Anthrax Detection: Agencies Need to Validate Sampling Activities 
in Order to Increase Confidence in Negative Reults (05-APR-05,	 
GAO-05-493T).							 
                                                                 
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.				 
-------------------------Indexing Terms------------------------- 
REPORTNUM:   GAO-05-493T					        
    ACCNO:   A20780						        
  TITLE:     Anthrax Detection: Agencies Need to Validate Sampling    
Activities in Order to Increase Confidence in Negative Reults	 
     DATE:   04/05/2005 
  SUBJECT:   Chemical and biological agents			 
	     Disease detection or diagnosis			 
	     Diseases						 
	     Health hazards					 
	     Infectious diseases				 
	     Interagency relations				 
	     Laboratories					 
	     National preparedness				 
	     Postal facilities					 
	     Postal service					 
	     Strategic planning 				 
	     Terrorism						 
	     Test facilities					 
	     Testing						 
	     Disease surveillance				 
	     Homeland security					 
	     Anthrax						 

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GAO-05-493T

                 United States Government Accountability Office

Testimony before the Chairman,

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

For Release on Delivery Expected at 2:00 p.m.

Tuesday, April 5, 2005 ANTHRAX DETECTION

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

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

GAO-05-493T

[IMG]

April 2005

ANTHRAX DETECTION

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

                                 What GAO Found

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

                                       5

Sample analysis

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

The results of the agencies' testing in 286 postal facilities were largely
negative-no anthrax was detected. But negative results do not necessarily
mean that a facility is free from anthrax. In addition, agencies'
detection activities (for example, sample collection and analytical
methods) were not validated. 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, and (3)
funded some new research. 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 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
anthrax and other biothreat agents.

                 United States Government Accountability Office

April 5, 2005

Mr. Chairman and Members of the Subcommittee:

We are pleased to participate in this hearing by presenting our assessment
of the federal agencies'-U.S. Postal Service (USPS), Centers for Disease
Control and Prevention, and Environmental Protection Agency (EPA)-
activities conducted to detect anthrax in postal facilities in 2001. My
statement is based on our report, entitled Anthrax Detection: Agencies
Need to Validate Sampling Activities in Order to Increase Confidence in
Negative Results, which was issued on March 31, 2005.1

As you know, in September and October 2001, contaminated letters laced
with Bacillus anthracis, or anthrax spores,2 were sent through the mail to
two senators, Thomas Daschle and Patrick Leahy, and members of the media.
The postal facilities in New Jersey and Washington, D.C., that processed
the senators' letters became heavily contaminated.3 Other mail routed
through these facilities, as well as additional ones in the postal
network, also became contaminated. In addition, numerous federal
facilities in the Washington, D.C., area were later found to be
contaminated. The letters led to the first cases of anthrax disease
related to bioterrorism in the United States. 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 has been recognized for a considerable time.
Long before the anthrax 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

1GAO, Anthrax Detection: Agencies Need to Validate Sampling Activities in
Order to Increase Confidence in Negative Results, GAO-05-251 (Washington,
D.C.: March 31, 2005). www.gao.gov.

2"Anthrax" in this testimony 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.

3Anthrax 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.

Background

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.4

Before I discuss our assessment, let me first present some background.
(See appendix I for a discussion of our scope and methodology.)

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.

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,
facility was sampled for the last time. Four contractors conducted USPS
sampling.

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.

4According 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.

The federal agencies involved in the response in the postal facilities had
differing responsibilities. The Centers for Disease Control and Prevention
(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. The Environmental Protection Agency
(EPA) has been the nation's lead agency for responding to a release of
hazardous substances into the environment.

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 superseded by the Homeland Security
Act of 2002, which transferred many of its functions to the Department of
Homeland Security (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.

In addition, the Laboratory Response Network (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. These laboratories
function as first responders that can perform standard initial tests to
rule out, but not definitively confirm, anthrax.

Now I will discuss our assessment of the following federal agencies'
activities: (1) federal agencies' activities to detect anthrax
contamination in the postal facilities; (2) the results of the federal
agencies' testing in the postal facilities; and (3) 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.

  Federal Agencies' Activities to Detect Anthrax Contamination in the Postal
  Facilities

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).

                      Figure 1: Agency Sampling Activities

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 judgments. 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,

                                       5

Sample analysis

Of living organisms in preliminary
tests
and
confirmatory
tests

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

Neither these activities nor the overall process has been validated for
anthrax testing. Consequently, the agencies had only limited information
available for reliably choosing one method over another and no information
on the limits of detection to use when evaluating negative results. In
addition, 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.

Activity 1: Sampling Strategy Development

Agencies Primarily Used a Targeted Strategy

when the source of potential contamination is not definitive, the basic
question-Is this building contaminated?-will remain unanswered.

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. In
the beginning, in each USPS 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. CDC's and EPA's site-specific
strategies were primarily discretionary. The number of samples CDC
collected varied by facility, ranging from a low of 4 to a high of 202.
The number of samples EPA collected ranged from a low of 4 to a high of
71.

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. CDC's assumptions for this
strategy are that at the outset, (1) a scenario where all locations have
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 the 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.

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

Incorporating Probability Sampling Would Allow Greater Confidence in
Negative Results

sampling. We disagree with this interpretation. Probability sampling is
statistically based and does not depend solely on empirical criteria
regarding the details of possible contamination.

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. 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, high-efficiency particulate air (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.

The situation in 2001 was unique, and the agencies were not fully prepared
to deal with environmental contamination. 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.

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

Activity 2: Collecting Samples

The agencies used a variety of sample collection methods. 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. 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 sample collection 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. Single
methods were involved in 304 sampling events-that is, CDC and USPS
collecting dry swab samples (185) and CDC and others collecting
premoistened swabs (119).5 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.

