Report to the Chairman, Subcommittee 

on National Security, Emerging 

Threats, and International Relations, 

House Committee on Government 

Reform, House of Representatives 

United States Government Accountability Office

GAO 

March 2005 ANTHRAX 

DETECTION 

Agencies Need to 

Validate Sampling 

Activities in Order to 

Increase Confidence 

in Negative Results 

GAO-05-251 
What GAO Found

United States Government Accountability Office

Why GAO Did This Study

Highlights

Accountability Integrity Reliability

www.gao.gov/cgi-bin/getrpt?GAO-05-251.

To view the full product, including the scope 

and methodology, click on the link above. 

For more information, contact Keith Rhodes 

at (202) 512-6412 or rhodesk@gao.gov. 

Highlights of GAO-05-251, a 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 

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. 

1

Sampling 

strategy 

development

2

Sample 

collection

3

Sample 

transportation

5

Sample 

analysis

4

Sample 

extraction

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. 

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 the Subcommittee 

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. 

What GAO Recommends

GAO recommends that the 

Department of Homeland Security 

(DHS) develop a coordinated 

approach to working with federal 

agencies, so that appropriate 

validation studies of various 

activities involved in detecting 

anthrax are conducted. The DHS 

Secretary should also ensure that 

an agreed-on definition of 

validation is developed; 

appropriate investments are made 

to explore improved sampling 

strategies; and agencies’ policies, 

procedures, and guidelines reflect 

the results of all these efforts. DHS 

stated that while it has the overall 

responsibility for coordination, 

EPA and HHS have the lead roles in 

responding to biological attacks. 

DHS said that it would coordinate 

with EPA to ensure that 

appropriate investments are made 

to explore improved sampling. 
Page i GAO-05-251 Anthrax Detection 

Letter 1

Results in Brief 3 

Background 9 

Agencies Were Challenged by Problems Associated with Sampling 

Activities 17 

The Five Activities Involved Many Variables That Can Affect 

Results 45 

The Sampling Results Were Largely Negative 46 

Lack of Validation Raises Questions about the Reliability of 

Negative Results 56 

Agencies Have Taken Some Steps to Prepare for Future Incidents 68 

Conclusions 79 

Recommendations for Executive Action 82 

Agency Comments and Our Evaluation 83 

Appendix I Objectives, Scope, and Methodology 92 

Appendix II Information on Sampling Events in Facilities with 

Positive Results 96 

Appendix III Comments from the Centers for Disease Control 

and Prevention 101 

Appendix IV Comments from the Department of Homeland 

Security 104 

Appendix V Comments from the United States Postal Service 108 

Appendix VI Comments from the Association of Public Health 

Laboratories 110 

Contents 
Page ii GAO-05-251 Anthrax Detection 

Appendix VII Comments from the Department of Defense 114 

Tables 

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

October 2001 through April 2002 27 

Table 2: Total Samples Collected and Sampling Events 46 

Table 3: Delivery Bar Code Sorting Machines Sampled, Wallingford 

Facility, 2001 52 

Table 4: Agency Evaluations, October 2001 through February 2002 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 
Page iii GAO-05-251 Anthrax Detection 

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. 
Page 1 GAO-05-251 Anthrax Detection 

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. 

2

Anthrax 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. 

United States Government Accountability Office

Washington, DC 20548 
Page 2 GAO-05-251 Anthrax Detection 

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 

• describe and assess federal agencies’ activities to detect anthrax 

contamination in the postal facilities; 

• assess the results of the federal agencies’ testing in the postal facilities; 

and 

• 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 

 3

According 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. 

4

GAO, 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). 
Page 3 GAO-05-251 Anthrax Detection 

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. 

CDC, EPA, and USPS, the federal agencies involved in sampling the postal 

facilities in 2001 to detect anthrax, undertook several activities: (1) 

 5

For 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. 

Results in Brief 
Page 4 GAO-05-251 Anthrax Detection 

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 

 6

These 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.” 

