BW-ch18.pdf

Laboratory Identiication of Biological Threats

Chapter 18

LABORATORY IDENTIFICATION OF

BIOLOGICAL THREATS

ERIK A. HENCHAL, P h D * ; GEORGE V. LUDWIG, P h D † ; CHRIS A. WHITEHOUSE, P h D ‡ ; and JOHN M. SCHERER, P h D §

INTRODUCTION

THE LABORATORY RESPONSE

Role of the Military Clinical and Field Laboratories

Military Field Laboratories

Laboratory Response Network

Biosafety and Biosecurity in the Military Clinical and Field Medical Laboratories

IDENTIFICATION APPROACHES

Specimen Collection and Processing

Clinical Microbiological Methods

Antibiotic Susceptibility Testing

Immunodiagnostic Methods

Molecular Detection Methods

EMERGING THREATS

BIOFORENSICS

FUTURE APPROACHES

Early Recognition of the Host Response

Joint Biological Agent Identification and Diagnostic System

SUMMARY

* Colonel, US Army (Ret); formerly, Commander, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

† Deputy Principal Assistant for Research and Technology, US Army Medical Research and Materiel Command, 504 Scott Street, Suite 204, Fort Detrick,

Maryland 21702; formerly, Science Director, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

‡ Microbiologist, Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

21702; formerly, Microbiologist, US Army Dugway Proving Ground, Dugway, Utah

§ Lieutenant Colonel, Medical Service Corps, US Army; Chief, Division of Diagnostic Systems, US Army Medical Research Institute of Infectious

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702; formerly, Chief, Biological Threat Assessment, 520th Theater Army Medical Laboratory,

Aberdeen Proving Ground, Maryland

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Medical Aspects of Biological Warfare

INTRODUCTION

The ability of military laboratories to identify and

confirm the presence of biological threats has signifi-

cantly improved over the past decade. Identification

approaches have advanced from classical identification

methods performed in only a few reference laboratories

to complex integrated diagnostic systems that are matur-

ing as part of the Joint Biological Agent Identification

and Diagnostic System (JBAIDS) for field laboratories.

During the Persian Gulf War (1990–1991), deployed

field laboratories and environmental surveillance units

depended significantly on immunoassay methods with

limited sensitivity and specificity. Because of intensive

efforts by scientists at military reference centers, such as

the US Army Medical Research Institute of Infectious

Diseases (USAMRIID), the Naval Medical Research

Center, the Armed Forces Institute of Pathology, and

the US Air Force Institute for Operational Health, re-

searchers are better prepared to identify and confirm

the presence of the highest priority biological threats to

human health (Exhibit 18-1). 1,2 However, the biological

threat is more complicated than ever before. Future

diagnostic and identification systems will depend on

an integrated set of technologies, including new immu-

nodiagnostic assays and rapid gene analysis methods

to detect a broad spectrum of possible biological mark-

ers for diagnosing biological threats (see Exhibit 18-1). 2

The combination of several diagnostic approaches will

improve reliability and confidence in laboratory results,

which may shape medical treatment or response after

an attack. Military and civilian clinical laboratories are

now linked into a laboratory response network (LRN)

for bioterrorism sponsored by the Centers for Disease

Control and Prevention (CDC). 3 Together, these efforts

have improved the national preparedness, but continu-

ing research and development are needed to improve the

speed, reliability, robustness, and user friendliness of the

new diagnostic technologies. This chapter will review

the agent identification approaches and state-of-the art

diagnostic technologies available to protect and sustain

the health of soldiers and other military personnel.

THE LABORATORY RESPONSE

Role of the Military Clinical and Field Laboratories

be used. However, a medical laboratory may not be

available for short duration operations in which the

health service element is task organized for a specific

mission. In this case, medical laboratory support should

be provided by a facility outside the area of opera-

tions. 4 Army medical treatment facilities in a theater of

operations have limited microbiology capabilities un-

less supplemented with a microbiology augmentation

set (M403), which is fielded with an infectious disease

physician, a clinical microbiologist, and a laboratory

technician. The M403 set contains all of the necessary

equipment and reagents to identify commonly en-

countered pathogenic bacteria and parasites, evaluate

bacterial isolates for antibiotic sensitivity, and screen for

some viral infections. Although this medical set does

not contain an authoritative capability for definitively

identifying biological warfare agents, it supports ruling

out common infections. Specimens requiring more com-

prehensive analysis capabilities are forwarded to the

nearest reference or confirmatory laboratory. After the

Persian Gulf War, all of the military services recognized

a need to develop additional deployable laboratory

assets to support biological threat identification and

preventive medicine efforts (described below).

