CLASSIFICATION OF PREAMENDMENTS IVDs FOR THE IDENTIFICATION OF BACILLUS
ANTHRACIS AND YERSINIA PESTIS IN HUMAN SPECIMENS

**PANEL INFORMATION PACKAGE**

MARCH 7, 2002

At the March 7 meeting the Microbiology Devices Advisory Panel will be
asked to recommend the classification of certain products that were used
before 1976 (preamendments) and were overlooked at the time other
preamendments products were classified, back in the early 1980s. At this
meeting the panel will not be asked to discuss any particular product or
submission. The panel will be asked to discuss and make recommendations
on the classification of a category of products, i.e., in vitro
diagnostic products for the identification of *B. anthracis* and *Y.
pestis* that were available before 1976. The classification (Class I,
II, or III) will be based on the review by the panel of the old devices'
safety and effectiveness. These products, when classified, will serve as
predicate devices for new products with similar intended uses that do
not raise new questions of safety and effectiveness.

Factors taken into consideration to classify a device include: the
population for whom the device is intended, conditions of use,
risk/benefit assessment, reliability of the devices based on valid
scientific evidence, and level and types of controls (general or
special) that can offset risks. FDA is providing you with information
about the preamendments *B. anthracis* and *Y. pestis* in vitro
diagnostic products that have been identified. This information includes
the history of the products, how they were used, factors known to affect
the use of these products, and types of controls that are likely to
minimize the risks associated with their use. FDA has also included
proposed language for the classification regulation describing these
products.

GENERAL BACKGROUND

*Bacillus anthracis* and *Yersinia pestis* are bacterial pathogens that
cause disease primarily in animals, and also in humans in contact with
infected animals. Vaccination of animals and high-risk workers had
virtually eliminated human anthrax by the 1970s and human anthrax was
rarely reported in the U.S. before October 2001. Natural cases of human
plague are rare (averaging 8/year) and limited to rural isolated areas
of the southwest, and along the Pacific Coast. Case incidence has
increased since 1975 possibly due to increased transmission to humans as
residential areas encroach upon formerly rural areas, and increased
transmission by domestic cats. Both agents are on the "Critical
Biological Agents" list as agents likely to be used as biological
weapons.

Products for the laboratory diagnosis of these organisms were not
classified with other preamendments products. Several products were
available preamendments although not widely used in many clinical
laboratories in the U.S. These products were used extensively for
surveillance and control activities and for identifying agents in
specimens from animals.

FDA has identified specific bacteriophages and certain antibody and
antigen reagents as preamendments. Please refer to the attached
summaries and key publications for information on these products.

RISKS ASSOCIATED WITH THE USE OF THE OLD DEVICES

The risks that are specifically associated with the use of preamendments
bacteriophages, antibody and antigen reagents for the identification of
*B. anthracis* and *Y. pestis* are those associated with:

-   Laboratory errors (including incorrect procedures, misinterpretation
    of results, inappropriate use) that can cause false negative and
    false positive results;

-   Functional reliability of the test reagents, including their
    expected performance, quality and integrity; and

-   Biosafety and risks to laboratory workers with handling cultures and
    control materials.

The diagnosis of anthrax and plague are both made on the basis of
clinical findings, history of exposure, and identification of the
organism by the clinical laboratory. Failure to identify *B. anthracis*
or *Y. pestis* from a culture isolate or directly from human specimens
may delay the diagnosis and treatment of anthrax or plague (false
negative). A false negative result may delay recognition of the
infection and the investigation of a common source that can affect
others, and in the case of plague, delay identification of patient
contacts. A false positive result may delay the diagnosis of the
patient's true condition and lead to inappropriate or unnecessary
treatment. A false positive result could also lead to unnecessary public
health activities.

False negative or positive test results can be caused by inability of
the test to characterize all strains of the organism, cross-reactivity
with other species (product performance), or user error (from improper
procedures or interpretation of test results) in the laboratory.
Preparation or manufacturing of products may also affect performance of
laboratory testing. Although use of the appropriate test control
materials in the laboratory may identify problems with product
manufacturing, laboratory quality control testing may not always be
adequate or feasible to recognize manufacturing problems.

