SENTINEL LEVEL CLINICAL LABORATORY GUIDELINES

FOR

SUSPECTED AGENTS OF BIOTERRORISM

AND

EMERGING INFECTIOUS DISEASES

Bacillus anthracis and Bacillus cereus biovar anthracis

American Society for Microbiology (ASM)

Revised August 2017

For last revision, see website below

https://www.asm.org/Articles/Policy/Laboratory-Response-Network-LRN-Sentinel-Level-C

ASM Subject Matter Experts:

James W. Snyder, Ph.D. Steven D. Mahlen, Ph.D.

University of Louisville Affiliated Laboratory, Inc.

Louisville, KY Bangor, ME

[email protected] [email protected]

ASM Sentinel Level Laboratory Protocol Working Group APHL Advisory Committee

Vickie Baselski, Ph.D.

University of Tennessee at

Memphis

Memphis, TN

vbaselsk[email protected]u

David Craft, Ph.D.

Penn State Milton S. Hershey

Medical Center

Hershey, PA

Dcraft1@hmc.psu.edu

Peter H. Gilligan, Ph.D.

University of North

Carolina Hospitals/

Clinical Microbiology and

Immunology Labs

Chapel Hill, NC

[email protected]c.edu

Larry Gray, Ph.D.

TriHealth Laboratories and

University of Cincinnati

College of Medicine

Cincinnati, OH

Larry_gray@trihealth.com

Major Todd Kijek, Ph.D.

US Army Medical Research

Institute for Infectious Diseases

Ft. Detrick, MD

[email protected]y.mil

Michael J. Loeffelholz, Ph.D.

Department of Pathology

Univ. Texas Medical Branch

Galveston, TX

mjloeffe@utmb.edu

Judith C. Lovchik, Ph.D.

Indiana State Department of

Health Laboratories

Indianapolis, IN

[email protected]N.gov

Scott W. Riddell, Ph.D.

Department of Pathology

SUNY Upstate Medical

University

Syracuse, NY

[email protected]du

Barbara Robinson-Dunn, Ph.D.

Department of Clinical

Pathology

Beaumont Health System

Royal Oak, MI

BRobinson-

Dunn@beaumont.edu

Michael A. Saubolle, Ph.D.

Banner Health System

Phoenix, AZ

Mike.Saubolle@bannerh

ealth.com

Susan L. Shiflett

Michigan Department of

Community Health

Lansing, MI

ShiflettS@michigan.gov

Alice Weissfeld, Ph.D.

Microbiology Specialists Inc.

Houston, TX

alice@microbiologyspecialist.

com

David Welch, Ph.D.

Medical Microbiology

Consulting

Dallas, TX

dfw@gmx.us

Mary K. York, Ph.D.

MKY Microbiology

Consultants

Walnut Creek, CA

marykyork@gmail.com

Coordinating Editor:

Steven D. Mahlen, Ph.D.

Affiliated Laboratory, Inc.

Bangor, ME

smahlen@emhs.org

Administrative Support

Kimberly E. Walker, Ph.D.

American Society for

Microbiology

kwalker@asmusa.org

Patricia Blevins, MPH

San Antonio Metro Health

District Laboratory

Patricia.blevins@sanantonio.gov

Erin Bowles

Wisconsin State Laboratory of

Hygiene

bowlesej@mail.slh.wisc.edu

Christopher Chadwick, MS

Association of Public Health

Laboratories

christopher.chadwick@aphl.org

Mary DeMartino, BS,

MT(ASCP)SM

State Hygienic

Laboratory at the

University of Iowa

mary[email protected]u

Harvey Holmes, PhD

Centers for Disease

Control and Prevention

[email protected]