7

A 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.
Page 5 GAO-05-251 Anthrax Detection 

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. 

 8

We 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. 

9

Earlier, 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.
Page 6 GAO-05-251 Anthrax Detection 

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. 
Page 7 GAO-05-251 Anthrax Detection 

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.” 
Page 8 GAO-05-251 Anthrax Detection 

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 
Page 9 GAO-05-251 Anthrax Detection 

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 response to the incident in the American Media Incorporated building 

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. 

Background 

The Environmental 

Sampling Response 
Page 10 GAO-05-251 Anthrax Detection 

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.
Page 11 GAO-05-251 Anthrax Detection 

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. 
Page 12 GAO-05-251 Anthrax Detection 

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. 

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 

USPS Mail System 
Page 13 GAO-05-251 Anthrax Detection 

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. 

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. 

Federal Agencies Involved 

in Anthrax Detection in 

Postal Facilities Had 

Differing Responsibilities 
Page 14 GAO-05-251 Anthrax Detection 

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. 

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. 

The Laboratory Response 

Network 
Page 15 GAO-05-251 Anthrax Detection 

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). 
Page 16 GAO-05-251 Anthrax Detection 

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® 

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 

Terms Associated with 

Sampling Methods and 

Procedures 
Page 17 GAO-05-251 Anthrax Detection 

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 
Page 18 GAO-05-251 Anthrax Detection 

Figure 1: Agency Sampling Activities 

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).

Activity 1: Sampling 

Strategy Development 

1

Sampling 

strategy 

development

Includes sample 

size, locations 

to collect 

samples from, 

method of 

collection 

2

Sample 

collection

From surfaces 

or air or both, 

using specified 

methods

3

Sample 

transportation

From facility to 

laboratory, 

under 

appropriate 

conditions

5

Sample

analysis

Of living 

organisms in

4

Sample 

extraction

At laboratory in 

preparation for 

analysis preliminary 

tests

and

confirmatory 

tests

·

·

Source: GAO analysis of CDC, EPA, and USPS data.
Page 19 GAO-05-251 Anthrax Detection 

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).
Page 20 GAO-05-251 Anthrax Detection 

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). 
Page 21 GAO-05-251 Anthrax Detection 

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.” 

Agencies Primarily Used a 

Targeted Strategy 
Page 22 GAO-05-251 Anthrax Detection 

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. 
Page 23 GAO-05-251 Anthrax Detection 

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. 

According to CDC, a targeted sampling strategy may be effective in 

detecting contamination in a facility when sufficient site-specific 

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. 

Incorporating Probability

Sampling Would Allow Greater 

Confidence in Negative Results 
Page 24 GAO-05-251 Anthrax Detection 

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 
Page 25 GAO-05-251 Anthrax Detection 

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. 
Page 26 GAO-05-251 Anthrax Detection 

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. 

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.

Activity 2: Collecting 

Samples 
Page 27 GAO-05-251 Anthrax Detection 

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

 Agency Methods at different 

sampling events 

Sampling 

eventa

CDC CDC and EPA USPSb FBI NJDHSS Unknown 

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. 

a

Sampling 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. 

b

Early, 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 
Page 28 GAO-05-251 Anthrax Detection 

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.” 
Page 29 GAO-05-251 Anthrax Detection 

Figure 2: Three Sample Collection Methods 
Page 30 GAO-05-251 Anthrax Detection 

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. 
Page 31 GAO-05-251 Anthrax Detection 

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. 

Agencies transported samples by land or air to laboratories for extraction 

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.

Activity 3: Transporting 

Samples 
Page 32 GAO-05-251 Anthrax Detection 

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). 
Page 33 GAO-05-251 Anthrax Detection 

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. 
Page 34 GAO-05-251 Anthrax Detection 

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. 

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 20TM, a surfactant) in phosphate-buffered 

saline before plating. 

