Laboratory Safety Practices Associated With Potential Agents of Biocrime or Bio Terrorism

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10.1128/JCM.41.7.2801-2809.2003.

2003, 41(7):2801. DOI:J. Clin. Microbiol.David L. SewellPotential Agents of Biocrime or BioterrorismLaboratory Safety Practices Associated with

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JOURNAL OF CLINICAL MICROBIOLOGY, July 2003, p. 28012809 Vol. 41, No. 70095-1137/03/$08.000 DOI: 10.1128/JCM.41.7.28012809.2003Copyright 2003, American Society for Microbiology. All Rights Reserved.

MINIREVIEW

Laboratory Safety Practices Associated with Potential Agents ofBiocrime or BioterrorismDavid L. Sewell*

Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center, and Department of Pathology,Oregon Health and Sciences University, Portland, Oregon 97239

The anthrax incident of 2001 in the United States clearlydocumented the threat posed by the intentional release of aninfectious agent in a susceptible population. It also demon-strated that clinicians and clinical microbiology laboratoriesare key to the early detection of disease, identification of theputative agent, and notification of appropriate authorities. Tobe effective in this role, laboratories must be prepared for apossible biocrime or bioterrorism event. Preparation requiresthat laboratories have an awareness of the potential agents thatmay be used, laboratory techniques for the early identificationof these agents, procedures for the management of the event,and knowledge of the safety precautions necessary to safelyhandle these infectious agents (7). Once prepared, laboratorypersonnel must constantly be alert for the possible isolation ofthese agents during the routine manipulations of cultures atthe bench (10). With the exception of smallpox virus and viralhemorrhagic fever (VHF) agents, most of the biothreat agentsare occasionally isolated from patients who have been natu-rally infected. To ensure a safe work environment, the labora-

tory must implement and strictly adhere to the routine safetypractices that minimize risk to laboratory personnel (8, 9).

LABORATORY PREPAREDNESS

On a national level the Centers for Disease Control andPrevention (CDC) have developed a Bioterrorism Prepared-ness and Response Program that addresses a unified publichealth response to bioterrorism events (http://www.bt.cdc.gov).The program addresses disease surveillance; rapid laboratorydiagnosis of biological agents; epidemiologic investigations;communication among local, state, and federal public healthauthorities; preparedness planning; and readiness assessment.Part of the program includes the development of the Labora-

tory Response Network to integrate multilevel laboratories(local, state, federal, military, veterinary, and environmental).The Laboratory Response Network provides for the rapididentification of biological agents and a capacity to respond tobioterrorism and other public health emergencies. The classi-fication of laboratories is based on their presumed role in abioterrorism event that is related to their safety and technical

capability (11). Originally, this model contained four levels oflaboratories (levels A through D) but these have evolved toessentially three levels: (i) Sentinel Laboratories (formerlylevel A), whose role is to recognize, rule out, and refer organ-isms, (ii) Reference Laboratories (formerly levels B and C),

whose role is confirmatory testing, and (iii) two National Lab-oratories (formerly level D), i.e., the CDC and U.S. ArmyMedical Research Institute of Infectious Diseases. Most com-mercial and clinical laboratories are classified as Sentinel Lab-oratories.

ROLE OF THE SENTINEL LABORATORIES

The early recognition of a biocrime or bioterrorism event bythe laboratory rests on microbiologists rapidly recognizing po-tential agents of bioterrorism (11). The agents that are likelierto be used in these incidents are not commonly encountered inmost clinical microbiology laboratories (Table 1) (http://www.bt.cdc.gov). Because many of these agents grow in conven-tional culture media and are not easily recognized by labora-tory personnel, the ordering physician or autopsy pathologistshould notify the laboratory if the diagnosis is suspected. Thesepotential agents of bioterrorism have been placed into threecategories based upon (i) the ability of the agent to be easilydisseminated, (ii) the ability to be transmitted from person toperson, (iii) the ability to cause significant morbidity and mor-tality, and (iv) the ability to cause public panic and socialdisruption (http://www.bt.cdc.gov/Agent/agentlist.asp). Cate-gory A agents pose the greatest threat because they can beeasily disseminated or transmitted from person to person, arehighly infectious, have the potential for major public healthimpact, and require special action for public health prepared-

ness (1). The characteristics of category A agents and Brucellaspp. and their associated disease states are listed in Table 2(http://www.bt.cdc.gov). Category B agents are moderatelyeasy to disseminate and have moderate morbidity and lowmortality. Category C agents consist of emerging pathogensthat could be engineered for mass dissemination in the future.Because most of these agents are rarely isolated and are oftendifficult to rapidly identify, clinical microbiology laboratoriesmust employ diagnostic test protocols or algorithms for rulingout suspicious organisms encountered during the routine ma-nipulation of cultures. These protocols are available on theCDC (http://www.bt.cdc.gov) and American Society for Micro-biology (http://www.asmusa.org/pcsrc/biodetection.htm) web-