Kara MacKeil

Association of Public

Health Laboratories

kara.MacKeil@aphl.org

Chris Mangal, MPH

Association of Public

Health Laboratories

Chris.Mangal@aphl.org

Amanda Moore, BS

South Carolina Department of

Health and Environmental

Control

[email protected]ov

James Rudrik, PhD,

Michigan Department of

Community Health

RudrikJ@michigan.gov

Maureen Sullivan, MPH

Minnesota Department

of Health

Maureen.sullivan@state.mn.us

PREANALYTICAL CONSIDERATIONS

I. PRINCIPLE

A. Introduction

Bacillus anthracis, the agent of anthrax, is a zoonotic disease that is transmissible

to humans through consumption or handling of contaminated products, is an

aerobic, spore-forming, nonmotile, large Gram-positive bacterium (4). Bacillus

cereus biovar anthracis strains were identified in the early 2000’s in Cameroon

(strains CA) and Côte d’Ivoire (strains CI) (1,17). These strains were recovered

from gorillas and chimpanzees with anthrax-like disease. The organism has since

been recovered from an elephant and goats in other countries of Africa (1). B.

cereus biovar anthracis CA strains are non-hemolytic, motile, and resistant to

penicillin G, while B. cereus biovar anthracis CI strains are non-hemolytic,

motile, and sensitive to pencillin G (17); some B. cereus biovar anthracis strains

may exhibit weak beta-hemolysis upon extended incubation (48 h) and may be

more hemolytic when incubated in CO

at 48 h (see Table 1). B. anthracis and B.

cereus biovar anthracis strains are classified as Tier I, Category A agents because

of their suitability for use as biothreat agents in an attack or commission of a

biocrime. At this time no human infections caused by B. cereus biovar anthracis

have been described. This procedure describes steps to recognize, presumptively

identify, and rule out these organisms from clinical specimens in Sentinel Clinical

Laboratories. Such laboratories are defined as those who are certified to perform

high complexity testing under the Clinical Laboratory Improvement Amendment

of 1988 (CLIA’88) by the Centers for Medicare and Medicaid Services (CMS) for

the applicable Microbiology specialty. Laboratory in-house testing includes

Gram stains, and at least one of the following: lower respiratory tract, wound, or

blood cultures.

Sentinel clinical laboratories are not required to register with the Select Agent

Program to conduct diagnostic testing for Select Agents, both Tier I and non-Tier

1. Testing for Select Agents may be performed by laboratories as long as the

laboratory follows the policies listed in the reporting section of this document

when a Select Agent cannot be ruled out. Consult with your designated LRN

Reference Laboratory or refer to the CDC Division of Select Agents and Toxins

website at http://www.selectagents.gov for questions.

NOTE: To rule out B. anthracis and B. cereus biovar anthracis requires a

combination of morphological assessment including Gram stain characteristics,

and three simple, conventional tests. Automated systems and manual multi-test

kit identification methods have no place in the identification of this organism due

to the danger of misidentification and its close relationship to other species within

the Bacillus genera.

Table 1. Comparison of B. anthracis, B. cereus, and B. cereus biovar anthracis

characteristics (adapted from reference 17)

Characteristic

anthracis

B. cereus

B. cereus biovar

anthracis CI

B. cereus biovar

anthracis CA

Hemolysis

Motility

+/-

Gamma-phage

susceptibility

Penicillin G

Capsule

Absent in

vitro

1: CI = Côte d’Ivoire strains, from chimpanzees

2: CA = Cameroon strains, from gorillas/chimpanzees

3: Hemolysis: + = beta hemolytic on sheep blood agar; - = non-hemolytic

4: Motility: + = motile; - = non-motile. +/- = B. cereus biovar anthracis strains are usually motile,

including those recovered from gorillas, chimpanzees, and elephants; B. cereus biovar anthracis

goat strains from Democratic Republic of the Congo were non-motile (3).

5: Gamma phage susceptibility: + = susceptible; - = resistant.

6: S= susceptible; R = resistant

B. Geographic distribution

Anthrax is endemic in southern Europe, parts of Africa, Australia, Asia, and

North and South America. Its persists in arid deserts of the Middle East, Asia,

Africa, Australia and South America with the majority of cases reported from

Iran, Turkey, Pakistan, and Sudan. Although relatively rare in the United States,

over 500 cases in Texas were reported in White Tailed Deer since 2006. Cases

have also been reported in livestock in South Dakota, Nebraska, Arkansas,

Mississippi, Louisiana, and California.