Military clinical and field laboratories play a critical

role in the early recognition of biological threats. For

the purposes of this chapter, a biological threat is any

infectious disease entity or biological toxin intention-

ally delivered by opposing forces to deter, delay, or

defeat US or allied military forces in the accomplish-

ment of the mission. Biological agents can also be used

in bioterrorism scenarios to create terror or panic in

civilian and military populations to achieve political,

religious, or strategic goals. Although the principal

function of military clinical and field laboratories is

to confirm the clinical diagnosis of the medical officer,

laboratory staff also provide subject matter expertise in

theaters of operation on the handling and identification

of hazardous microorganisms and biological toxins.

Because these laboratories have a global view of disease

in the theater, they play an important sentinel role by

recognizing unique patterns of disease. Military field

laboratory personnel may also evaluate environmental

samples and veterinary medicine specimens as part of a

comprehensive environmental or preventive medicine

surveillance system in a theater of operations.

Military Field Laboratories

Army

If a complete medical treatment facility is part of a

deployment, its intrinsic medical laboratory assets can

Army medical laboratories (AMLs) are modular,

task-organized, and corps-level assets providing

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Laboratory Identiication of Biological Threats

EXHIBIT 18-1

REGULATED BIOLOGICAL SELECT AGENTS AND TOXINS

US DEPARTMENT OF HEALTH AND HUMAN

SERVICES SELECT AGENTS AND TOXINS

Abrin

Cercopithecine herpesvirus 1 (Herpes B virus)

Coccidioides posadasii

Conotoxins

Crimean-Congo hemorrhagic fever virus

Diacetoxyscirpenol

Ebola virus

Lassa fever virus

Marburg virus

Monkeypox virus

Reconstructed replication competent forms of the 1918

pandemic influenza virus containing any portion of the

coding regions of all eight gene segments (Reconstructed

1918 Influenza virus)

Ricin

Rickettsia prowazekii

Rickettsia rickettsii

Saxitoxin

Shiga-like ribosome inactivating proteins

South American Haemorrhagic Fever viruses

Flexal

Guanarito

Junin

Machupo

Sabia

Tetrodotoxin

Tick-borne encephalitis complex (flavi) viruses

Central European Tick-borne encephalitis

Far Eastern Tick-borne encephalitis

Kyasanur forest disease

Omsk hemorrhagic fever

Russian Spring and Summer encephalitis

Variola major virus (Smallpox virus) and Variola minor

virus (Alastrim)

Yersinia pestis

Eastern equine encephalitis virus

Francisella tularensis

Hendra virus

Nipah virus

Rift Valley fever virus

Shigatoxin

Staphylococcal enterotoxins

T-2 toxin

Venezuelan equine encephalitis virus

US DEPARTMENT OF AGRICULTURE SELECT

AGENTS AND TOXINS

African horse sickness virus

African swine fever virus

Akabane virus

Avian influenza virus (highly pathogenic)

Bluetongue virus (Exotic)

Bovine spongiform encephalopathy agent

Camel pox virus

Classical swine fever virus

Cowdria ruminantium (Heartwater)

Foot-and-mouth disease virus

Goat pox virus

Japanese encephalitis virus

Lumpy skin disease virus

Malignant catarrhal fever virus (Alcelaphine herpesvi-

rus type 1)

Menangle virus

Mycoplasma capricolum / M.F38/ M mycoides Capri (con-

tagious caprine pleuropneumonia)

Mycoplasma mycoides mycoides (contagious bovine pleu-

ropneumonia)

Newcastle disease virus (velogenic)

Peste des petits ruminants virus

Rinderpest virus

Sheep pox virus

Swine vesicular disease virus

Vesicular stomatitis virus (Exotic)

OVERLAP SELECT AGENTS AND TOXINS

Bacillus anthracis

Botulinum neurotoxins

Botulinum neurotoxin producing species of Clostridium

Brucella abortus

Brucella melitensis

Brucella suis

Burkholderia mallei (formerly Pseudomonas mallei )

Burkholderia pseudomallei (formerly Pseudomonas

pseudomallei )

Clostridium perfringens epsilon toxin

Coccidioides immitis

Coxiella burnetii

US DEPARTMENT OF AGRICULTURE PLANT

PROTECTION AND QUARANTINE (PPQ)

SELECT AGENTS AND TOXINS

Candidatus Liberobacter africanus

Candidatus Liberobacter asiaticus

Peronosclerospora philippinensis

Ralstonia solanacearum race 3, biovar 2

Schlerophthora rayssiae var zeae

Synchytrium endobioticum

Xanthomonas oryzae pv. oryzicola

Xylella fastidiosa (citrus variegated chlorosis strain)

Reproduced from: US Department of Health and Human Services and US Department of Agriculture Select Agents and Toxins, 7 CFR

Part 331, 9 CFR Part 121, and 42 CFR Part 73. Available at: http://www.cdc.gov/od/sap/docs/salist.pdf. Accessed February 23, 2006.

