other
    interested congressional members and committees. In addition, the report
    will be available at no charge on GAO's Web site at http://www.gao.gov.

    Other staff who contributed to this report included Hazel Bailey, Heather
    Balent, Venkareddy Chennareddy, Jack Melling, Penny Pickett, Laurel Rabin,
    Mark Ramage, and Bernard Ungar.

    If you or your staff have any questions about this report or would like
    additional information, please contact me at (202) 512-6412, or Sushil
    Sharma, PhD., DrPH, at (202) 512-3460. We can also be reached by e-mail at
    rhodesk@gao.gov and sharmas@gao.gov.

    Sincerely yours,

    Keith A. Rhodes, Chief Technologist Center for Technology and Engineering
    Applied Research and Methods

    Appendix I: Objectives, Scope, and Methodology

    To describe and assess federal agencies' activities to detect anthrax
    contamination in postal facilities in 2001, we interviewed officials from
    the agencies involved in sampling the facilities or analyzing the samples
    that were collected from the facilities. Among them were the United States
    Postal Service (USPS); the U.S. Army Medical Research Institute of
    Infectious Diseases (USAMRIID) in the Department of Defense (DOD); the
    Centers for Disease Control and Prevention (CDC) and its National Center
    for Infectious Diseases (NCID) and National Institute for Occupational
    Safety and Health (NIOSH), which are within the Department of Health and
    Human Services (HHS); the Agency for Toxic Substances and Disease Registry
    (ATSDR), also within HHS; the Environmental Protection Agency (EPA); and
    the U.S. Army Corps of Engineers.

    We interviewed officials from public health laboratories and private
    sector laboratories that analyzed some of the samples from the postal
    facilities, as well as officials from the Association of Public Health
    Laboratories (APHL), which was involved in an agreement with USPS under
    which public health laboratories analyzed samples USPS contractors
    collected from the facilities. Finally, we interviewed experts,
    technicians, and researchers, including scientists who have worked on
    microbial detection in indoor environments. These scientists are Robert
    Hamilton, Johns Hopkins University School of Medicine; Paul Keim,
    University of Arizona; George Ludwig, USAMRIID; Jeff Mohr, U.S. Army
    Dugway Proving Ground; and Linda Stetzenbach, University of Nevada at Las
    Vegas. We did not review sampling techniques used by the Federal Bureau of
    Investigation (FBI), in view of the ongoing criminal investigation.

    We performed literature searches and reviewed studies and scientific
    literature, including anthrax on surface and air sampling methods for
    detecting biological substances on surfaces and in the air. We also
    performed literature searches and reviewed agency, association, and
    industry documentation on sampling activities-the five activities we
    identified. In particular, we looked at the development and key elements
    of sampling plans through to laboratory analysis of samples, including the
    types of sampling approaches; federal regulations for transporting
    infectious substances by land, sea, and air; and extraction and analytic
    procedures used by the Laboratory Response Network (LRN) laboratories and
    others to confirm the presence of hazardous substances in samples,
    including biological substances such as anthrax. We also looked at
    potential variables that could be associated with all these activities.

    We focused our data collection on sampling activities agencies performed
    to detect whether anthrax was present. We also interviewed local public

    Appendix I: Objectives, Scope, and Methodology

    health officials, USPS managers, and others associated with the primary
    facilities. We conducted site visits to some postal facilities that were
    affected, as well as some state public health and private sector
    laboratories involved in analyzing the samples.

    To assess the results of the federal agency testing in the facilities, we
    analyzed CDC, EPA, and USPS data on some initial sampling activities
    during October 2001 through April 2002, when the Wallingford facility was
    sampled for the final time. Throughout our review, we contacted agency and
    public health officials to discuss the data they provided and to clarify
    any inconsistencies we identified. CDC, EPA, and USPS provided us with
    some data on the FBI testing. The data we report on the FBI sampling
    events were included in the information CDC, EPA, and USPS provided us.