Currently, B. cereus biovar anthracis strains have only been found in certain

African countries, including Cameroon and Côte d’Ivoire. These strains are

known to cause an anthrax-like disease in gorillas and chimpanzees, and have

been isolated from other animals, including elephants and goats. B. cereus biovar

anthracis strains are genetically similar to B. anthracis and produce all of the

primary B. anthracis virulence factors, thus, they are now considered to be select

agents in the United States.

The use of B. anthracis as a bioterrorism agent to inflict disease and death

following contact with or inhalation of spores has dominated recorded history for

centuries (6, 19). Primarily used by many countries, including the United States,

for military purposes in the conduct of biowarfare, it gained notoriety in the

commission of biocrimes, determined to be hoaxes, throughout the 1990s.

Although the LRN was created in late 1999, its role and responsibilities in

preparing for, and responding to bioterrorism increased dramatically during and

after the 2001 outbreak. The lessons learned from this national event

demonstrated the serious need for training Sentinel Level Clinical Laboratories

and preparing them to play a key role in the LRN.

Three naturally occurring cases of anthrax have been reported within the past five

years, one case respectively of gastrointestinal, cutaneous, and inhalational (20,

22, 23). These incidences are examples in which Sentinel Level Clinical

Laboratories will be challenged by ensuring that all suspicious Bacillus spp., have

been ruled out based on the use of the LRN designated tests and algorithm for B.

anthracis and B. cereus biovar anthracis discussed in this protocol.

C. Diseases and Clinical Presentation

Anthrax is a zoonotic disease that occurs most frequently in herbivorous animals

(e.g., cattle, sheep, and goats), which acquire endospores from contaminated soil.

Human disease is less common and results from contact with infected animals or

with commercial products derived from them, such as wool and hides. Infection

can occur in one of three forms:

1. Cutaneous, responsible for >95% of naturally occurring cases, is initiated when

the bacterium or spores enter the skin through cuts or abrasions, such as when

handling contaminated hides, wool, leather, or hair products (especially goat

hair) from infected animals (2, 14, 22). Skin infection begins as a raised itchy

bump or papule that resembles an insect bite. Within 1 to 2 days, the bump

develops into a fluid-filled vesicle, which ruptures to form a painless ulcer

(eschar), usually 1 to 3 cm in diameter with a characteristic black necrotic area

in the center. Pronounced edema if often associated with lesions to the releases

of edema toxin, a major virulent factor produced by the organism. Lymph

glands in the adjacent area may also swell. Approximately 20% of untreated

cases of cutaneous anthrax result in death either because the infection becomes

systemic or because respiratory distress caused by edema in the cervical and

upper thoracic regions. Deaths are rare following appropriate antimicrobial

therapy, with lesions becoming sterile within 24 h and resolving several weeks

later. There are a few case reports of transmission by insect bites, presumably

after the insect fed on an infected carcass (24).

2. Gastrointestinal anthrax may occur 1 to 7 days following consumption of

contaminated undercooked meat from infected animals and is characterized by

acute inflammation of the intestinal tract. Initial signs of nausea, loss of

appetite, vomiting, and fever are followed by abdominal pain, vomiting of

blood, and severe bloody diarrhea. If not treated, the mortality rate ranges from

25 to 60%. Pharyngeal lesions may also occur from ingestion of contaminated

food (20).

3. Inhalational anthrax results from the inhalation of B. anthracis spores and can

occur following an intentional aerosol release as was evident in the 2001

anthrax biocrime. Depending on the quantity of aerosolized spores, the

incubation period ranges from 1 to 6 days. Aerosolization of anthrax spores is

regarded as the most likely method to be used in a bioterrorism or biocrime

event (5, 9, 10, 15). Though the minimum infectious inhaled dose has not been

specifically determined, the U. S. Department of Defense (DoD) estimates that

the 50% lethal dose for humans is between 8,000 and 10,000 spores (8).