The quality control materials used by the laboratory will necessarily be
limited by restrictions on maintaining these organisms and the rarity of
appropriate specimen materials. Because handling these organisms poses a
risk to laboratory workers, use of these products and the needed
laboratory control materials would be restricted to laboratories with
the appropriate biosafety facilities and training.

CONTROLS

During the classification proceedings, the panel may recommend special
controls, if general product controls (registration/device listing, good
manufacturing practices, premarket notification, records and reports)
are not considered sufficient. Special controls include, but are not
limited to: postmarket surveillance, performance standards, testing
guidelines, and device tracking. The following controls may be
applicable to these products:

-   The product labeling includes indications, warnings and precautions.
    The warnings and precautions listed in the product labeling are
    written to make the user aware of potential risks associated with
    the use of the device, and to minimize the impact of these risks in
    case they occur.

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-   CDC has developed and disseminated organism-specific practice
    guides. They provide guidance for specimen collection, culture
    methods, and identification criteria. They also give specific
    instructions for transferring isolates and specimens, and for
    notifying local and state health departments. CDC also provides
    laboratory training and proficiency programs. These documents are
    available at the ASM website
    (http://www.asmusa.org/pcsrc/biodetection.htm)

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-   Laboratories are required to notify Departments of Health when *B.
    anthracis* and *Y. pestis* are suspected and to refer suspicious
    isolates to expert laboratories within the national Laboratory
    Response Network.

-   Biosafety guidelines and restrictions for use in laboratories with
    adequate facilities, and training and experience with the
    identification of unusual pathogens.

-   Appropriate manufacturing controls such as pre-release testing can
    reduce risks associated with false negative and false positive
    results (described above).

## *Bacillus species* Preamendments reagents

# Background

*Bacillus anthracis* is one of the oldest known pathogens, causing
anthrax disease in humans and mammals, especially grazing herbivores.
Humans can be infected accidentally through contact with infected
animals or their products. Human cases, primarily cutaneous, are not
uncommon in parts of the world where *B. anthracis* is highly endemic
and animal anthrax vaccine is not widely used (Iran, Iraq, Turkey,
Pakistan and sub-Saharan Africa). Remaining endemic areas in the U.S.
(e.g., Texas, Oklahoma) closely parallel cattle trails of the 1800s. In
the 1950s and 1960s the majority of cases were related to the
manufacture of textiles in which imported fibers were used.

Decreased use of imported contaminated fibers and immunization of
textile workers dramatically reduced human cases in the U.S. Anthrax is
now rare in the U.S., with only three cases reported from 1984-1993. *B.
anthracis* is the bacterial agent believed most likely to be used as a
biological weapon and the first deaths since 1975 occurred in 2001 from
criminal dissemination.

*Bacillus cereus* causes gastrointestinal disease that is generally
self-limiting. B. cereus can also rarely cause nongastrointestinal
infections, particularly with IVDU and immunosuppressed individuals.
Rare nongastrointestinal infections are associated with trauma or
surgery, particularly implants, catheters and shunts. Other Bacillus
species are occasionally reported to cause gastrointestinal and
nongastrointestinal disease. The probability of incorrect species
identification confounds many of these reports.

*Bacillus* species are ubiquitous in nature and persist under adverse
conditions due to resistance of their spores. As a result, *Bacillus*
species are frequently encountered in clinical laboratories as
"contaminants," but also as significant pathogens. Cultured specimens
from which Bacilli may be recovered include blood, CSF, other sterile
body fluids, sputum, skin lesions, and biopsy material.