Activity 4: Extracting 

Samples 
Page 35 GAO-05-251 Anthrax Detection 

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. 
Page 36 GAO-05-251 Anthrax Detection 

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. 

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.

Activity 5: Analyzing 

Samples 
Page 37 GAO-05-251 Anthrax Detection 

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 

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. 

Laboratory Protocols Were 

Adapted for Analyzing Samples 

Procedure 

After 18 to 24 hours, look at culture plate. Is there growth? If “yes,” do any of 

the organisms look like anthrax? If so, select some of them for testing.

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.

Test

Preliminary 

Confirmatory

Source: GAO analysis of CDC, DOD, LRN, and USPS documents.
Page 38 GAO-05-251 Anthrax Detection 

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.
Page 39 GAO-05-251 Anthrax Detection 

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. 
Page 40 GAO-05-251 Anthrax Detection 

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. 
Page 41 GAO-05-251 Anthrax Detection 

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. 

Agencies Encountered 

Problems with Preliminary 

Analytic Methods 
Page 42 GAO-05-251 Anthrax Detection 

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.
Page 43 GAO-05-251 Anthrax Detection 

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. 
Page 44 GAO-05-251 Anthrax Detection 

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. 
Page 45 GAO-05-251 Anthrax Detection 

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 Five Activities 

Involved Many 

Variables That Can 

Affect Results 
Page 46 GAO-05-251 Anthrax Detection 

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 7,040 182

CDC 2,313 119

CDC and EPA (Florida) 183 10

FBI 225 11

State health department 20 1

Othera 26 1

Total 9,807 324

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

a

The 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. 

The Sampling Results 

Were Largely Negative 
Page 47 GAO-05-251 Anthrax Detection 

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 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. 

Testing in the Three 

Primary Facilities 

Revealed More Positive 

Results Than in the 

Wallingford Facility 
Page 48 GAO-05-251 Anthrax Detection 

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

Note: Numbers in parentheses are total samples collected and total positive results for a particular 

sampling effort (e.g., 29/14). 

a

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

b

USPS also had a hazardous materials unit collect two HHAs samples in Brentwood on 

October 18, 2001, which were negative. 

c

The 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 

Primary facilities: processed the letters containing anthrax Received mail from Trenton

Wallingforda

(350,000 sq. ft.)

Morgan

(2.1 million sq. ft.) 

Trenton

(282,000 sq. ft.)

Brentwood

(684,000 sq. ft.)

Oct. 18

Oct. 23

USPSb

Dry swabs

(29/14)

CDC 

Wet wipes, 

HEPA vacuum 

sock, and air 

samples 

(165/35)

Oct. 18

Oct. 21

FBIc

Premoistened 

swabs

(25/14)

CDC 

Premoistened 

swabs 

(57/20)

Oct. 22

Oct. 25

USPS 

Dry swabs 

(146/4)

CDC 

Dry swabs 

(56/7)

Nov. 11

Nov. 21

Nov. 25

Nov. 28

USPS 

Dry swabs 

(53/0)

USPS 

Dry swabs 

(64/0) 

CDC

Premoistened 

swabs

(60/0)

CDC 

Swabs, 

wet wipes, 

HEPA vacuum, 

and bulk

(202/6)

Positive results

Negative results

Source: GAO analysis of CDC and USPS data.
Page 49 GAO-05-251 Anthrax Detection 

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. 

USPS and CDC sampled facilities that processed mail from the primary 

facilities to determine whether any other facilities had become 

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. 

The Majority of the 

Facilities’ Test Results 

Were Negative 
Page 50 GAO-05-251 Anthrax Detection 

Figure 5: Test Results Were Largely Negative 

The following discusses results for some of the positive facilities, 

excluding the primary ones: 

• 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. 

• 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.