Following inhalation, the spores enter pulmonary macrorophages and are

carried to the mediastinal lymph nodes. Germination and vegetative growth

result in the production of an antiphagocytic capsule and a toxin comprised of

three proteins, edema factor, lethal factor, and protective antigen, which play

major roles in the virulence and resultant infectious and clinical manifestation

(7). Disease onset is gradual and nonspecific. Fever, malaise, and fatigue may

be present initially (prodromal stage) and be accompanied by a nonproductive

cough and chest discomfort. These initial symptoms are often followed by a

short period of severe respiratory distress with labored breathing (dyspnea),

perfuse sweating (diaphoresis), high-pitched whistling respiration (stridor), and

cyanosis (bluish skin color). Fatal sepsis with generalized hemorrhage, massive

hemorrhagic mediastinitis, and necrosis ensue. Shock and death usually occur

within 24 – 36 h following the onset of respiratory distress, and in later stages if

therapy is not initiated within 48 h following the onset of symptoms, with

mortality reaching 95 – 100% (4, 7, 15, 27). Physical findings are usually

nonspecific. The chest X-ray is often pathognomic (disease-specific), revealing

a widened mediastinum (not always present) with pleural effusions with

infiltrates commonly absent. A single case of inhalational anthrax should alert

all healthcare workers to the possibility of a bioterrorism or biothreat event (5).

Person-to-person transmission of inhalational anthrax has not been confirmed

(3, 9).

D. CDC CASE DEFINITION (http://wwwn.cdc.gov/nndss/)

A clinically compatible illness with one of the following:

• Culture and identification of B. anthracis from clinical specimens by the

LRN

• Demonstration of B. anthracis antigens in tissues by

immunohistochemical staining using both B. anthracis cell wall and

capsule monoclonal antibodies

• Evidence of a four-fold rise in antibodies to protective antigen between

acute and convalescent sera or a four-fold change in antibodies to

protective antigen in paired convalescent sera using CDC quantitative

anti-PA immunoglobulin G (IgG) ELISA testing

• Documented anthrax environmental exposure and evidence of B. anthracis

• DNA (e.g., by LRN-validated polymerase chain reaction) in clinical

specimens collected from a normally sterile site (e.g., blood or CSF) or

lesion of other affected tissue (skin, pulmonary, reticuloendothelia, or

gastrointestinal).

II. SAFETY CONSIDERATIONS

A. According to the 5

Edition of the BMBL, unless you are working with high

concentrations of this organism or performing procedures that produce aerosols,

B. anthracis can be handled using BSL-2 practices (25). Do not process

nonclinical (environmental or animal) specimens in hospital or commercial

reference laboratories; restrict processing to human clinical specimens only.

Nonclinical specimens should be directed to the designated LRN Reference

Laboratory.

B. All patient specimens can be handled using BSL-2 practices. BSL-3 precautions,

wearing gloves and gown and working in a certified Class II biosafety cabinet

(BSC) are recommended when performing activities having a high potential for

aerosol production. Subcultures should be performed in a BSC and plates should

be taped/shrink sealed, and incubated in 5 – 10% CO

. All additional testing

should be performed only in the BSC while wearing gloves to prevent acquiring

infection through the skin.

C. Decontamination of laboratory surfaces is easily accomplished using a fresh

solution of 10% bleach. In addition, pipettes, needles, plastic loops, and

microscopic slides should be soaked in 10% bleach or 10 – 30% formalin for 24

h before being autoclaved. Phenolics are not sporocidal at the usual working

dilutions (12, 25).

III. MATERIALS

1. Media

a. Standard liquid blood culturing system with manual or instrument

detection

b. BAP

c. CHOC

d. MAC (or EMB)

e. Semi-solid motility medium

2. Reagents

a. Appropriate disinfectant such as 10% bleach

b. Gram stain reagents

c. Catalase (3% hydrogen peroxide)

3. Equipment and supplies

a. Biosafety cabinet

b. Personal Protective Equipment (PPE; gloves, solid front gown)

c. 35 - 37° C incubator (ambient air, 5 – 10% CO

)

d. Light microscope with 100x objective and 10x eyepiece

e. Microscope slides and cover slips

f. Pipettes, inoculating loops

g. Blood culture instrument (optional)

NOTE: Refer to General Introduction and Recommendations section for a listing of

biochemical test and associate procedures.