* Mailing address: Pathology and Laboratory Medicine ServiceP5PATH, Veterans Affairs Medical Center, 3710 SW Veterans Rd.,Portland, OR 97239. Phone: (503) 273-5363. Fax: (503) 721-7823.E-mail: [email protected].

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sites and appear in other publications (12). To be effective,these protocols should be incorporated directly into the benchprocedure to ensure rapid recognition and referral of any sus-picious isolate to a Reference Laboratory (levels B and C) and,most importantly, to ensure safe handling of the agent (10).Protocols for identification of smallpox virus and botulismtoxin are not listed because the Sentinel Laboratories (level A)should not process these specimens but should rather send the

smallpox virus specimens to the CDC and the botulism toxinspecimens to the Reference Laboratory. The CDC maintainsan emergency telephone number ([770] 488-7100) with instruc-tions for the collection and shipment of specimens. SentinelLaboratories (level A) should not accept or process environ-mental or animal specimens for biocrime or bioterrorismagents. These specimens, after consultation with the ReferenceLaboratory and the Federal Bureau of Investigation, should beforwarded directly to the Reference Laboratory (levels B andC) that has the safety and technical capability to process them.

LABORATORY SAFETY MEASURES

The category A and B agents have all been associated withlaboratory-acquired infections (9, 13). However, most occupa-tionally acquired infections caused by these microorganismsoccurred because laboratory personnel did not recognize therisk associated with these infectious agents and failed to followroutine biosafety level 2 (BSL2) safety practices, especiallypractices for containment of aerosols. To minimize risk, man-agement must implement a safety program that assesses riskassociated with laboratory practices, provides appropriate con-tainment facilities and personal protective equipment (PPE), writes safety procedures, and supports exposed individualswith medical care. It is the individuals responsibility to under-stand the risk associated with handling infectious agents and toemploy safe practices to minimize exposure to other laboratory

workers and to prevent contamination of the laboratory. Lab-oratories should institute the safety recommendations speci-fied in the CDC and National Institutes of Healths publicationBiosafety in Microbiology and Biomedical Laboratories (9),which can be accessed on the CDCs website (http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm).

It is likely that the first patients seen in a hospital followingrelease of a bioterrorism agent will be routinely evaluated.

These early cultures pose the greatest risk to clinical microbi-ologists because the agent may not be initially recognized.Once an outbreak is identified, Sentinel Laboratories withoutadequate staffing or safety facilities to handle the volume ofspecimens may choose to restrict receipt of specimens (http://www.asmusa.org/pasrc/intro.htm). The agents that pose thegreatest risk are transmitted by aerosols produced by routinelaboratory practices, such as pipetting, flaming inoculationloops, subculturing blood bottles, streaking agar plates, soni-cation, homogenization, mixing bacterial suspensions, expel-ling bubbles from syringes, and centrifugation (8). Specimensshould be handled in a class II biological safety cabinet (BSC).Laboratory personnel should wear protective eyewear, closed

laboratory coats with cuffed sleeves, and gloves stretched overthe cuffs; avoid touching mucosal surfaces with their hands;and remove gloves and coat and wash hands prior to leavingthe laboratory. Benches should be decontaminated after use,and waste material should be placed in appropriate biohazardcontainers (2, 8, 9).

To categorize the risk associated with an infectious agentand define the appropriate safety practices for handling theagent, the CDC and National Institutes of Health have pro-posed four BSLs (9). The BSLs imply increased occupationalrisk from exposure to an agent and need for additional con-tainment for work with that agent. The guidelines for assigningmicroorganisms to a BSL are as follows: BSL1, well-character-

TABLE 1. Categories of potential agents and diseases

Category Agent(s) Disease

A Bacillus anthracis AnthraxClostridium botulinum toxin BotulismYersinia pestis PlagueVariola major virus Smallpox

Francisella tularensis TularemiaFiloviruses (Ebola and Marburg viruses) VHF

Arenaviruses (Lassa and Machupo viruses)

B Brucella species Brucellosis Burkholderia mallei Glanders Burkholderia pseudomallei MelioidosisChlamydia psittaci PsittacosisCoxiella burnetii Q fever