393

Medical Aspects of Biological Warfare

comprehensive preventive medicine laboratory sup-

port to theater commanders. AMLs are capable of test-

ing environmental and clinical specimens for a broad

range of biological, chemical, and radiological hazards.

For biological agents, the laboratory uses a variety of

rapid analytical methods, such as real-time PCR, elec-

trochemiluminescence (ECL), enzyme-linked immuno-

sorbent assay (ELISA), and more definitive analyses

involving bacterial culture, fatty acid profiling, and

necropsy and immunohistochemistry. 2 AMLs have

significant “reach back” capability to reference labo-

ratories in the continental United States (CONUS) for

support. The largest of the service laboratories, AMLs

can identify “typical” infectious diseases including

endemic disease threats and they contain redundant

equipment for long-term or split-base operations. The

laboratory contains all of the necessary vehicles and

equipment to move and maintain itself in the field.

Laboratory Response Network

The response to future biological threats will

require the entire military laboratory network. The

logistical and technical burden of preparing for all

possible health threats will be too great for the mili-

tary clinical or field laboratories, which have limited

space and weight restrictions. The most important

role of these laboratories is to “listen to the hoof beats”

of medical diagnosis, rule out the most common of

threats, and alert the public health network about

suspicious disease occurrences. The military LRN

consists of the front-line medical treatment facility

clinical laboratories or deployed AMLs backed by

regional medical treatment facilities or military refer-

ence laboratories with access to more sophisticated

diagnostic capabilities. The clinical laboratories in the

regional medical centers or large medical activities

are the gateways into the civilian LRN sponsored by

the CDC. At the top of the military response pyramid

are research laboratories, such as USAMRIID (Fort

Detrick, Md) and the Naval Medical Research Center

(Silver Spring, Md). Other laboratories, such as the

Armed Forces Institute of Pathology (Washington,

DC) and the US Air Force Institute for Operational

Health (San Antonio, Texas) also provide reference

laboratory services for endemic infectious diseases.

Military research laboratories are best used to solve

the most complex and difficult diagnostic problems,

because usually they are not organized to perform

high-throughput clinical sample processing and

evaluation. Sentinel laboratories are generally sup-

ported by the network’s designated confirmatory

laboratories but may communicate directly with

national laboratories when hemorrhagic fevers or

orthopoxviruses (ie, smallpox virus) are suspected.

The network of military laboratories with connections

to federal and state civilian response systems provides

unparalleled depth and resources to the biological

threat response (Figure 18-1).

Navy

The Navy’s forward deployable preventive medicine

units (FDPMUs ) are medium-sized mobile laboratories

using multiple rapid techniques (polymerase chain

reaction [PCR] and ELISA) for identifying biological

warfare agents on the battlefield. The FDPMUs are

also modular and have the ability to analyze samples

containing chemical and radiological hazards. These

laboratories specialize in identifying biological threat

agents in concentrated environmental samples (high

confidence), but they can also identify endemic infec-

tious disease in clinically relevant samples.

Air Force

Air Force biological augmentation teams (AFBATs )

use rapid analytical methods (such as real-time PCR)

to screen environmental and clinical samples for threat

agents. The teams are small (two persons), easily

deployed, and designed to fall in on preexisting or

planned facilities. The units are capable of providing

early warning to commanders of the potential presence

of biological threat agents.

The theater commander, in conjunction with the

theater surgeon and nuclear, biological, and chemical

officer, must decide which and how many of these

laboratories are needed, based on factors such as the

threat of a biological attack, the size of the theater, the

number of detectors and sensitive sites in the theater,

and the confidence level of results needed. A critical but

little understood concept is that the rapid recognition

of biological warfare threats must be fully integrated

with preventive medicine activities and the response

to endemic infectious diseases.