IV. QUALITY CONTROL

Perform quality control of media and reagents according to package inserts, most

recent CLSI document M22, and CLIA standards, using positive and negative

controls. Do not use B. anthracis or B. cereus biovar anthracis as a control organism

due to its infectious nature. Examine culture plates for contamination, poor

hemolysis, cracks, and drying. Quality control of biochemical tests using a positive

and negative reacting organism should generally be performed on each lot of reagent.

For frequency of quality control, refer to manufacturer guidelines and state and

federal regulation. Refer to biochemical test section (located in the General

Introduction and Recommendations Section) of procedures and quality control

organisms for each test.

V. SPECIMEN COLLECTION

References (9, 13, 16, 18, 26)

* additional specimens that may be requested by your designated LRN Reference Laboratory are: plasma, pleural fluid, serum

A. Rejection of specimens

1. Use established laboratory criteria for rejection of specimens

A. Collection and Transport of Clinical Specimens for Laboratory Rule-Out Testing*

Cutaneous

Vesicular (early) stage

Eschar (late) stage

• Unroof vesicle and aspirate fluid or collect with two sterile swabs

(dacron)

• Insert swab (dacron) beneath the edge of the eschar, rotate swab or

obtain an aspirate

• Transport specimens at room temperature

Gastrointestinal

• Stool (> 5 grams…pecan size), collect and transport in a leak proof

sealed container

• Collect blood (late stage of infection) directly into an appropriate

blood culture bottle (aerobic and anaerobic)

•

Transport specimens and bottles at room temperature

Inhalational

• Sputum

• Blood: collect directly into an appropriate blood culture bottle (aerobic

and anaerobic)

• Cerebral Spinal Fluid only if signs of meningitis occur

• Transport specimens and bottles at room temperature

Postmortem

Tissue

• Tissue pieces should be collected and kept moist

• Transport in sterile container at room temperature within 1 hour of

collection

2. Environmental or non-clinical specimens are not to be processed by Sentinel

Laboratories; contact your designated LRN Reference Laboratory directly.

ANALYTICAL CONSIDERATIONS

VI. SPECIMEN PROCESSING

A. Cutaneous

1. Plate cutaneous, tissue, and stool specimens onto BAP and MAC (or EMB).

With the exception of stool specimens, prepare smears for Gram stain.

B. Respiratory

1. Plate respiratory specimens onto BAP, CHOC, and MAC (or EMB). Prepare

smear for Gram stain.

C. Blood

1. Aseptically inoculate liquid blood culture bottles with appropriate blood volume

per manufacturer’s instructions.

D. Cerebrospinal fluid (CSF)

1. Prepare a cytospin smear for Gram stain

2. Centrifuge CSF specimens at 1,500 x g for 15 min using a clinical centrifuge

equipped with appropriate biocontainment tube holders. Open the

biocontainment tube holders only in a BSC. Plate the resultant sediment onto

BAP and CHOC; aspirate the remaining sediment and inoculate TSB or

equivalent.

VII. INCUBATION AND EXAMINATION OF CULTURES

A. Incubate all plated specimens, except MAC (or EMB), at 35 to 37°C in 5-10% CO

;

MAC (or EMB) is incubated in ambient air at 35 to 37°C.

B. Examine all cultures within 18-24 h of incubation. Growth of B. anthracis or B.

cereus biovar anthracis may be observed as early as 8 h following incubation. This

can be helpful when looking for B. anthracis or B. cereus biovar anthracis from

mixed cultures (sputum, stool).

VIII. CULTURE IDENTIFICATION

If your laboratory uses matrix-assisted laser desorption/ionization time-of-flight (MALDI-

TOF) mass spectrometry for bacterial identification and if the manufacturer provides your

facility with an alternate tube extraction method, it is recommended that the resulting

extract be filtered using a 0.2 µ (or less) filter. This additional step is recommended to

reduce the risk of laboratory contamination with viable bacteria and spores.

Using automated systems, including MALDI-TOF technology, may result in exposure to

dangerous pathogens, and could result in erroneous identification, Ex: Bacillus anthracis

misidentified as B. cereus; Yersinia pestis misidentified as Y. pseudotuberculosis.

The rule out or referral of B. anthracis or B. cereus biovar anthracis is accomplished by

following the algorithm below and the associated tests.