Rickettsia prowazekii Typhus feverFood safety threats (e.g. Salmonella spp., Shigella spp., Escherichia coli O157)Water safety threats (Cryptosporidium parvum and Vibrio cholerae)

Alphaviruses (VEE, EEE, and WEE)a EncephalomyelitisToxins (e.g., ricin and staphylococcal enterotoxin B)

C Bunyaviruses (hantavirus and Crimean-Congo virus) Hemorrhagic feverFlaviviruses (yellow fever and dengue viruses) Yellow fever and dengue

Nipah virus EncephalomyelitisTickborne viruses Hemorrhagic fever or encephalitis

a VEE, Venezuelan equine encephalomyelitis virus; EEE, Eastern equine encephalomyelitis virus; and WEE, Western equine encephalomyelitis virus.

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TABLE 3. Summary of BSL1 and 2 precautions containmenta of infectious agents

BSLd Description of agent(s)

Primary containmentb

Secondary containment facilitiesc

(secondary barriers)Practices or techniques to followSafety equipment(primary barriers)

1 Well-characterized agents(e.g. Bacillus subtilis) notknown to consistentlycause disease in healthyadults and of minimalpotential hazard tolaboratory personnel andenvironmente

BSL1 practices:1. Laboratory personnel specifically

trained to perform any procedureconducted in the laboratory

2. Supervision by a scientist with generaltraining in microbiology

3. Limited access to laboratory whileexperiments are in progress

4. Washing of hands after handling ofcultures and before exit from laboratory

5. Eating, drinking, insertion of contactlenses or application of cosmetics, andstorage of food in the laboratoryprohibited

6. Mouth pipetting prohibited7. Sharps policy instituted8. Creation of aerosols minimized during

all procedures9. Work surfaces decontaminated after

spills and at end of day10. Waste disposal policy instituted

11. Biohazard sign (with name of agent(s)and name and phone number ofsupervisor) posted at entrance wheninfectious agents are present

12. Insect and rodent control program ineffect

For BSL1, none requiredbut the following arerecommended:

1. Work performed onopen bench top

2. Lab coats, gowns, oruniforms worn to pro-tect street clothes

3. Gloves worn if skinon hands is broken orif a rash is present(alternatives to pow-dered latex glovesshould be available)

4. Protective eyewearworn for proceduresin which splashes areanticipated

5. Persons wearing con-tact lenses also weargoggles or face

shields

At least a sink for washing handsbut also the following:

1. Laboratory is not necessarilyseparated from the generaltraffic patterns of the building

2. Laboratory should have doorsfor access control

3. Laboratory designed to becleaned easily (carpets andrugs are not appropriate)

4. Bench tops impervious towater and resistant tomoderate heat, organicsolvents, acids, alkalis orchemicals used todecontaminate the worksurfaces

5. Furniture can supportanticipated weight load anduses, with spacing betweencabinets, benches, and

equipment accessible forcleaning

6. Windows fitted with screens

2 Agents associated withhuman disease (e.g.,Bacillus anthracis,Shigella spp., andYersinia pestis)f

BSL1 practices plus the following:1. Laboratory personnel are specifically

trained to handle pathogenic agentsand are directed by competent scientists

2. Policies and procedures allow onlypersons meeting specific entry and/ortraining requirements to enterlaboratory

3. Individuals at increased risk ofacquiring infection are allowed limitedaccess to or are restricted from entering

the laboratory area3. Posted biohazard sign (as above) lists

biosafety level, required immunization,required personal protective equipment,and any procedures required for exitinglaboratory

4. Immunizations or tests for agents inlaboratory (e.g., hepatitis B virus

vaccine or TB skin testing) areprovided

5. Personnel receive appropriate trainingin safety precautions, exposureprevention, and sharps precautionsand receive annual updates forprocedure/policy changes

6. Biosafety manual defining infectiouswaste handling or decontamination andmedical surveillance policies is available

7. Decontamination policy for worksurfaces, spills, and contaminatedequipment is established

8. An accident policy involving anaccidental or overt exposure toinfectious materials is established thatrequires immediate reporting to thelaboratory director for documentation;medical evaluation, surveillance, andany necessary treatment

For BSL2:1. Properly maintained

BSCg (Class I or IIbut preferably ClassII) for all manipula-tions involvingsplashes or aerosolsof infectious materials