Differentiation between *Bacillus* species isolates in the laboratory
can be difficult. For clinical purposes, the differentiation of *B.
anthracis* from *B. cereus* is most important, for clinical diagnosis
and for recognizing need for enhanced biosafety measures. A large number
of phenotypic tests can be used, but often only a single one separates
different species. The principal distinguishing characteristics between
*B. cereus* and *B. anthracis* are motility, hemolysis on blood agar,
and gamma phage susceptibility. No one characteristic is sufficient to
discriminate the different species, since *B. cereus* can be nonmotile,
*B. anthracis* can be weakly hemolytic, and rare strains other than *B.
anthracis* can be susceptible to lysis by gamma bacteriophage.

Preamendments reagents for the laboratory differentiation of *Bacillus
anthracis* from other *Bacillus* species include antibodies specific to
organism capsule or other cellular components, organism-specific
bacteriophages, and antigens for serological detection of antibodies to
capsular, somatic, or other antigens.

# Bacteriophage gamma

Description: a specific bacterial virus used in a culture plating method
to distinguish B. anthracis from B. cereus and other Bacillus species.
The bacteriophage can rarely lyse other Bacillus sp. and can fail to
lyse rare strains of B. anthracis.

# 

In 1951, McCloy reported isolation of a bacteriophage that lysed strains
*of B. anthracis*, the parent *B. cereus* strain W, 2 of 56 other *B.
cereus* strains and none of 18 other *Bacillus* species. Brown and
Cherry (1955) subsequently isolated gamma-phage from the original W
bacteriophage of McCloy. Brown et al showed (1958) the phage
specifically lysed vegetative *B. anthracis* (122 strains) and not *B.
cereus* strains tested (115 strains); 8 of 70 *B. cereus* var.
*mycoides* (all dissociant strains) were lysed.

Buck et al (1963) reported that 63 of 74 (85%) *B. anthracis* strains
and a strain of B. megaterium out of 64 other species tested were lysed
by the gamma-phage. USAMRIID recently validated performance showing that
only 2 known phage-refractory *B. anthracis* strains were not lysed (out
of 50 tested) and that a rare *B. cereus* *and B. mycoides* out of 50
non-*B. anthracis* species were lysed \[see Abshire et al, 2001. ASM
poster\].

The following are reported factors that may contribute to false positive
or negative results with the gamma phage testing:

-   Differences in behavior of variant phage strains

-   Media used may affect visualization of lysis

-   Extended or shortened incubation

-   Altered phage titer and stability

-   Heavy inocula

-   Laboratory and technologist inexperience interpreting partial lysis

-   Not using phage results n conjunction with other microbiological
    observations (growth characteristics, Gram reaction, catalase,
    hemolysis and motility)

[Fluorescent antibody detection of *B. anthracis* capsule]{.underline}

*Description: A fluorescein-labeled antibody against B. anthracis
capsule is used to microscopically visualize specific binding with
cultured bacteria. The test can be performed with culture growth or can
be done on specimens that have Gram positive bacteria resembling B.
anthracis. Fluorescence with capsules is presumptive evidence for
identification of B. anthracis that must be confirmed with other
testing.*

Cultured isolates of B. anthracis produce capsules when grown on
bicarbonate agar with serum in the presence of CO~2~. Vegetative
organisms in specimens from patients or animals infected with *B.
anthracis* are also usually encapsulated. Virulent B. anthracis strains
can lose ability to encapsulate when recovered from environmental
sources or maintained in laboratory culture media.

The polypeptide-poly-D-glutamic acid capsule of B. anthracis can be
visualized by certain stains (M'Fadyean, Giemsa, India ink, and
toluidine blue have been described). Cherry & Freeman (1959) reported
using immunofluorescence to identify *B. anthracis*. *B. subtilis* and
*B. megaterium* also produce similar capsules that are positive by the
immunofluorescent antisera prepared against encapsulated *B. anthracis*.