    Because multiple agencies conducted sampling in the postal facilities,
    there were multiple sources of information. As a result, we combined the
    information CDC, EPA, and USPS provided us into a single database for
    analysis. In addition, because multiple sampling events were conducted in
    some USPS facilities, we reported the data as numbers of "sampling events"
    rather than numbers of facilities sampled. Finally, if there was more than
    one sampling event in a particular facility, we included information on
    each event. For example, in Wallingford, on November 11, 2001, through
    November 28, 2001, there were four separate sampling events before anthrax
    was eventually detected. USPS sampled two times, with negative results;
    CDC sampled two times, with positive results the second time.

    Data for USPS sampling typically came from contractor-generated anthrax
    sampling reports. Data for CDC and EPA sampling came from multiple
    sources, as did data for facilities that had positive samples. When there
    was more than one data source for a particular sampling event, we compared
    these sources for consistency. To deal with inconsistencies among sources,
    we reported data that were supported by greater detail and that were
    reported more often across these sources. The most frequent
    inconsistencies involved facility name, number of samples collected, or
    date of sampling event.

    For USPS sampling events, we determined the collection method, generally
    by reviewing and analyzing laboratory documentation and USPS written
    sampling plans. From interviews with USPS officials, as well as officials
    from some of the laboratories that analyzed the samples, we corroborated
    that USPS generally used dry swabs. For CDC and EPA, we determined the
    collection methods they used by reviewing and analyzing

    Appendix I: Objectives, Scope, and Methodology

    documentation they provided, as well as other data sources, such as agency
    chronologies, published information on the anthrax investigation, and
    interviews with agency laboratory officials.

    When appropriate, we used our best judgment on all the information
    provided in order to complete information that was missing. For example,
    information on some facilities that CDC sampled was incomplete. Therefore,
    we assumed that the sample collection method and reason for sampling in
    those particular facilities were the same as the method and reason for
    other facilities sampled by CDC in the same area at about the same time.
    CDC, in light of missing information, agreed with our assumptions. We made
    similar assumptions for the FBI sampling events, for which information was
    not obtained. For example, we assumed that the FBI used premoistened swabs
    in the New Jersey facilities because data from USPS and interviews with
    New Jersey public health officials indicated that the FBI had used
    premoistened swabs in these facilities. To the extent possible, we
    improved the accuracy of the data provided to us by corroborating it with
    published data. We relied on sources such as articles in CDC's journal,
    Emerging Infectious Diseases, and on interviews with CDC, EPA, USPS,
    USAMRIID, U.S Army Corps of Engineers, and public health officials.

    To assess the reliability of the CDC, EPA, and USPS data, we (1) compared,
    for obvious errors and accuracy, all sources of information on anthrax
    sampling events in USPS facilities; (2) interviewed officials- from CDC,
    EPA, USPS, and laboratories-who were knowledgeable about the data; (3)
    reviewed related documentation, including articles in peer reviewed
    journals; and (4) worked with agency officials to correct data problems we
    discovered. We also worked with USPS officials to correct for missing
    data, and discrepancies as well as differing facility names, before
    completing our analysis. For other discrepancies, such as inconsistent
    numbers of samples collected, we used our best judgment, which we based on
    all the information provided to us, including contractor-generated
    sampling reports and agency summaries of sampling events. We generally
    used the data in the source with the most specific details of the sampling
    event. We also provided agencies, for their review, with copies of our
    final data set.

    After we determined that the data were sufficiently reliable for the
    purposes of this report, we concluded that any remaining discrepancies-
    such as inconsistencies in the multiple data sources, that is, the number
    of samples collected and the collection methods used during every sampling
    event-were likely to be minimal. For example, more samples may have

    Appendix I: Objectives, Scope, and Methodology

    been collected or USPS may have used wipes as a collection method more
    often than we identified. However, such discrepancies do not have a
    material impact on our results. We did not independently verify test
    result data or laboratory or contractor compliance with testing or
    sampling protocols provided to us by USPS and other federal agencies.

    To assess whether the agencies' activities were validated and to describe
    their validation processes, we interviewed agencies' officials and experts
    from CDC (including NCID and NIOSH), EPA, and USPS, as well as APHL,
    ASTDR, DOD, Johns Hopkins University, the University of Nevada at Las
    Vegas, the U.S. Army Dugway Proving Ground, USAMRIID, and selected public
    health laboratories and private sector laboratories. We performed
    literature searches and reviewed validation procedures and criteria. We
    also reviewed validation methodology, for other types of substances, by
    recognized validation authorities. To determine the federal agencies'
    actions to address anthrax testing issues, we reviewed the related
    policies, procedures, and guidelines they issued after fall 2001;
    interviewed various officials, experts, and researchers; and reviewed
    literature and other relevant documents.