2. Personal protectiveequipment, includingthe following:

a. Protective labora-tory clothing (thisclothing is removedand left in the lab-oratory area priorto exit, and it iseither disposable orlaundered by theinstitution; itshould never betaken home)

b. Gloves are wornwhen hands maycontact potentiallyinfectious materi-als, surfaces, orequipment; dispos-able gloves are not

to be reused, washed, or used totouch clean sur-faces (telephones,etc.); hands are

washed followingglove removal

c. Face protection(goggles, mask,face shield, orsplatter guard) isused for antici-pated splashes orsprays of hazardousmaterials for ma-nipulations outsidethe BSC

BLS1 requirements plus thefollowing:

1. Autoclave available2. Doors for facilities that house

restricted agents can belocked

3. Laboratory separated fromgeneral traffic patterns andaway from public areas

4. Sinks for handwashingequipped with foot, knee, or

automatic faucet operation5. BSC optimally located for

containment6. Eyewash station readily

available

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ized agents that are not known to consistently cause disease inhealthy adult humans and are considered a minimal potentialhazard to laboratory personnel and the environment; BSL2,agents of moderate potential hazard to personnel and theenvironment; BSL3, indigenous or exotic agents that causeserious or potentially lethal disease as a result of exposure bythe inhalation route; and BSL4, dangerous and exotic agentsthat pose a high individual risk of aerosol-transmitted labora-tory infections and life-threatening disease.

Most clinical microbiology laboratories (Sentinel Laborato-ries or level A laboratories) function at BSL2 and employBSL2 laboratory practices (Table 3) (http://www.bt.cdc.gov) (5,8, 11). In general, clinical specimens containing these agentscan be handled at BSL2 because few organisms are present.The exposure risk increases when the infectious agent is am-plified in culture or when laboratory practices that may gener-ate aerosols are used to manipulate the culture. In these situ-ations, BSL3 practices should be employed (Table 4) (http: //www.bt.cdc.gov). BSL2 practices require that all manipula-tions that produce an aerosol be performed in a class II BSC,whereas BSL3 practices specify that all manipulations shouldbe performed in a BSC. Routine manipulations of cultures

(e.g., preparation of bacterial suspension in saline) may poten-tially produce aerosols. Aerosol production during the routinemanipulation of cultures of Neisseria meningitidis may havecaused laboratory-acquired infections that resulted in death(3). Laboratories should assess the risk of aerosol productionfor all procedures, including automated instrumentation (8).Certain procedures such as sonication or vortexing of bacterialsuspensions of unidentified organisms are likely to produce anaerosol and should be performed in a BSC (http://www.asmusa.org/pasrc/intro.htm). All procedures that produce aerosolsshould be performed in a BSC or discontinued. Once an agentis identified as a BSL3 pathogen or when a specimen is sub-mitted to rule out a BSL3 agent, the material should be labeled

as BSL3 and all subsequent manipulations should be per-formed in a BSC or in a BSL3 laboratory where available.After laboratory practices that produce aerosols are identifiedand contained or discontinued, the risk associated with han-dling BSL3 agents will be minimized.

The potential release of BSL4 agents, such as smallpox virusor VHF agents, poses a critical risk to health care and labora-tory personnel. When infection by these agents is suspected,the CDC and the state health department should be notified.Instructions for the collection and shipment of specimens di-rectly to the CDC can be obtained during this communication.Many state public health care practitioners have received train-ing in the recognition of infection caused by these agents. TheBSL4 agents are extremely hazardous and when amplified

should be contained in a BSL4 facility at the CDC or the U.S.Army Medical Research Institute of Infectious Diseases. Theunsuspected amplification of a BSL4 agent in a routine virol-ogy laboratory is a potential risk. Virology laboratories shouldassess this potential, and laboratory workers should becomefamiliar with the cell lines that support the growth of BSL4agents and the cytopathic effect produced in each cell line(http://www.bt.cdc.gov). All virology laboratories should have aBSC, and their members should be trained in the correct use of

a BSC (8) (http://www.cdc.gov/od/ohs/biosfty/bsc/bsc.htm). Inthe case of smallpox, the laboratory and facility should imple-ment a clinical pathway for patients with vesicular or pustularrash illness (http://www.idsociety.org). Because most clinicianshave little or no clinical experience diagnosing patients in-fected with VHF agents, a high degree of suspicion is alsorequired. Adherence to standard precautions and handlingclinical specimens in a BSC should minimize exposure risk tolaboratory workers from smallpox virus and VHF agents.

