STRATEGIES FOR THE DETECTION OF UNKNOWN

BIOLOGICAL AGENTS

Dr. Peter J. Stopa

US Army Edgewood Chemical Biological Center

Aberdeen Proving Ground, MD

OBJECTIVES FOR UNKNOWNS DETECTION

Differentiate between natural and artificial events.Implement and maintain protective postures.Confirm whether forces have been attacked.Initiate treatment in the absence of a specific biological agent hazard.

Potential Classification Parameters

ClassifyPathogen/non-Pathogen

Aerosol Additives

Naturalvs.

Man-made Bacteria

Encapsulants

Virus

Protein Peptide Toxin

Non-Protein Toxin

Bio/non-Bio

Natural vs. Man-made

Parameter Test Rationale Status

Particles Particle-size Analysis

Most man-made aerosols are > 3 um

Commercial

Particle-shape Analysis

May see uniform distribution of similar particle shapes.

Commercial

Elemental Analysis

Spectrometric analysis

May see “unnatural” elements or ratios of elements appear.

Commercial

Aerosol Additives

Parameter Test Rationale Status Silica Particles Silica Analysis Silica particles can

be used to improve aerosol dissemination.

Commercial – Various commercial manufacturers have silica tests available.

Protein Content Standard Protein Determinations

Protein content in air is low. Could be used as an additive or agent itself.

Available (Field) – various protein detection kits are routinely used.

Bio/non-Bio Determination

Pathogen/non-pathogen Determination

Bacterial Specific Detection

Viral Specific Detection

Protein /Peptide Specific Detection

Non-Protein ToxinSpecific Detection

Parameter Test Rationale Status Virulence Determination

Binding to receptors. Sentinel Cell Assay

A variety of toxins bind to gangliosides. Measure binding to a sentinel cell.

Available (Lab) –.

Molecular Weight Determination

Mass Spectrometry Scope of potential physiological active materials is great. Only MS can probably address.

Demonstrated in the laboratory.

Enzymatic Activity

Substrate Analyses A variety of toxins inhibit specific enzymatic processes.

Demonstrated in the laboratory.

Determination of Appropriate Equipment

How do you determine what you need?

Need to identify: The end user. How they operate. Constraints. Performance Criteria. How important are each of these factors in

determining the best equipment?) (Weighting Factors)

A Computer-based Approach

Can identify each of the parameters and subsequent weighting factors and plug them into a statistical model.Identify appropriate candidate technologies and score them according to your criteria.Obtain the final results.See how they fared and why.Make purchasing decisions.

Biological DetectionMore difficult than chemical. No vapor pressure – sampling for detection is more

difficult. Present in low concentrations. May be hard to discriminate an intentional release

against background levels of similar “natural” biological materials.

Technologies not as mature. Clinical personnel sometimes do not understand “the

field”. Most confirmatory technologies are slow (i.e., culture). Microbiologists are reluctant to adopt instrumented

methods (need to see it or smell it to believe it).

Biological Detection Process

Collect samples

Chemical Property Characterization

Generic detection Biological agent

characteristics

Specific identification Identification kits Reference laboratory

Chemical Properties

From July/August 2003 Homeland First Response

Chemical Properties

From July/August 2003 Homeland First Response

Premise:

• Biological materials can be viable only within a pH range of 5-9.

• Turbidity and density in water may give an indication of particle size and solubility.

• Presence of Protein may indicate a biological agent.

Chemical Tests

WHY GENERIC DETECTION?

Rapidly provide the Incident Commander enough information to justify the response to a suspected biological terrorist threat.Minimize response resources to false threats.Evaluate if the sample can be further analyzed with immunoassay tickets on site or sent to a laboratory for confirmation.Should not rely on immunoassay tickets alone. Limited library - some materials that a terrorist may

use are not on the threat list. For ticket results to be valid, the sample must be

within certain concentration, pH, and ionic strength guidelines.

Biological Properties

Generic detectors that detect biological properties are relatively rapid, inexpensive, and easy to use.

More information than the chemical property test alone –i.e., is it LIVE?

Confirmatory identification must be performed in the laboratory.

Generic Detection Strategy

Use various properties to detect the presence of a biological material in a suspect sample. Luminescence – ATP –

viability (cell battery). DNA – present in all

biological materials. (blueprint).

Protein – also present in all biological materials. (building block).

Particle size/concentration.

Biological Detection Instruments

Particle analyzer

Fluorometer (DNA)

Luminometer (ATP)

Colorimeter (protein)Particle Analyzer

Colorimeter

Fluorometer

Luminometers

                                     

Integrated System

Expected Results for Generic Detection

SAMPLE ATP DNA PROTEIN

Bacteria + + ? (1)

Toxin - ? (2) +

Virus - + ? (1)

(1) Possibly from culture media.

(2) If protein concentration is high enough (> 10 ug/ml), will probably see contaminating DNA from protein source.

Identification

Immunoassay TicketsAnd Readers Biological Laboratory

Field Instruments(PCR)

Comments

Experience from Fall 2001 showed that no single technology was 100% accurate.

Both IA platforms and PCR platforms missed samples for various reasons.

Culture was able to detect in all of the samples – but TOO slow to be of value in the field.

Need redundant capabilities.

CONCLUSIONS

Tests and equipment exist for use by first responders for field screening of biological samples.

Additional discussion is needed among the policy makers, field responders, and reference laboratories to develop or accept a workable system.