The following are reported factors that may contribute to false positive
or negative results with fluorescent antibody testing:

-   Capsular and cell wall antigens of B. anthracis are shared by other
    species

-   Preparing high titer antiserum in animals is difficult and a safety
    concern

-   Spore surface antigens are not species-specific

-   Growth conditions affect encapsulation

NOTE: Hyperimmune antiserum prepared in rabbits against either live
virulent bacteria or formalin-killed virulent bacteria was commonly used
for the Ascoli precipitin test. This test was first described in 1911.
The procedure is primarily used for detecting *B. anthracis* antigens in
tissues of animals suspected to be infected. It was also used to test
hides and other animal products for evidence of anthrax contamination.
The test is not highly specific but was useful in that other *Bacillus*
species would not likely have proliferated and deposited precipitating
antigens as would *B. anthracis*.

[*B. anthracis* antigens for antibody detection]{.underline}

Antibody detection can be used for confirmation of anthrax if the
patient survives, and early antibiotic treatment does not abrogate
antibody expression. Such serological testing is most useful for
monitoring responses to anthrax vaccines and for epidemiological
investigations. Whole cell antigen preparations are not specific to B.
anthracis. Protective antigen was first identified by Gladstone (1946)
in cell-free culture filtrates.

A skin test preparation (Anthraxin) has been used since 1962 in the
former USSR. Anthraxin also contains antigens that are not highly
specific for B. anthracis.

FDA is continuing to look for information relevant to the classification
of antigen products. If these become available they will be forwarded to
you at a later date.

[References provided]{.underline}:

-   Abshire TG, JE Brown, CM Allan, SL Redus, JD Teska, JW Ezzell.
    Validation of the use of gamma phage for identifying Bacillus
    anthracis, 2001. C122, 102^nd^ ASM Ann Meeting poster.

-   Brown, ER and WB Cherry. Specific identification of Bacillus
    anthracis my means of a variant bacteriophage, 1955. J inf Dis
    96:34-39.

-   Brown, ER, MD Moody, EL Treece, CW Smith. Differential diagnosis of
    Bacillus cereus, Bacillus anthracis and Bacillus cereus var.
    mycoides, 1958. J Bacteriol 75: 499-509.

-   Buck, CA, RL Anacker, FS Newman, A Eisenstark. Phage isolated from
    lysogenic Bacillus anthracis, 1963. J Bacteriol 85:1423-1430.

-   Parry, JM, PCB Turnbull, JR Gibson. A Colour Atlas of Bacillus
    species, 1983. pp 59-91. Wolfe Medical Publications Ltd, London.

# 

# ***Yersinia pestis* Preamendments reagents**

# 

# Background

*Yersinia pestis* causes disease in humans and animals. Plague is
primarily a zoonotic disease with reservoirs worldwide. It has been
described since Biblical times. In the US, most cases are contracted via
fleabites from infected rural wild rodents (squirrels, mice, rabbits,
prairie dogs) and more recently domestic cats. Enzootic disease is
limited to areas west of the Rocky Mountains.

Naturally-occurring human disease is not common (390 cases were reported
in the US from 1947 to 1996), and is rarely seen outside enzootic areas
except for travelers. Case incidence has increased since 1975 possibly
due to increased transmission as residential areas encroach upon
formerly rural areas, and increased transmission by domestic cats. *Y.
pestis* is also on the list of "Critical Biological Agents" as having
potential to be used as a biological weapon.

Naturally acquired infections are primarily bubonic or septicemic with
some developing pneumonia secondary to the initial route of infection.
Primary pneumonic plague results from aerosol transmission between an
infected human or animal with plague (2% of the 390 cases from
1947-1996). Pneumonic plague has occurred from cat-transmitted disease.
Intentional dissemination of plague would most probably occur by
aerosolization and cause primary pneumonic plague. Plague is fatal in
50% of infected humans, if untreated. Septicemic and pneumonic plague
are clinically indistinguishable from other syndromes.

Reagents and methods for bacteriologic diagnosis of *Y. pestis* are
referred to in the medical literature, including pitfalls of a
presumptive identification \[see Bibel & Chen, 1976\]. A serologic
response may take 10 to 14 days to develop and can provide retrospective
evidence of infection. In the 1970s when preamendments products were
first classified, clinical laboratories throughout the U.S. did not
encounter the organism often and specific reagents were mostly used at
certain public health laboratories and reference laboratories. Classical
staining with Gram, Giemsa, Wright, or Wayson stains can suggest *Y.
pestis*, but is nonspecific as other bacteria can have the "safety-pin"
appearance, and not uncommonly *Y. pestis* will not have this
appearance. Fluorescent antibody against Fraction 1 capsular antigen
directly on clinical specimens can be presumptive evidence when clinical
history and presentation is suggestive.