Biosafety and Biosecurity in the Military Clinical

and Field Medical Laboratories

Biosafety Considerations

Specific guidelines for handling hazardous agents

are contained in “Biosafety in Microbiological and

Biomedical Laboratories” published by the US De-

partment of Health and Human Services (DHHS). 5

By avoiding the creation of aerosols and using certain

safety practices, most bacterial threats can be handled

using standard microbiological practices at biosafety

level (BSL) 2. BSL-2 conditions require that laboratory

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Laboratory Identiication of Biological Threats

personnel have specific training in handling patho-

genic agents and are directed by competent scientists.

Access to BSL-2 laboratories is restricted when work

is being conducted and safety precautions are taken

with contaminated sharp items. Procedures that may

create infectious aerosols are conducted only in bio-

logical safety cabinets or other physical containment

equipment. When samples must be processed on a

bench top, laboratory personnel must use other pri-

mary barrier equipment, such as plexiglass shields,

protective eyewear, lab coat and gloves, and work in

low-traffic areas with minimum air movement. BSL-3

conditions, which consist of additional environmental

controls (ie, negative pressure laboratories) and pro-

cedures, are intended for work involving indigenous

or exotic agents that may cause serious or potentially

lethal disease from inhalational exposure. Limited

prophylactic vaccines and therapeutics may be avail-

able to treat exposed personnel in case of an accident.

BSL-4 conditions are reserved for the most dangerous

biological agents for which specific medical interven-

tions are not available and an extreme risk for aerosol

exposure exists. BSL-4 requires the use of negative

pressure laboratories and one-piece, positive-pressure

personnel suits ventilated by a life support system.

Laboratory personnel should incorporate universal

bloodborne pathogen precautions and follow the

guidelines outlined in federal regulation 29 Code of

Federal Regulations (CFR) 1910.1030, “Occupational

Exposure to Blood-borne Pathogens.” 6 Specific pre-

cautions for each of the highest priority biological

threats can be found in the Basic Protocols for Level

A (Sentinel) Laboratories (http://www.bt.cdc.gov or

http://www.asm.org).

Biosurety

The 2001 anthrax letter attacks, which resulted in

22 cases of cutaneous or inhalational anthrax and

five deaths, raised the national concern about the

safety and security of laboratory stocks of biological

threats in government, commercial, and academic

laboratories. 7 As a result, the DHHS promulgated new

regulations (42 CFR, Part 73) that provided substantial

controls over access to biological select agents and

toxins (BSATs), required registration of facilities, and

established processes for screening and registering

laboratory personnel. 8 DHHS and the US Department

of Agriculture (USDA) identified over 80 biological

agents that required these regulatory controls (see

Exhibit 18-1). In addition to federal regulations, the

US Department of Defense (DoD) directed additional

controls for access to BSATs and required the establish-

ment of biosurety programs. These actions were taken

to foster public trust and assurance that BSATs are

handled safely and securely in military laboratories.

Among the services, the Army has established the most

comprehensive set of draft regulations (AR 50-XX) with

implementing memoranda.

At USAMRIID the framework for the military bio-

surety program was derived from the DoD’s experi-

ence with chemical and nuclear surety programs. 9-11

These surety programs incorporate reliability, safety,

and security controls to protect particular chemical and

nuclear weapons. The DoD biological surety program

applies many of the same controls as the chemical and

nuclear surety programs to medical biological defense

research and exceeds the standards of biosecurity pro-

grams in other federal and nonfederal laboratories.

Every military facility that stores and uses BSATs

must be registered not only with the CDC (see 42 CFR

Part 73) but also with the DoD. 8,9 In the case of Army

laboratories, registrations are completed through

the Assistant Secretary of the Army (Installation and

Environment). Army clinical laboratories, especially

those participating in the LRN triservice initiative,

are coordinated through the Army Medical Command

health policy and services. Not all clinical laboratories

need to be registered. However, unregistered laborato-

ries must follow the 42 CFR 73 “Clinical Laboratories

Exemption,” which states that clinical laboratories

identifying select agents have 7 days to forward or

destroy them. The transfer of BSAT cultures requires

the exchange of transfer documents (ie, CDC/APHIS

Form 2) between CDC-registered facilities.

Laboratory directors who supervise activities that

stock BSATs must be prepared to implement a variety of

stringent personnel, physical security, safety, and agent-

inventory guidelines. The law established penalties of

Level A Laboratories

First Responders

SAFER • HEALTHIER • PEOPLE TM

Reference Laboratories

National Laboratories

Fig. 18-1. The network of military laboratories with connec-

tions to federal and state civilian response systems provides

unparalleled depth and resources to the biological threat

response.

395

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