LABORATORY SAFETY PRACTICES WITH

SPECIFIC AGENTS

(i) Bacillus anthracis. The agent may be present in a varietyof clinical specimens, including blood, cerebrospinal fluid(CSF), pleural fluid, sputum, wound exudates, and, rarely,urine and feces. In clinical specimens, the B. anthracis cells areprimarily vegetative, and not easily transmitted. The primaryhazards to laboratory personnel are direct and indirect contactof intact and broken skin with cultures and accidental paren-teral inoculation. The recent case of cutaneous anthrax in alaboratory worker manipulating an environmental sample il-lustrates this potential risk (4). Exposure to infectious aerosolsis an additional hazard associated with the screening of envi-ronmental samples. Most Sentinel Laboratories (level A) are

not trained or equipped for work with environmental samples.Samples should be transported to a Reference Laboratory(levels B and C) for testing. Clinical specimens and subsequentcultures can be handled under BSL2 practices, containmentequipment, and facilities. BSL3 conditions are required for work involving production quantities, concentrations of cul-tures, or activities with a high potential for aerosols, includingmanipulation of suspect powders.

(ii) Francisella tularensis. F. tularensis is a common cause oflaboratory-acquired infection from aerosol exposure. How-ever, most cases occur in research facilities that handle largequantities of liquid cultures. The agent may be present innearly all clinical specimens. Laboratory hazards include directcontact of skin or mucous membranes with infectious material,

a Containment refers to safe methods for managing infectious materials in the laboratory environment; its purpose is to reduce or eliminate exposure of laboratoryworkers, other persons, and the outside environment to potentially hazardous agents.

b Primary containment is the protection of personnel and the immediate laboratory environment from exposure to infectious agents. The most important elementof containment is strict adherence to standard microbiological practices and techniques.

c Secondary containment is the protection of the environment external to the laboratory from exposure to infectious materials, provided by facility design andoperational practices.

d Risk assessment factors, such as pathogenicity, route of transmission, agent stability, infectious dose, organism concentration, specimen origin, animal study data,availability of prophylaxis, medical surveillance, and technical proficiency, are but a number of elements that contribute to the establishment of a given biosafety level.

eAppropriate for undergraduate and secondary educational training and teaching laboratories.f

BSL2 recommendations and Occupational Safety and Health Administration requirements focus on the prevention of percutaneous, ingestive, and mucousmembrane exposure(s) to clinical materials.

g BSC, Biological Safety Cabinet.

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TABLE 4. Microbiology biosafety

Agent

BSL for: Specimen types posingrisk in case of

exposure

Recommended precautions for level A laboratories

Specimenhandling

Culturehandling

BSL2 BSL3 BSL4

Bacillus anthracis 2 2 Blood, skin lesion

exudates, CSF,pleural fluid,sputum, and,rarely, urine andfeces

Activities may include

collection of clinicalmaterial and handlingof only diagnosticquantities ofinfectious cultures

Activities may include those

with high potential foraerosol or dropletproduction

Brucella spp.a 2 3 Blood, bonemarrow, CSF,tissue, semen,and occasionallyurine

Activities limited tocollection, transportand plating of clinicalmaterial

All activities involvingmanipulation of cultures

Clostridiumbotulinumb

2 2 Food specimens,clinical material(serum, gastric,and fecal

specimens), andenvironmentalsamples (soil andsurface water)

Activities with materialsknown or potentiallycontaining toxinallowed but must be

handled in aBiological SafetyCabinet (Class II)

with a lab coat,disposable surgicalgloves, and a faceshield (as needed)

Activities with highpotential for acrosol ordroplet production

Francisella tularensisc 2 3 Skin lesionexudates,respiratorysecretions, CSF,blood, and urine,as well as tissuesfrom infectedanimals andfluids frominfectedarthropods

Activities limited tocollection, transport,and plating of clinicalmaterial

All activities involvingmanipulations of cultures

Yersinia pestisd 2 2 Bubo fluid, blood,sputum, CSF,feces, and urine

Activities may includecollection of clinicalmaterial and handlingof only diagnosticquantities ofinfectious cultures

Activities with highpotential for aerosol ordroplet production

Smallpoxe 4 4 Lesion fluid orcrusts, respiratorysecretions, andtissue

Specimen collectionand/or transport

VHFf 4 4 Blood, urine,

respiratory andthroat secretions,semen, and tissue

Specimen collection

and/or transport

a Laboratory-acquired brucellosis has occurred via sniffing of cultures; aerosols generated by centrifugation; mouth pipetting; accidental parenteral inoculations; andsprays into eyes, nose, and mouth; and, finally, by direct contact with clinical specimens.