23

263

Facilities testing positive

Facilities testing negative

Source: GAO analysis of CDC, EPA, and USPS data.
Page 51 GAO-05-251 Anthrax Detection 

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. 
Page 52 GAO-05-251 Anthrax Detection 

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 testing CDC outbreak investigation 

Machine Nov. 11, 2001 Nov. 21, 2001 Nov. 25, 2001 Nov. 28, 2001

Dec. 2, 2001 

(characterization 

sampling)a 

Total 

samples

1 1 8 9

2 1 8 9

3 8 8

4 11 (1) 48 (1) 59

5 2 12 14

6 1 2 3 23 48 (1) 77

7 2 12 14

8 8 8

9 1 8 9

10b 8 (4) 52 (30) 60

11 1 8 (1) 52 (3) 61

12 8 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 CodesTM, 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 CodesTM that included the 

customer’s town was found positive. 
Page 53 GAO-05-251 Anthrax Detection 

USPS precautionary testing CDC outbreak investigation 

Machine Nov. 11, 2001 Nov. 21, 2001 Nov. 25, 2001 Nov. 28, 2001

Dec. 2, 2001 

(characterization 

sampling)a 

Total 

samples

13 1 8 9

Total 1 6 8 130 (6) 200 (35) 345

Source: GAO analysis of CDC data. 

Note: Numbers in parentheses indicate positive results. 

a

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

b

About 3 million anthrax spores were found in one sample. 

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. 

Interpreting the Test 

Results
Page 54 GAO-05-251 Anthrax Detection 

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). 
Page 55 GAO-05-251 Anthrax Detection 

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. 
Page 56 GAO-05-251 Anthrax Detection 

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 
Page 57 GAO-05-251 Anthrax Detection 

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. 

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. 

No Activity or Process Was 

Validated 
Page 58 GAO-05-251 Anthrax Detection 

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. 
Page 59 GAO-05-251 Anthrax Detection 

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. 

Validation Is 

Independently 

Administered 
Page 60 GAO-05-251 Anthrax Detection 

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. 

Nonvalidated Sampling 

Strategies Could Be Based 

on False Assumptions 
Page 61 GAO-05-251 Anthrax Detection 

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

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. 

1

Includes sample size, 

locations to collect 

samples from, method 

of collection 

2

From surfaces or air 

or both, using specified 

methods

3

From facility to 

laboratory, under 

appropriate conditions

5

Of living organisms in

4

At laboratory in 

preparation for 

analysis ·

·

preliminary tests

and

confirmatory tests

Sampling

strategy 

development

Sample

collection

Sample

transportation

Sample

extraction

Sample

analysis

. . . techniques for 

sampling, transporting, 

extracting, and analysis 

will not be known, and 

thus the strategy may 

be based on false 

assumptions . . .

. . . performance 

characteristics of the 

methods for sampling 

surfaces will not be 

known . . .

. . . an adequate 

balance between 

safety and sample 

integrity cannot be 

ensured . . .

. . . the basic 

capabilities and skills 

staff need may not be 

understood or

required . . .

. . . tests may not be 

reproducible and 

therefore results 

cannot be trusted

Sampling activities: The overall process

Without validation

Sampling activities

Activities without validation

Source: GAO analysis of CDC, EPA, and USPS data.
Page 62 GAO-05-251 Anthrax Detection 

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). 

Agencies’ Advice Cannot 

Be Definitive without 

Validation 
Page 63 GAO-05-251 Anthrax Detection 

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. 
Page 64 GAO-05-251 Anthrax Detection 

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.
Page 65 GAO-05-251 Anthrax Detection 

Table 4: Agency Evaluations, October 2001 through February 2002

Date Agency Objective and method Conclusion 

October 

2001a

 

CDC To learn more about the potential for a contaminated 

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. 

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. 

October 

2001b

 

EPA To learn the extent of indoor secondary aerosolization 

of anthrax spores under active and inactive office 

conditions. 

Collected surface samples (swabs and 

microvacuums), air samples (Anderson 6-stage 

sampler and 2-stage sampler), and open agar plates. 

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. 