A. Gram stain suspicious colonies from agar plates and blood cultures in a BSC. B.

anthracis and B. cereus biovar anthracis are large (1 to 1.5 by 3 to 5um) Gram-

positive rods. Vegetative cells seen on Gram-stained smears of clinical specimens

often occur in short chains of two to four cells that are encapsulated. Gram stains

from colonies grown on BAP appear as long chains of nonencapsulated Gram-

positive bacilli. Endospores are not commonly seen in direct smears of clinical

specimens. If present, the spores are oval and located centrally or subterminally and

do not cause swelling of the vegetative cell (see Fig 1).

Fig. 1. Gram stain of blood culture containing B. anthracis

Photo courtesy of Dr. James Rudrick, Michigan Department of Community Health

B. Both B. anthracis and B. cereus biovar anthracis grow well on BAP and CHOC, but

not on MAC (or EMB). Colonies are round with irregular edges, flat or slightly

convex with a ground glass appearance. There are often “comma-shaped” projections

from the edge of the colony, producing the “Medusa head” shape. The colonies are

nonhemolytic on BAP and have a tenacious consistency that when teased with a loop,

the growth will stand up like beaten egg whites. Some B. cereus biovar anthracis

strains may exhibit weak hemolysis upon extended incubation (48 h), particularly

when incubated in CO

Fig. 2. Colonies of B. anthracis on Blood Agar

Photo courtesy of APHL

C. Perform all testing in a BSC. Refer to the Biochemical Test Procedures listed in the

Introduction and General Recommendations Section.

1. Catalase – should be positive

2. Motility in semi-solid medium– B. anthracis is usually negative; B. cereus

biovar anthracis strains are usually motile (Table 1).

NOTE: Performing a catalase test is potentially hazardous due to the potential for the creation of

an aerosol thus it is recommended to perform this test in a BSC. It is recommended to perform

motility in semi-solid medium instead of a wet mount since results are less subjective in semi-

solid medium.

D. Presumptive identification of B. anthracis and B. cereus biovar anthracis (see flow

chart below)

NOTE: Confirmatory identification is made by an LRN Reference Laboratory; refer to

http://www.bt.cdc.gov/lrn/biological.asp

1. Direct Gram stain: large, Gram-positive rods; Spores are not normally

observed in smears from clinical specimens.

2. Colonies on BAP are nonhemolytic, project a ground glass appearance, and

may have “Medusa head” characteristics. If endospores are present, they are

oval-shaped and located either centrally or subterminally.

Bacillus anthracis and B. cereus biovar anthracis Identification Flowchart

Note: Biochemical test procedures and quality control instructions can be found at the end of the

General Recommendation and Biochemical Testing Procedures document.

Major Characteristics of Bacillus anthracis and B. cereus biovar anthracis

Gram Stain Morphology: Large, Gram-positive rods.

Spores may be found in cultures grown in 5% CO

but not usually in clinical samples

Colony Morphology: Ground glass appearance, non-pigmented, gamma hemolytic (no

hemolysis) on BAP (some strains of B. cereus biovar anthracis may be weakly hemolytic

after 48 h of incubation)

No growth on MAC (or EMB)

Catalase positive?

Bacillus anthracis and B.

cereus biovar anthracis are

ruled out. Continue with

routine identification.

Bacillus anthracis/B. cereus biovar anthracis not

ruled out.

Call the LRN Reference Level Laboratory and send

suspected agent.

Report: Possible Bacillus anthracis or B. cereus biovar

anthracis submitted to LRN Reference Level

Laboratory for confirmatory testing.

Gamma hemolytic (no hemolysis)

Yes

POST ANALYTICAL CONSIDERATIONS

IX. REPORTING, NOTIFICATION, AND TRANSFER

A. B. anthracis or B. cereus biovar anthracis is suspected and cannot be ruled out if the

isolate fulfills the following characteristics:

• Direct Gram staining reveals large Gram-positive rods

• Grows on BAP as non-pigmented, odorless, white colonies having a ground glass

appearance with edges that are slightly undulate (“Medusa heads”; comma-

shaped)

• Does not grow on MAC (or EMB)

• Nonhemolytic on BAP

• Catalase – positive

• Motility – B. anthracis strains are nonmotile, B. cereus biovar anthracis strains

are motile

B. Notification and submission of cultures if B. anthracis or B. cereus biovar anthracis

cannot be ruled out based on the above characteristics.