b The toxin may be present in the specimen types listed and is extremely poisonous. Exposure to toxin is the primary laboratory hazard since absorption can occurwith direct contact with skin, eyes, or mucous membranes, including the respiratory tract. The toxin can be neutralized by 0.1 M sodium hydroxide. C. botulinum isinactivated by a 1:10 dilution of household bleach. Contact time is 20 min. If material contains both toxin and organisms, the spill must be sequentially treated withbleach and sodium hydroxide for a total contact time of 40 min.

c Laboratory-acquired tularemia infection has been more commonly associated with cultures than with clinical materials or animals. Direct contact of skin or mucousmembranes with cultures, parenteral inoculation, ingestion, and aerosol exposure have resulted in infection.

d Special care should be taken to avoid the generation of aerosols.e Ingestion, parenteral inoculation, and exposure of mucous membranes to droplet or aerosol form, and exposure of broken skin to infectious fluids or tissues are

the primary hazards to laboratory workers.fExposure of respiratory passages to infectious aerosols, exposure of mucous membranes to infectious droplets, and accidental parenteral inoculation are the primary

hazards to laboratory workers.



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TABLE 5. Vaccines

Disease oragent

Immunity bynatural exposure

Vaccine typeVaccine efficacy in case of

aerosol exposureComments

Anthrax Yesa For humans, cell-free culturefiltrate of an avirulent,nonencapsulated derivativeof a bovine isolate

designated V770; foranimals, a sporesuspension of an avirulent,nonencapsulated livestrain

Two-dose efficacy(against 200 to 500LD50

e in monkeys)

Required for level A Laboratory: NoLaboratory-acquired cases: None reported

since the late 1950s, at which time thehuman vaccine was introduced

Immunity: Combined vaccine efficacyagainst both forms of anthrax(inhalational and cutaneous) 93%

Brucellosis Yes For humans, no vaccineavailable in the UnitedStates; for animals, in1996, RB51, a live,attenuated strain of B.

abortus replaced the S19strain, which was also alive, attenuated vaccine

No vaccine Required for level A Laboratory: Noneavailable

Laboratory-acquired cases: Brucellosis isthe most commonly reported bacterialinfection acquired in laboratories; oneof the largest reported incidencesinvolved 45 cases with 1 death;protection is based on adherence toBSL3 precautions

Immunity: Studies of humans demonstrate

that immunity is acquired after activeinfection; both cellular and humoralresponses are required

Botulism No Pentavalent (ABCDE)toxoidc

Three-dose efficacy,100% (against 25 to250 LD50 inprimates)

Required for level A Laboratory: NoLaboratory-acquired cases: There has

been one report of laboratory-associated botulism

Immunity: In cases of foodborneexposure, immunity does not developeven with severe disease, and itsrepeated occurrence has been reported

Tularemia Partial Live, attenuated vaccine 80% protectionagainst 1 to 10 LD50

Required for level A Laboratory: NoLaboratory-acquired cases: Over the past

50 years, tuleremia has been the third-

most-common bacterial infectionacquired in laboratories, mostly inresearch laboratories

Immunity: Multiple episodes ofreinfection have been documentedamong vaccinated laboratory personneland in unimmunized individuals

Plague Partial Suspension of killed(formalin-inactivated)Y. pestis organisms

Has yet to bemeasured preciselyin controlledstudies. At least two

vaccinated personscontractedpneumonic plaguefollowing exposure

toY. pestis

Required for level A Laboratory: NoLaboratory-acquired cases: Few

laboratory-associated cases have beenreported; since 1936, only three cases ofpneumonic plague have beendocumented

Immunity: Indirect evidence, mainly fromthe military, indicates that the plague

vaccine is effective for preventing flea-borne transmission of disease

Smallpox Yes Vaccinia (smallpox) vaccined

(grown in the skin ofvaccinated bovine calves)

Vaccine protectsagainst large dosesin primates

Required for level A Laboratory: NoLaboratory-acquired cases: noneImmunity: If a smallpox sample is

handled at a level A laboratory,vaccination within 3 days postexposureis considered effective in preventingserious infection and death; vacciniaimmune globulin may also beconsidered but may compromisepostexposure vaccination efficacy

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accidental parenteral inoculation or ingestion, and exposure toinfectious aerosols or droplets through manipulation of cul-

tures. The greatest risk to laboratory personnel is associatedwith manipulation of cultures. The recommended laboratoryprecautions for handling clinical specimens are BSL2 practices,containment equipment, and facilities. BSL3 conditions arerecommended for all activities involving cultures.