December 

2001c

 

CDC, 

ATSDR, 

and 

USPS 

To compare the relative efficiency of sampling 

methods used to collect spores from contaminated 

surfaces. 

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. 

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. 

February 

2002d

 

CDC 

and 

USPS 

To compare air sampling methods side by side. 

Evaluated air sampling methods (mixed cellulose, 

polytetrafluoroethylene, gelatin-coated, dry filters, and 

Anderson single-stage cascade impactors) for anthrax 

spores before and after the operation of mail sorting 

equipment in the Trenton P&DC. 

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. 

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

a

P. M. Dull and others, “Bacillus anthracis Aerosolization Associated with a Contaminated Mail Sorting 

Machine,” Emerging Infectious Diseases 8 (2002): 1044–47. 

b

C. 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. 

c

W. T. Sanderson and others, “Surface Sampling Methods for Bacillus anthracis Spore 

Contamination,” Journal of Emerging Infectious Diseases 8 (2002): 1145–50. 
Page 66 GAO-05-251 Anthrax Detection 

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). 

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). 

Lack of Validation 

Highlighted Experts’ and 

Agencies’ Different 

Opinions 
Page 67 GAO-05-251 Anthrax Detection 

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®), 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 97-

3981 (Research Triangle Park, N.C.: Mar. 1997). 
Page 68 GAO-05-251 Anthrax Detection 

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. 

Agencies Have Taken 

Some Steps to 

Prepare for Future 

Incidents 
Page 69 GAO-05-251 Anthrax Detection 

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. 
Page 70 GAO-05-251 Anthrax Detection 

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.
Page 71 GAO-05-251 Anthrax Detection 

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.
Page 72 GAO-05-251 Anthrax Detection 

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. 
Page 73 GAO-05-251 Anthrax Detection 

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.” 
Page 74 GAO-05-251 Anthrax Detection 

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: 

• 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 

• 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.

Ongoing Studies May Help 

Validate Individual 

Activities but Are Not 

Aimed at the Process as a 

Whole 
Page 75 GAO-05-251 Anthrax Detection 

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. 

• 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. 

• Several studies, funded by TSWG, focused on the sensitivity and 

specificity of sample collection methods for detecting anthrax and 

other biological substances.128 

• 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. 
Page 76 GAO-05-251 Anthrax Detection 

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. 

• 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: 

• 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. 
Page 77 GAO-05-251 Anthrax Detection 

• 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. 

• Quantitative results based on standardized sample collection and 

analytic methods do not yet give information that relates to health risk 

to individuals. 

• 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. 
Page 78 GAO-05-251 Anthrax Detection 

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 

underrepresented the degree of contamination.132

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. 

Test Reliability Remains 

Uncertain Despite 

Agencies’ Progress 
Page 79 GAO-05-251 Anthrax Detection 

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: 

• What sampling strategies can provide a known level of confidence that 

anthrax will be detected in facilities, especially those that are not 

heavily contaminated? 

• 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? 

• Do transportation conditions affect the integrity of samples? 

• How effective are the various methods for extracting material from 

samples for analysis? 

• Do laboratories have the capability and capacity to analyze different 

types of samples? 

• How should validation be defined? 

• What changes to agency policies, procedures, and guidance should be 

made to reflect lessons learned and the results of the validation 

process? 

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 

Conclusions 
Page 80 GAO-05-251 Anthrax Detection 

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 
Page 81 GAO-05-251 Anthrax Detection 

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. 
Page 82 GAO-05-251 Anthrax Detection 

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. 