1. Generate a report to the attending physician that B. anthracis or B.

cereus biovar anthracis cannot be ruled out and the isolate has been

referred to your designated LRN Reference Laboratory for confirmatory

identification.

2. Do not attempt full identification and susceptibility testing in the

Sentinel Level Clinical Laboratory.

3. Immediately notify your designated LRN Reference Laboratory who

will provide the referring laboratory with guidance and

recommendations for retaining the specimen or isolate and submission

for confirmatory identification.

4. Preserve original specimens pursuant to a criminal investigation and

transfer to your designated LRN Reference Laboratory in accordance

with state and local requirements. In particular, the appropriate material,

including blood culture bottles, tubes, plates, and clinical specimens

(e.g., aspirates, biopsies, sputum specimens) should be documented and

either submitted to the LRN Reference Laboratory or retained until the

Reference Laboratory confirms the identification.

5. Do not ship specimens or cultures to the LRN Reference Laboratory

without prior arrangements.

6. Notify other public health authorities (e.g., state public health

department Epidemiologist/Health Officer) as required by local and state

communicable disease reporting requirements. The state public health

laboratory/department will notify law enforcement officials (state and

federal), such as the local FBI, as appropriate.

7. Within the hospital setting, immediately notify the infection control

preventionist(s) and/or infectious disease service so that the patient can

be treated appropriately, infectious precautions can be taken, and a

further investigation of the patient’s history can be made (10).

8. Consult with the LRN Reference Laboratory regarding additional

clinical specimens that may be submitted for testing.

9. Initiate documentation showing the specimen identification control,

notification and transfer to your designated LRN Reference Laboratory,

and documentation of all plates and tube cultures which will need to be

destroyed or transferred once identification has been completed.

C. Sentinel Level Clinical Laboratories should consult with their designated LRN Reference

Laboratory prior to or concurrent with testing if B. anthracis or B. cereus biovar

anthracis is requested by the physician or a bioterrorist event is suspected. Obtain

guidance from the state public health laboratory as appropriate (e.g., requests from local

law enforcement or other local government officials). The FBI and state public health

laboratory/state public health department will coordinate the transfer of

isolates/specimens to a higher level LRN laboratory as appropriate.

D. If B. anthracis or B. cereus biovar anthracis is ruled out, proceed with efforts to identify

using established criteria.

E. If other cases are suspected or there is a laboratory exposure, collect appropriate samples

and submit them to your designated LRN Reference Laboratory for additional testing.

X. Select Agent reporting and compliance

1. Reporting of all identified Select Agents is still required, even though Sentinel

laboratories are not required to register under the Select Agent Rule.

2. The laboratory must complete Form 2 within one week (7 days) following

notification of the confirmed identification. For further guidance and access to

the necessary forms, consult with your designated LRN Reference Laboratory or

refer to the CDC Division of Select Agents and Toxins website at

www.selectagents.gov

3. Reporting all identified Select Agents is required by completing Form 4 A within

7 days of confirmed identification. If the isolate is from a Proficiency test sample,

Form 4 B is to be completed within 90 days of receipt of the sample.

4. Your designated LRN Reference Laboratory will advise you with completion of

required forms (e.g., Forms 2, 3, and 4). Always refer to www.selectagents.gov

for the latest guidance and versions of these forms.

XI. SUMMARY/SPECIAL CONSIDERATIONS

A. Antimicrobial Susceptibility

1. Antimicrobial susceptibility testing of B. anthracis or B. cereus biovar

anthracis is neither needed nor appropriate for Sentinel Level Laboratories

to perform. Interpretive breakpoints have not been established by the

CLSI.

2. Quinolones (e.g. ciprofloxacin, levofloxacin), doxycline, and penicillin are

currently the only FDA-approved antibiotics for the treatment of anthrax

(2, Center for Biosecurity of UPMC, www.upmc-biosecurity.org).