(iii) Brucella spp. Brucellosis is the most commonly reportedlaboratory-acquired infection, owing in part to the low infec-tious dose. The agent is present in blood, CSF, semen, and,rarely, urine. Laboratory hazards include direct contact of skinor mucous membranes, accidental parenteral inoculation oringestion, and exposure to aerosols generated during manipu-lation of cultures. As with F. tularensis, BSL2 conditions sufficefor handling clinical specimens and BSL3 conditions are rec-

ommended for all activities involving cultures.(iv) Yersinia pestis. Y. pestis is a rare cause of infection in

laboratory personnel. The agent may be present in bubo fluid,blood, sputum, CSF, feces, and urine. The primary hazards tolaboratory personnel are direct contact with cultures and in-fectious materials, autoinoculation or ingestion, and exposureto aerosols or droplets produced by manipulation of cultures.BSL2 conditions are recommended for specimen and culturehandling, while BSL3 practices are recommended for activitieswith a high potential for droplet or aerosol production.

(v) Smallpox virus. Smallpox virus is highly transmissibleand presents a significant risk to laboratory personnel. Theagent is present in lesion fluid or crusts, respiratory secretions,

and tissue. The primary hazards to laboratory personnel areingestion, parenteral inoculation, and droplet or aerosol expo-sure of mucous membranes or broken skin with infectiousmaterial. If a patient is identified as high or moderate risk forsmallpox, physicians should immediately contact the statehealth department before collection of specimens (http://www.idsociety.org). Only recently vaccinated personnel wearing ap-propriate protective equipment should collect specimens. Alltesting of specimens from patients at high risk for smallpoxshould be performed by a National Laboratory (level D). Con-sult the CDC [phone, (770) 488-7100] for specific instructionson the shipment of specimens. Once smallpox is confirmed ina geographical area, additional cases can be diagnosed clini-cally. Testing specimens on patients not at moderate or high

risk for smallpox can be performed under BSL2 conditions. Ifsmallpox cannot be ruled out through testing, contact the local

or state health department.(vi) VHF agents. The hemorrhagic fever viruses also pose a

significant risk to laboratory personnel, and laboratory-ac-quired infections from handling these agents are documented.The agents may be present in blood, urine, respiratory andthroat secretions, semen, and urine. The primary hazards inthe laboratory setting include accidental parenteral inocula-tion, mucous membrane exposure to infectious droplets, andexposure to infectious aerosols. Specimens from patients withsuspected VHF should be referred, after consultation, to aNational Laboratory for testing. At a minimum, laboratorystaff including autopsy personnel should be alerted to the po-tential diagnosis and only designated personnel should handle

specimens. When possible, point-of-care analyzers should beused at the bedside. Specimens transported to the laboratoryshould be identified, double bagged, and hand carried at pre-determined times. No specimens should be sent in pneumatictubes. Serum samples should be pretreated with Triton X-100or heated at 60C for 1 h before testing to inactivate the virus.When handling specimens, laboratory personnel should wearappropriate PPE and process specimens in a BSC followingBSL3 practices (http://www.idsociety.org).

(vii) Botulism. Botulinum toxin may be present in food,serum, gastric contents, feces, and environmental samples.Toxin can be absorbed after direct contact with skin or mucousmembranes, including the respiratory tract. Materials should

be handled in a BSC while using BSL2 practices, includinggloves and a face shield. Manipulations producing aerosolsrequire BSL3 conditions. Sentinel Laboratories (level A)should collect appropriate specimens and, after consultation,ship the specimens to a Reference Laboratory.

IMMUNIZATION OF LABORATORY PERSONNEL

The availability of vaccines for biocrime or bioterrorismagents and CDCs recommendations for immunization of lab-oratory personnel are shown in Table 5 (http://www.bt.cdc.gov). There are no generally available vaccines against brucel-losis or VHF at this time, but some investigational vaccines arebeing evaluated for VHF (http://www.idsociety.org). Immuni-

TABLE 5Continued

Disease oragent

Immunity bynatural exposure

Vaccine typeVaccine efficacy in case of

aerosol exposureComments

VHF ?b None available No vaccine Required for level A Laboratory: NoLaboratory-acquired cases: Exposure of

skin and/or mucous membrane to virus-laden material, i.e., blood, cell cultures,

body fluid/secretions, has beenresponsible for most recognized casesamong humans

Immunity: To be determined

a Some degree of immunity is evident following cutaneous anthrax, i.e., the dose generally considered to be lethal is too low to produce an immune response.b Immunity to Lassa fever reinfection occurs following infection, but the length of protection is unknown.c Distributed by the CDC under an investigational new drug protocol and used to protect laboratory workers at high risk, i.e., those actively working with C. botulinum

or the toxins.d Distributed by the CDC.e LD50, 50% lethal dose.