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 

Recommendations for 

Executive Action 
Page 83 GAO-05-251 Anthrax Detection 

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 

Agency Comments 

and Our Evaluation
Page 84 GAO-05-251 Anthrax Detection 

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 
Page 85 GAO-05-251 Anthrax Detection 

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. 
Page 86 GAO-05-251 Anthrax Detection 

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. 
Page 87 GAO-05-251 Anthrax Detection 

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 
Page 88 GAO-05-251 Anthrax Detection 

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 
Page 89 GAO-05-251 Anthrax Detection 

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. 
Page 90 GAO-05-251 Anthrax Detection 

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 

Page 91 GAO-05-251 Anthrax Detection 

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 
Appendix I: Objectives, Scope, and 

Methodology 

Page 92 GAO-05-251 Anthrax Detection 

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 

Page 93 GAO-05-251 Anthrax Detection 

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 

Page 94 GAO-05-251 Anthrax Detection 

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 

Page 95 GAO-05-251 Anthrax Detection 

Samples collected Positive samples

Agencya

Sampling 

completed Number and type General locationb

 Number and type General location 

Trenton P&DC (primary facility) 

FBI 10/18/01 25 premoistened 

swabs 

Machinery, corners of facility 14 positive 

premoistened swabs 

Machinery 

NJDHSS 10/18/01 20 premoistened 

swabs 

Common areas (lobby, locker 

room, lunchroom) 

 0 NA 

CDC and 

NJDHSS 

10/21/01 57 premoistened 

swabs 

Machinery, workstations, offices, 

common areas, air-handling unit 

 20 positive 

premoistened swabs 

Desk, floor, 

machinery, 

workstations, airhandling unit 

CDC and 

NJDHSS 

11//9/01 27 premoistened 

swabs 

8 HEPA vacuum 

Machinery, rafter, pipe, airhandling unit  19 positive 

premoistened swabs 

4 positive HEPA 

vacuum 

Machinery, airhandling units 

Brentwood P&DC (primary facility) 

USPS 10/18/01 29 dry swabs Machinery, government mail area 14 positive dry swabs Machinery, 

government mail 

area 

USPS 10/18/01 2 HHAs Machine filter 0 NA 

CDC 10/23/01 114 wet wipes 

39 HEPA vacuum 

12 air 

No information 8 positive wet wipes 

27 positive HEPA 

vacuum 

0 positive air 

No information 

Morgan Station P&DC (primary facility) 

USPS 10/22/01 146 dry swab 

2 controls 

Machinery, vacuums,c manual 

cases 

 4 positive dry swabs Machinery, 

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, 

vacuums,c

 machines 

 1 positived

 No information 

Boca Raton Main Post Office 

FBI 10/12/01 6 premoistened 

swabs 

1 bulk 

3 controls 

Cases, flats, sort area, canvas 

bag 

 2 positive 

premoistened swabs 

(results for bulk not 

reported) 