Specimens collected for culture include blood, bubo aspirates, sputum,
CSF or skin lesion scrapings. The organism is identified from culture
growth by morphology, preferential growth at 27-28° C, biochemical
tests, fluorescent antibody testing, and susceptibility to a specific
bacteriophage. Rapid biochemical identification methods are generally
not useful and may cause erroneous results, as the organism is slow
growing and relatively inactive biochemically. Automated microbiological
test systems may not be able to differentiate *Y. pestis* from other
similar Gram-negative bacteria, most notably *Y. pseudotuberculosis*, a
closely related enteric pathogen commonly found in environmental
samples.

FDA has identified three products that were overlooked when pre-1976
products were first classified. These products are used to identify Y.
pestis or to provide serological evidence of infection with Y. pestis.
The following sections describe the preamendments history of these three
products, their performance characteristics, and factors associated with
inaccurate results.

[Bacteriophage]{.underline}

*Description: a specific bacterial virus used in a culture plating
method to distinguish Y. pestis from Y. pseudotuberculosis. The
procedure is performed at 20-25° C, as the bacteriophage can lyse Y.
pseudotuberculosis at 37°C, but not at the lower temperature. Lysis at
22-25° C provides presumptive evidence that a culture isolate is Y.
pestis.*

In 1950, the WHO Expert Committee of Plague recommended using a specific
bacteriophage for identification of *Y. pestis* (P. pestis at that
time). Cavanaugh and Quan described a method for using Phage H
lyophilized on filter paper strips (Rapid identification of P. pestis
\[1953, AJClinPathol 13:619-620\]. CDC and public health laboratories
used this method for many years. The preparation of the gamma phage
reagent is well described \[Bahmanyar & Cavanaugh, 1976. Plague Manual.
WHO\].

Test methods that have been described, assess specific lysis at both
temperatures (room temperature and *37°C*) and compare it to that of
control organisms.

The following are reported factors that may contribute to false positive
or negative results:

-   Differences in behavior of variant phage strains

-   Incubation temperatures critical for correct results

-   Media used may affect visualization of lysis

-   Mixed cultures may overgrow and mask lysis

-   Altered phage titer and stability

-   Laboratory and technologist inexperience

-   Not using in conjunction with other microbiological observations
    (growth characteristics, Gram reaction, growth at 28°, catalase,
    oxidase and urease, DFA)

[Fluorescent Antibody Reagent]{.underline}

Description: A fluorescein-labeled antibody against Fraction 1-antigen
that is used to microscopically visualize specific binding with cultured
bacteria. The test can be performed with culture growth or can be done
on specimens which have Gram negative bacteria resembling Y. pestis.
Presence of F1 antigen is presumptive evidence of Y. pestis that must be
confirmed with other testing.

Lederle Laboratories, Inc. prepared rabbit anti-plague serum against
whole *Y. pestis* cells for testing done by WRAIR. WHO described a
method for preparing anti-F1 serum used in a direct immunofluorescence
assay.

Antigen expression is optimal when cultures are grown from 33-37° C. No
cross-reactivity is seen with Y. pseudotuberculosis or E. coli.