    We conducted our review from May 2003 through November 2004 in Washington,
    D.C., and in Atlanta, Georgia; Las Vegas, Nevada; Miami, Florida; New
    York, New York; Salt Lake City, Utah; Trenton, New Jersey; and
    Wallingford, Connecticut. We conducted our review in accordance with
    generally accepted government auditing standards.

    Appendix II: Information on Sampling Events in Facilities with Positive
    Results

    Sampling Samples collected Positive samples Agencya completed Number and
    type General locationb Number and type General location

    Trenton P&DC (primary facility) Brentwood P&DC (primary facility) Morgan Station
                                P&DC (primary facility)

                                   25   Machinery,
         FBI   10/18/01  premoistened   corners of    14 positive    Machinery
                                         facility
                            swabs                     premoistened
                                                      swabs
    NJDHSS                      20  Common areas        0             NA
               10/18/01  premoistened (lobby, locker
                            swabs          room,
                                        lunchroom)
                                      Machinery,
    CDC and                     57 workstations,                 Desk, floor,
               10/21/01  premoistened offices,        20 positive
                                      common areas,   premoistened
    NJDHSS               swabs     air-handling    swabs          machinery,
                                      unit
                                                                    workstations,
                                                                             air-
                                                                   handling unit
                                        Machinery,
    CDC and                     27  rafter, pipe,               Machinery,
               11//9/01  premoistened      air-       19 positive  air-
    NJDHSS               swabs      handling unit  premoistened handling units
                                                      swabs
                        8 HEPA vacuum                  4 positive
                                                          HEPA
                                                         vacuum

    USPS 10/18/01  29 dry swabs  Machinery, government  14 positive dry swabs
                                    mail area                   Machinery,
                                                                  government mail
                                                                             area
    USPS 10/18/01     2 HHAs        Machine filter              0 NA
                                                          8 positive wet wipes No
    CDC  10/23/01 114 wet wipes     No information     information
                     39 HEPA vacuum                          27 positive HEPA
                         12 air                                   vacuum
                                                              0 positive air

                                           c

    USPS 10/22/01	146 dry swab Machinery, vacuums, manual 4 positive dry swabs
    Machinery, 2 controls cases manual cases

     CDC 10/25/01 56 dry swabs Machines and other locations 7 positive dry swabs No
                                      information

                                 Blue Lake Post Office

    FBI 10/14/01	29 premoistened swabs

    6 vacuum

    3 controls

                              Cages, bins, box, sort area,

    d

                               1 positive No information

    c

                                    machinesvacuums,

                              Boca Raton Main Post Office

    FBI 10/12/01	6 premoistened Cases, flats, sort area, canvas 2 positive
    Cases and sort swabs bag premoistened swabs area 1 bulk (results for bulk
    not

    reported)3 controls

    CDC and EPA

    10/15/01 23 premoistened swabs

    1 control

                             Letter throwback, case, cage,

    1 positive premoistened swab

                                          Case

                                           c

                                         vacuum

    Appendix II: Information on Sampling Events in Facilities with Positive Results

    Sampling Samples collected Positive samples Agencya completed Number and
    type General locationb Number and type General location

                                   Dulles Post Office

    CDC 10/25/01	11 premoistened Sorting area, tray, camera 1 positive Sorting
    table swabs monitor, mailboxes, TV screen, premoistened swab 1 control
    cart, window, computer screen

                                   Friendship Station

    CDC 10/24/01	32 premoistened Elevator, cart, cases, dock, bins, 1 positive
    Composite from swabs sort station, computer monitor premoistened swab
    cases

    5 controls

      Greenacres Post Office Indianapolis Critical Parts Center & Repair Facility
    Jackson Main Post Office Kansas City Stamp Fulfillment Services Lake Worth Post
                                         Office