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zation of laboratory personnel against the other agents is notrecommended.

DECONTAMINATION

Standard disinfectants (e.g., 0.5% household bleach) areconsidered adequate for cleaning surfaces potentially contam-

inated by bioterrorism agents (6). As with all biological spills,personnel should wear gloves, protective eyewear, and PPEduring the cleanup process. Respiratory protection should beworn when aerosolization is suspected. Following direct expo-sure, clothing and body surfaces should be washed with soapand water. All laundry that is potentially contaminated withsmallpox virus or VHF agents should be incinerated or auto-claved prior to washing in hot water containing bleach.

SECURITY

All of these agents are classified as select agents and areregulated by the federal government (4a). The regulation is

available at http://www.cdc.gov/od/sap. Laboratories that han-dle select agents must meet the regulations that went intoeffect on 7 February 2003. For most Sentinel Laboratories, thatmeans that none of the select agents may be kept in the lab-oratory unless the regulations are met. As a minimum, theCDC must be informed whenever a select agent is recoveredfrom a clinical specimen and the destruction of the agent mustbe documented.

REFERENCES

1. Centers for Disease Control and Prevention. 2000. Biological and chemicalterrorism: strategic plan for preparedness and response. Recommendations

of the CDC Strategic Planning Workgroup. Morb. Mortal. Wkly. Rep.49(RR-4):114.

2. Centers for Disease Control and Prevention. 2001. Update: investigation ofanthrax associated with intentional exposure and interim public healthguidelines, October 2001. Morb. Mortal. Wkly. Rep. 50:889893.

3. Centers for Disease Control and Prevention. 2002. Laboratory-acquiredmeningococcal diseaseUnited States, 2000. Morb. Mortal. Wkly. Rep.51:141144.

4. Centers for Disease Control and Prevention. 2002. Update: cutaneous an-

thrax in a laboratory workerTexas, 2002. Morb. Mortal. Wkly. Rep. 51:482.4a.Federal Register. 2002. Possession, use, and transfer of select agents and

toxins; interim final rule. Fed. Regist. 240:7688576905.4b.Garner, J. S., et al. 1996. Guideline for isolation precautions in hospitals.

Infect. Control Hosp. Epidemiol. 17:5380.5. Gilchrist, M. J. R., W. P. McKinney, J. M. Miller, and A. S. Weissfeld. 2000.

Cumitech 33, Laboratory safety, management and diagnosis of biologicalagents associated with bioterrorism. Coordinating ed., J. W. Snyder. ASMPress, Washington, D.C.

6. Hawley, R. J., and E. M. Eitzen, Jr. 2000. Bioterrorism and biological safety,p. 567578. In D. O. Fleming and D. L. Hunt (ed.), Biological safety:principles and practices, 3rd ed. ASM Press, Washington, D.C.

7. Miller, J. M. 2001. Agents of bioterrorism: preparing for bioterrorism at thecommunity health care level. Infect. Dis. Clin. N. Am. 15:11271156.

8. NCCLS. 2001. Protection of laboratory workers from occupationally ac-quired infections. Approved standard M29-A2. NCCLS, Wayne, Pa.

9. Richmond, J., and R. W. McKinney (ed.). 1999. Biosafety in microbiologicaland biomedical laboratories, 4th ed. U.S. Government Printing Office,

Washington, D.C.10. Shapiro, D. S., and D. R. Schwartz. 2002. Exposure of laboratory workers to Francisella tularensis despite a bioterrorism procedure. J. Clin. Microbiol.40:22782281.

11. Snyder, J. W. 2003. Role of the hospital-based microbiology laboratory inpreparation for and response to a bioterrorism event. J. Clin. Microbiol.41:14.

12. Snyder, J. W., and A. S. Weissfeld. 2003. Laboratory detection of potentialagents of bioterrorism, p. 121128. In P. R. Murray, E. J. Baron, J. H.Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical micro-biology, 8th ed. ASM Press, Washington, D.C.

13. Voss, A., and E. Nulens. 2003. Prevention and control of laboratory-acquiredinfections, p. 109120. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A.Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology, 8th ed.ASM Press, Washington, D.C.


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Laboratory Safety Practices Associated With Potential Agents of Biocrime or Bio Terrorism