The following are reported factors that may contribute to false positive
or negative results:

-   Although specific for F1, fluorescence with FA reagents is not
    definitive for Y. pestis

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-   Expression of F1 antigen by different strains may vary

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-   F1 antigen can be lost with storage

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-   Organism density of prepared slides can affect interpretation
    (antigen overload)

[Fraction 1 Antigen for HA Tests]{.underline}

*Description: Fraction 1 purified antigen is a protein with
antiphagocytic properties from the capsular envelope of Y. pestis. It is
highly immunogenic in both human and mammalian hosts. This reagent is
used to sensitize sheep erythrocytes for hemagglutination (HA) testing
to detect antibody responses to F1 in human or other host sera.
Significant levels of human antibody to this antigen can be
retrospective confirmation of Y. pestis infection or can be presumptive
when a single serum sample is tested.*

The Fraction1 envelope antigen of *Y. pestis* was first isolated in 1947
by Baker et al; protein and glycoprotein components were identified and
characterized. This antigen was used for detecting specific antibody to
*Y. pestis* in complement-fixation (CF) and passive hemagglutination
(PHA) tests developed by Chen et al (1952) and Chen & Meyer (1954). The
indirect hemagglutination WHO standard method (described in 1965 by
Cavanaugh et al), was used extensively for serological surveys. These
tests were useful when *Y. pestis* could not be recovered from tissues,
bubo fluid or blood. They were also used in early plague immunization
studies.

In an evaluation with sera from 83 human plague patients, 78% were
positive by serological tests, while only 36% were bacteriologically
positive (Meyer, 1964). The passive HA (PHA) test was found to be 20-50
times more sensitive than the CF test. No cross-reactivity between *Y
pestis* and Y. *pseudotuberculosis* was found with either CF or PHA
tests.

In 1972, Marshall et al published findings from testing 307 immunized
and 75 nonimmunized individuals using agar-gel precipitin inhibition
test, CF and PHA. PHA was the most sensitive. The test is generally
reproducible within 2-fold dilutions. HA titers persisted for at least a
year in a pneumonic plague patient studied. The HA test was found to be
more reliable than the CF test for a variety of wild and domestic
mammals. Again, Marshal et al showed utility of hemagglutination testing
for evaluating titers of vaccinees. In this report, 572/576 sera had
titers within one dilution when retested after 7 months \[Marshal et al,
1974. Plague Immunization III. Serologic response to multiple
inoculations of vaccine. J of Inf Dis 129:S26-S29\].

The soluble F1 antigen in physiological saline is stable at 4° C for at
least 5 years. Purity and concentration of F1 was found to correlate
with the degree of specificity. Methods for preparation are published
\[Chen & Meyer, 1966; Rust et al, 1972\].

In the 1970's, F1-sensitized sheep red cells were available from Walter
Reed Army Institute of Research \[Manual of Clinical Immunology, 1976\].

The following are factors contributing to false positive or negative
results with the HA testing:

-   Impure antigen preparations

-   incorrect concentration of F1 preparation

-   Serum sample obtained too early (serological response may appear as
    early as day 5 and peaks around day 14, persisting at least to day
    40)

-   Occasional infections with nonencapsulated Y. pestis

-   Prozone effects

-   inability to differentiate between recent and past infection

-   subjectivity of endpoints

-   nonspecific reactivity of heterophiles

[References provided:]{.underline}

Bibel, D and T Chen. 1976, Diagnosis of plague: an analysis of the
Yersin-Kitasato Controversy. Bacteriol. Rev. 40:633-651.

Cavanaugh, D and S Quan. 1953, Rapid Identification of Pasturella
pestis. Am J Clin Path. 23:619-620.

Chen, T and K Meyer. 1966, An Evaluation of Pasturella pestis
Fraction-1-Specific Antibody for the Confirmation of Plague Infections.
Bull. Wld Hlth Org 34:911-918.

Marshall J and D Cavanaugh. 1972, Comparison of the reliability and
sensitivity of three serological procedures in detecting antibody to
Yersinia pestis. Appl Microbiol. 24:202-204.

Perry D and J Fetherston. 1997, Yersinia pestis -- etiologic agent of
plague. Clin Micro Rev 10:35-66.

Rust, JH, S Berman, W Habig, J Marshall, D Cavanaugh. 1972, Stable
Reagent for the Detection of Antibody to the Specific Fraction I antigen
of Yersinia pestis. Appl. Microbiol. 23: 721-724.