    CDC and 10/19/01 12 premoistened Clerk case, floor, Invalid-laboratory  NA
                                        bin, shelf
    EPA              swabs                              error
                          2 controls
    CDC and                          Case, bin, water                      Bin
               10/20/01 17 premoistened bottle, vehicle,   2 positive
    EPA              swabs           camera             premoistened swabs
                           1 control

    USPS 10/26/01 44 dry Computer, desk, workstation, 2 positive 1 positive on
                     swabs                               dry swabs          table
                            c mechanical room, vacuum,                  in repair
                            dust                                         area and
                            collector, air filtration,              1 positive on
                            fan, printer,
                                    repair area                        printer

    CDC 11/3/01 24 premoistened Machinery, sort      1 positive   Machinery
                                   station, vacuum,c       swab
                        swabs       fan, vehicle, bin,
                                        computer,
                                   workstation, phone,
                                   public area,
                                           dock
    FBI 11/9/01 15 premoistened    No information     Positive  No information
                        swabs

    USPS 10/28/01  26 dry swabs       Trays, sacks,      2 positive 1 positive
                                         machines, work      dry swabs on a
                                         area, pallets,                  sack and
                       2 controls         workstations                    control
                                                                           sample
    CDC  11/6/01  50 premoistened     No information         0          NA
                          swabs
                       5 controls
                                      Pallets, box, white
    USPS 11/28/01  52 dry swabs           powder             0          NA

    CDC and 10/16/01 3 premoistened Cases, accountables, ledge 2 positive Case
    and ledge EPA swabs premoistened swabs

    1 wet wipe

    2 controls

    Appendix II: Information on Sampling Events in Facilities with Positive Results

    Sampling Samples collected Positive samples Agencya completed Number and
    type General locationb Number and type General location

                                  Lucerne Post Office

    CDC and 10/28/01 8 premoistened Missort box, case 1 positive Missort box
    EPA swabs premoistened swab

                                    Pentagon Station

    CDC 10/30/01	17 premoistened PO boxes, lobby, carts, 2 positive PO boxes
    swabs computer, case, radio, cabinet premoistened swabs

    2 controls

               Princeton Main Post Office Princeton-Palmer Square Station

    FBI 10/27/01 23 premoistened No information    1 positive               No
                                                                      information
                         swabs                     premoistened swab
    CDC 10/27/01 14 premoistened No information         0              NA
                         swabs
                      8 HEPA vacuum

    CDC 11/3/01 19 premoistened Case, cart, window, TV,    1 positive     Case
                                           public
                        swabs      area, workspace,        premoistened swab
                                   cabinet, CRT
                                        screen, lobby
    FBI 11/9/01 15 premoistened     No information              0          NA
                        swabs

    Raleigh P&DC

    USPS 11/8/01 42 dry  Lobby, PO boxes, mailroom  1 positive dry Accountable
                    swabs                              swab
                           surfaces, machinery, cases,
                           air-                                        paper room
                           handling unit, flats,
                           vacuum,c
                             accountable paper room

                        Rocky Hill Post Office South Jersey P&DC

    CDC 11/3/01 15 premoistened Lobby, ledge,         1 positive  Ledge where
                                   missent mail, sort
                        swabs       area, CRT screen,   premoistened   large mail
                                        microwave       swab              carrier
                                                                      is placed
    FBI                            No information         0       No
           11/9/01 15 premoistened                                   information
                        swabs

    FBI  10/31/01 40 premoistened    CRT station only     1 positive       CRT
                                            location                      station
                          swabs            identified       premoistened
                                                            swab
    USPS                          Docks, machine, cases,      0          NA
            11/1/01     27 wipes             HVAC,
                                       maintenance shop,
                                           conference
                                          room, lobby
    CDC  11/11/01 56 premoistened     No information          0          NA
                          swabs
    USPS           60 HEPA vacuum   Docks, machinery,         0          NA
            2/14/02                       air-handling
                                       unit, boiler room

    Appendix II: Information on Sampling Events in Facilities with Positive Results

    Sampling Samples collected Positive samples Agencya completed Number and
    type General locationb Number and type General location

                        Southern Connecticut P&DC (Wallingford)

    USPS    11/11/01    53 dry swabs    Docks, vacuum,c common areas,   0   NA
                                           machinery, manual cases, air-
                                           handling unit
    USPS    11/21/01    64 dry swabs    Vacuum,c manual cases,          0   NA
                                           machinery
    CDC     11/25/01    60 premoistened Machinery, vacuums              0   NA
                         swabs