Cases and sort 

area 

CDC and 

EPA 

10/15/01 23 premoistened 

swabs 

1 control 

Letter throwback, case, cage, 

vacuumc

 1 positive 

premoistened swab 

Case 

Appendix II: Information on Sampling Events 

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

in Facilities with Positive Results 

Page 96 GAO-05-251 Anthrax Detection 

Samples collected Positive samples

Agencya

Sampling 

completed Number and type General locationb

 Number and type General location 

Dulles Post Office 

CDC 10/25/01 11 premoistened 

swabs 

1 control 

Sorting area, tray, camera 

monitor, mailboxes, TV screen, 

cart, window, computer screen 

 1 positive 

premoistened swab 

Sorting table 

Friendship Station 

CDC 10/24/01 32 premoistened 

swabs 

5 controls 

Elevator, cart, cases, dock, bins, 

sort station, computer monitor 

 1 positive 

premoistened swab 

Composite from 

cases 

Greenacres Post Office 

CDC and 

EPA 

10/19/01 12 premoistened 

swabs 

2 controls 

Clerk case, floor, bin, shelf Invalid–laboratory 

error 

NA 

CDC and 

EPA 

10/20/01 17 premoistened 

swabs 

1 control 

Case, bin, water bottle, vehicle, 

camera 

 2 positive 

premoistened swabs 

Bin 

Indianapolis Critical Parts Center & Repair Facility 

USPS 10/26/01 44 dry swabs Computer, desk, workstation, 

mechanical room, vacuum,c

 dust 

collector, air filtration, fan, printer, 

repair area 

 2 positive dry swabs 1 positive on table 

in repair area and 

1 positive on 

printer 

Jackson Main Post Office 

CDC 11/3/01 24 premoistened 

swabs 

Machinery, sort station, vacuum,c

fan, vehicle, bin, computer, 

workstation, phone, public area, 

dock 

 1 positive swab Machinery 

FBI 11/9/01 15 premoistened 

swabs 

No information Positive No information 

Kansas City Stamp Fulfillment Services 

USPS 10/28/01 26 dry swabs 

2 controls 

Trays, sacks, machines, work 

area, pallets, workstations 

 2 positive dry swabs 1 positive on a 

sack and control 

sample 

CDC 11/6/01 50 premoistened 

swabs 

5 controls 

No information 0 NA 

USPS 11/28/01 52 dry swabs Pallets, box, white powder 0 NA 

Lake Worth Post Office 

CDC and 

EPA 

10/16/01 3 premoistened 

swabs 

1 wet wipe 

2 controls 

Cases, accountables, ledge 2 positive 

premoistened swabs 

Case and ledge 
Appendix II: Information on Sampling Events 

in Facilities with Positive Results 

Page 97 GAO-05-251 Anthrax Detection 

Samples collected Positive samples

Agencya

Sampling 

completed Number and type General locationb

 Number and type General location 

Lucerne Post Office 

CDC and 

EPA 

10/28/01 8 premoistened 

swabs 

Missort box, case 1 positive 

premoistened swab 

Missort box 

Pentagon Station 

CDC 10/30/01 17 premoistened 

swabs 

2 controls 

PO boxes, lobby, carts, 

computer, case, radio, cabinet 

 2 positive 

premoistened swabs 

PO boxes 

Princeton Main Post Office 

FBI 10/27/01 23 premoistened 

swabs 

No information 1 positive 

premoistened swab 

No information 

CDC 10/27/01 14 premoistened 

swabs 

8 HEPA vacuum 

No information 0 NA 

Princeton-Palmer Square Station 

CDC 11/3/01 19 premoistened 

swabs 

Case, cart, window, TV, public 

area, workspace, cabinet, CRT 

screen, lobby 

 1 positive 

premoistened swab 

Case 

FBI 11/9/01 15 premoistened 

swabs 

No information 0 NA 

Raleigh P&DC 

USPS 11/8/01 42 dry swabs Lobby, PO boxes, mailroom 

surfaces, machinery, cases, airhandling unit, flats, vacuum,c

accountable paper room 

 1 positive dry swab Accountable 

paper room 

Rocky Hill Post Office 

CDC 11/3/01 15 premoistened 

swabs 

Lobby, ledge, missent mail, sort 

area, CRT screen, microwave 

 1 positive 

premoistened swab 

Ledge where 

large mail carrier 

is placed 

FBI 11/9/01 15 premoistened 

swabs 

No information 0 No information 

South Jersey P&DC 

FBI 10/31/01 40 premoistened 

swabs 

CRT station only location 

identified 

 1 positive 

premoistened swab 

CRT station 

USPS 11/1/01 27 wipes Docks, machine, cases, HVAC, 

maintenance shop, conference 

room, lobby 

 0 NA 

CDC 11/11/01 56 premoistened 

swabs 

No information 0 NA 

USPS 2/14/02 60 HEPA vacuum Docks, machinery, air-handling 

unit, boiler room 

 0 NA 
Appendix II: Information on Sampling Events 

in Facilities with Positive Results 

Page 98 GAO-05-251 Anthrax Detection 

Samples collected Positive samples

Agencya

Sampling 

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, 

machinery, manual cases, airhandling unit 

 0 NA 

USPS 11/21/01 64 dry swabs Vacuum,c manual cases, 

machinery 

 0 NA 

CDC 11/25/01 60 premoistened 

swabs 

Machinery, vacuums 0 NA 

CDC 11/28/01 4 swabs 

87 wet wipes 

90 HEPA vacuum 

21 HEPA 

compositee

 