USPS officials said that the choice of dry swabs was based on advice from
CDC and an APHL working group, which had coordinated with the head of LRN.
CDC stated that the reason for the use of swabs was an accommodation USPS
had reached with APHL. According to APHL officials, the working 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.

During our fieldwork, we tried to determine what specific advice CDC gave
the Association of Public Health Laboratories (APHL) on using dry swabs.
In responding to our inquiry, CDC did not specifically deny APHL's
statement that an official from CDC's National Center for Infectious
Diseases (NCID) told APHL that dry swabs could be used. However, an
official from CDC's National Institute for Occupational Safety and Health

5We 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.

(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."

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. 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. 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.6

Activity 3: Transporting Samples

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.
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

6The 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.

be in the package."7 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.

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

7Department 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.

conditions, once validated, would have to be standardized to ensure
reproducibility.

Activity 4: Extracting Samples

LRN protocols required that sample material be extracted with specific
extraction procedures and fluids (such as sterile saline or water) and
that the extracted fluid be subjected to specific analytic methods. For
the samples USPS collected under the APHL agreement, the extraction
methods included adding a sample processing solution to the conical tubes
containing the dry swabs before "plating." This process was adapted from
LRN protocols for extracting swabs. 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 of. Cotton is more retentive than some artificial fibers like rayon
and may be more difficult for extraction of spores for analysis. 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. CDC has
acknowledged that "the recovery efficiency of the analytical methods has
not been adequately evaluated."

The reproducibility of the results when an extraction fluid is used can
also be an issue. For example, a U. S. Army Medical Research Institute for
Infectious Diseases (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.8 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 to samples that may have contained few spores.
Without knowing the

8Using 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.

extraction efficiency, a false negative result may potentially be seen as
a true negative.

Activity 5: Analyzing Samples

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

In analyzing the postal samples, laboratories used a variety of methods
for preliminary and confirmatory testing. Preliminary tests included
colony morphology, Gram's stain, hemolysis, and motility tests.9 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.

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

9When 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.

procedures. In a few instances, results were also reported as number of
colony forming units (CFU) per gram of sample material.

According to CDC guidelines, LRN laboratories were to analyze samples by
appropriate LRN protocols. 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
LRN laboratory had to be referred to an appropriately qualified laboratory
for confirmatory testing.

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.

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, although hand-held assays (HHA)
were considered preliminary tests, concerns were raised that the negative
results might lead to a false sense of security.10 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

10See GAO, 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).

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. 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.

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. 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
polymerase chain reaction (PCR) for deoxyribonucleic acid (DNA)
amplification to identify anthrax. Any problems experienced with any of
these variables across any of these plans could affect the final result.

The Sampling Results The results of the CDC, EPA, and USPS testing in 286
postal facilities were largely negative. Of 286 facilities, 23 tested
positive. For 2 of these 23 Were Largely Negative 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.

Testing results differed between the primary facilities and Wallingford.
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.

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. However, the use of both wet
and dry swabs produced positive results in this facility.

USPS and CDC sampled facilities that processed mail from the primary
facilities to determine whether any other facilities had become
contaminated. 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.

For some of the positive facilities, excluding the primary ones:

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

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

  Agencies' Activities
  Were Not Validated

used again. However, Wallingford did not test positive until the fourth
testing. These results underscore the importance of retesting and cast
doubt on the efficiency of the testing.
Of the 263 facilities that tested negative, only 9 were sampled more than
once. 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.

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).

None of the agencies' activities to detect anthrax contamination in the
postal facilities were validated. Without validation, the sampling
activities
could have been based on false assumptions. 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.

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

Validating the overall process is important because operational and health
related 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, 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. 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 anthrax and other biothreat agents.

The lack of validated methods for assessing contamination in postal
facilities impeded the agencies in responding to the incidents. 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. 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.

The issues we have raised in this report 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

  Conclusions

  Recommendations for Executive Action

mean different solutions. Accordingly, efforts to develop sampling
strategies and to validate methods should address requirements specific to
those biothreat agents as well. However, since addressing other agents
would consume resources and time, 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.

Accordingly, in our report, we recommended 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) 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.

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.

In response, 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." 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) probabilitybased sampling
will be necessary when information on the source and path of potential
contamination is not definitive. In our view, probability

sampling will be necessary in order for DHS to achieve its goal of having
a "scientifically defensible sampling strategy and plan."

Mr. Chariman, this concludes my prepared statement. I will be happy to
answer any questions you or Members of the Subcommittee may have.

Contacts and 	If you or your staff have any questions about this report or
would like additional information, please contact me at (202) 512-6412, or

Acknowledgments 	Sushil Sharma, PhD., DrPH, at (202) 512-3460. We can also
be reached by e-mail at [email protected] and [email protected].

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

                       Appendix I: Scope and Methodology

To respond to your request, we interviewed officials from federal agencies
involved in sampling the postal facilities. The federal agencies included
the Centers for Disease Control and Prevention (CDC), the Environmental
Protection Agency (EPA). We also interviewed 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.

We reviewed documentation provided or developed by CDC, EPA, and USPS,
including sample collection strategies, guidance, environmental collection
and analytical methods and protocols. In addition, we reviewed and
analyzed 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 reviewed studies on sampling methods for
detecting biological substances, including anthrax, on surfaces and in the
air. We conducted our review from May 2003 through November 2004 in
accordance with generally accepted government auditing standards.

Although our study focused on anthrax testing relating to 2001 anthrax
incident, we believe that the issue we identified concerning the need for
validated methods and sound sampling strategies would apply to similar
incidents 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.
*** End of document. ***