3. Post-exposure prophylaxis for personnel (adults and children), including

laboratory personnel suspected of exposure to B. anthracis or B. cereus

biovar anthracis spores includes ciprofloxacin, levofloxacin (adults) or

doxycline. Once started, antibiotic therapy should be continued for 60

days post-exposure (www.upmc-biosecurity.org).

B. Select Agent Reporting and Compliance

1. Reporting of all identified Select Agents is still required, even if a

laboratory has not been previously registered.

2. If the organism is transferred following presumptive identification, the

laboratory must complete Form 2. For further guidance and access to the

necessary forms, consult with your designated LRN Reference Laboratory

or refer to the CDC Division of Select Agents and Toxins website:

http://www.selectagents.gov

3. Reporting all identified Select Agents is required by completing Form 4A

within 7 days of confirmatory identification. If the isolate is from a

Proficiency Test Sample, Form 4B is to be completed within 90 days of

receipt of the sample.

4. Your designated LRN Reference Laboratory will advise you with

completion of required forms (e.g. Forms 2, 3, and 4). Always refer to

www.selectagents.gov

for the latest guidance and versions of these forms.

C. Destruction

1. Once the identification of the isolate has been confirmed, the Sentinel

Clinical Laboratory Select Agent Regulations require that the residual

specimen and cultures of the isolate be destroyed or transferred to an

approved Select Agent entity within 7 days of notification of a confirmed

identification. Your designated LRN Reference Laboratory must advise

you on destruction or transfer of isolates.

2. Generally, all plates, tubes, and clinical material that contain the organism

should be autoclaved, incinerated on-site or submitted to the designated

LRN Reference Laboratory for disposal.

3. Alternatively, contaminated items should be soaked in 10% bleach or 10%

formalin for 24h.

D. Packing and Shipping

1. Refer to the ASM Packing and Shipping Guidelines

2. All materials sent to your designated LRN Reference Laboratory must be

shipped in compliance with IATA and DOT regulations.

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Supplemental Reading

• Centers for Disease Control and Prevention. 2000. Biological and chemical

terrorism: strategic plan for preparedness and response. MMWR, 49(RR-4):1-14.

• Gilchrist, M. J., W. P. McKinney, J. M. Miller, and A. S. Weissfeld. 2000. Cumitech

33, Laboratory Safety, Management, and Diagnosis of Biological Agents Associated with

Bioterrorism. Coordinating ed. J. W. Snyder. ASM Press, Washington, DC.

• Klietmann, W. F. and K. L. Ruoff. 2001. Bioterrorism: Implication for the clinical

microbiologist. Clin. Microbiol. Rev. 14:364-381.

• Sharp, S. E. and M. Loeffelholz. 2011. Biothreat Agents, pp 174-187. In J.

Versalovic, J. H. Jorgenson, M. L. Landry, and D. W. Warnock (ed), Manual of Clinical

Microbiology, 10

ed. American Society for Microbiology, Washington, DC.

REFERENCE ADDENDUM

1. Keller, P. M., V. Bruderer, and F. Müller. 2016. Restricted Identification of Clinical

Pathogens Categorized as Biothreats by MALD-TOF Mass Spectrometry. J. Clin.

Microbiol. 54:816.

2. Tracz, D. M., K. Antonation, and C. R. Corbett. 2015. Verification of a matrix-assisted

laser desorption ionization-time of flight mass spectrometry method for diagnostic

identification of high-consequence bacterial pathogens. J. Clin. Microbiol. 54:764-767.

3. Tracz, D. M., S. J. McCorrister, P. M. Chong, D. M. Lee, C. R. Corbett, and G. R.

Westmacott. 2013. A simple shotgun proteomics methods for rapid bacterial

identification. J. Microbiol. Methods. 94: 54 -57.

4. Tracz, D. M., S. J. Mcorrister, G. R. Westmacott, and C. R. Corbett. 2013. Effect of

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Microbiol. Methods. 92: 132 – 134.

5. Cunningham, S. A. and R. Patel. 2015. Standard matrix-assisted laser desorption

ionization-time of flight mass spectrometry reagents may inactivate potentially hazardous

bacteria. J. Clin Microb 53: 2788 – 2789.