10 controls 

Machinery, bins, columns, 

stockroom, vacuum 

 4 positive wet wipes 

2 positive HEPA 

vacuum 

Machinery 

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

vacuum 

High-bay area 

Southwest Station 

CDC 10/23/01 20 premoistened 

swabs 

Conference room, package 

window, lobby, desk, mail tote, 

slots, mail tub, bins 

 1 positive 

premoistened swab 

Bin 

Trenton Station E 

CDC 11/3/01 18 premoistened 

swabs 

Truck, dock, bin, safe, computer 

screen, fan, public area, sort 

area, janitor’s closet, vent 

 1 positive 

premoistened swab 

Bin 

FBI 11/16/01 15 premoistened 

swabs 

No information Positive No information 

West Palm Beach P&DC 

CDC and 

EPA 

10/24/01 21 premoistened 

swabs 

1 control 

Cases 0 NA 

CDC and 

EPA 

10/27/01 71 premoistened 

swabs 

4 controls 

Vacuum,c

 machinery, air filter 5 positive 

premoistened swabs 

Vacuum, 

machinery 

CDC and 

EPA 

10/30/01 15 premoistened 

swabs 

2 controls 

Machinery 2 positive 

premoistened swabs 

Machinery 

CDC and 

EPA 

11/3/01 8 premoistened 

swabs 

2 controls 

Machinery 0 NA 

USPS 11/11/01 38 dry swabs Office, machinery, manual cases, 

stamp room, filter 

 1 positive dry swab Machinery 

Source: GAO analysis of CDC, EPA, and USPS data. 
Appendix II: Information on Sampling Events 

in Facilities with Positive Results 

Page 99 GAO-05-251 Anthrax Detection 

Notes: “P&DC” stands for “processing and distribution center.” “NA” means not applicable. ”No 

information” means that no information was provided to GAO. 

a

We 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. 

b

Locations 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. 

d

Information on location and collection method was not provided. 

e

Twenty-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. 

f

High-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 

Page 100 GAO-05-251 Anthrax Detection 

Appendix III: Comments from the Centers for 

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

Disease Control and Prevention 

Page 101 GAO-05-251 Anthrax Detection 
Appendix III: Comments from the Centers for 

Disease Control and Prevention 

Page 102 GAO-05-251 Anthrax Detection 
Appendix IV: Comments from the Department 

of Homeland Security 

Page 103 GAO-05-251 Anthrax Detection 

Appendix IV: Comments from the 

Department of Homeland Security 
Appendix IV: Comments from the Department 

of Homeland Security 

Page 104 GAO-05-251 Anthrax Detection 
Appendix IV: Comments from the Department 

of Homeland Security 

Page 105 GAO-05-251 Anthrax Detection 
Appendix IV: Comments from the Department 

of Homeland Security 

Page 106 GAO-05-251 Anthrax Detection 
Appendix V: Comments from the United 

States Postal Service 

Page 107 GAO-05-251 Anthrax Detection 

Appendix V: Comments from the United 

States Postal Service 
Appendix V: Comments from the United 

States Postal Service 

Page 108 GAO-05-251 Anthrax Detection 
Appendix VI: Comments from the Association of Public 

Health Laboratories 

Page 109 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 

Page 110 GAO-05-251 Anthrax Detection 
Appendix VI: Comments from the Association of Public 

Health Laboratories 

Page 111 GAO-05-251 Anthrax Detection 
Appendix VI: Comments from the Association of Public 

Health Laboratories 

Page 112 GAO-05-251 Anthrax Detection 
Appendix VII: Comments from the 

Department of Defense 

Page 113 GAO-05-251 Anthrax Detection 

Appendix VII: Comments from the 

Department of Defense 

(460556) 
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