    CDC 11/28/01    4 swabs      Machinery, bins,     4 positive wet Machinery
                                        columns,                  wipes
                     87 wet wipes   stockroom, vacuum  2 positive HEPA
                    90 HEPA vacuum                          vacuum
                       21 HEPA
                     composite e
                     10 controls

    USPS 4/21/02 64 HEPA vacuum High bay areaf	3 positive HEPA High-bay area
    vacuum

                                   Southwest Station

    CDC 10/23/01 20 premoistened Conference room, package 1 positive Bin swabs
    window, lobby, desk, mail tote, premoistened swab slots, mail tub, bins

                         Trenton Station E West Palm Beach P&DC

    CDC 11/3/01  18 premoistened Truck, dock, bin,    1 positive      Bin
                                    safe, computer
                         swabs      screen, fan, public premoistened
                                        area, sort      swab
                                      area, janitor's
                                       closet, vent
    FBI                            No information      Positive             No
           11/16/01 15 premoistened                                   information
                         swabs

    CDC and 10/24/01 21 premoistened      Cases             0           NA
         EPA                 swabs
                           1 control
                                            Vacuum,c
    CDC and                           machinery, air
               10/27/01 71 premoistened      filter        5 positive   Vacuum,
         EPA                 swabs                       premoistened   machinery
                                                         swabs
                          4 controls
    CDC and 10/30/01 15 premoistened    Machinery       2 positive   Machinery
         EPA                 swabs                       premoistened
                                                         swabs
                          2 controls
    CDC and 11/3/01  8 premoistened     Machinery           0           NA
         EPA                 swabs
                          2 controls
                                        Office,
        USPS                            machinery,       1 positive dry
               11/11/01  38 dry swabs   manual cases,    swab           Machinery
                                          stamp room,
                                             filter

    Source: GAO analysis of CDC, EPA, and USPS data.

    Appendix II: Information on Sampling Events in Facilities with Positive
    Results

    Notes: "P&DC" stands for "processing and distribution center." "NA" means
    not applicable. "No information" means that no information was provided to
    GAO.

    aWe sought no information directly from the FBI on the sampling events it
    conducted. We received information from CDC, EPA, and USPS on the number
    and locations of samples collected and the methods used by the FBI.
    However, we did not receive complete information on all FBI sampling
    events. Therefore, we made assumptions about the method used during the
    FBI sampling events for which complete information was not provided, based
    on the method reported as having been used by the FBI during the same
    period and in the same geographic area.

    bLocations in the table do not reflect locations from which every sample
    was collected. Instead, these locations are intended to provide a general
    idea of the types of locations from which samples were collected.

    c"Vacuum" refers to samples collected from vacuums as well as from vacuum
    filters.

    dInformation on location and collection method was not provided.

    eTwenty-one of the HEPA vacuum samples collected during this sampling
    event were combined into four composite samples-combinations of more than
    one sample collected at various sampling locations. Analysis of composite
    samples yields an average value of concentration within a number of
    locations, and composite samples can be used to keep analysis costs down.

    fHigh-bay areas are elevated areas, including pipes, ducts, lights,
    joists, beams, and overhead conveyors.

    Appendix III: Comments from the Centers for Disease Control and Prevention

    Appendix III: Comments from the Centers for Disease Control and Prevention

    Appendix III: Comments from the Centers for Disease Control and Prevention

                             Appendix IV: Comments from the
                            Department of Homeland Security

    Appendix IV: Comments from the Department of Homeland Security

    Appendix IV: Comments from the Department of Homeland Security

    Appendix IV: Comments from the Department of Homeland Security

                         Page 107 GAO-05-251 Anthrax Detection

                         Page 108 GAO-05-251 Anthrax Detection

    Appendix VI: Comments from the Association of Public Health Laboratories

    Appendix VI: Comments from the Association of Public Health Laboratories

    Appendix VI: Comments from the Association of Public Health Laboratories

    Appendix VI: Comments from the Association of Public Health Laboratories

    Appendix VII: Comments from the Department of Defense

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