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How do I sample and analyze for anthrax?

Anthrax CollageEnvironmental sampling is an important tool for determining the presence of Bacillus anthracis spores in indoor environments. Sampling can help assess the extent and degree of contamination and the risk of exposure to building occupants and responders. Sample results also contribute to informed decisions on medical treatment and decontamination options and are ultimately used to determine the effectiveness of decontamination.

General Sampling Plan
SamplingThe purpose of a sampling plan is to create a road map to achieving defined sampling objectives.  An effective sampling plan will provide confidence that the results obtained are valid and indicative of the contamination present. The sampling plan should address each of the following questions: In evaluating methods that will achieve the sampling objectives, several other factors should be considered:
  • Laboratory capability (such as the laboratory's ability to handle the proposed sampling media),

  • Collection efficiency of the method, number of samples, and need for quantification,

  • Suitability of the sample-collection and analytical methods,

  • Cost effectiveness and efficiency of the sampling plan in meeting stated objectives, and

  • Utility of the sampling method to the owner and/or federal agency having jurisdiction over the project.

Specific Sampling Objectives
There are a variety of potential sampling objectives. The specific sampling objectives that may be applicable for an anthrax response may include:
  • Real-time Monitoring: Determine, in real-time, whether a release of spores is occurring or has occurred in a facility. Real-time instruments may be used to detect biological agents as they are released.

  • Preliminary Assessment of a Facility (Screening): Determine qualitatively whether any spores are present. Typically, composite samples of large areas and air volumes are obtained to maximize the likelihood of finding contamination.

  • Identification of Spores in a Bulk Material: Determine qualitatively if a bulk material, such as a powder in an envelope, is contaminated with anthrax. On-site analysis may be used for preliminary assessment, but laboratory analysis provides confirmation.

  • Determination of Contamination of an Article: Determine whether the surface of a small article is contaminated. Typically, composite surface samples of large articles and/or individual samples of small articles are collected.

  • Extent and Location of Contamination (Site Characterization): After anthrax is positively identified, further sampling is necessary to determine how far the contamination has spread. Sampling is performed to determine qualitatively, and if possible, semi-quantitatively, the extent and magnitude of contamination.

  • Effectiveness of Decontamination (Process Verification Sampling): Determine whether decontamination has reduced spores to a safe level. 

  • Post-Decontamination Sampling (Re-occupancy Verification Sampling): Final post-decontamination sampling is conducted inside and outside of the exclusion zone to verify that the originally contaminated environment has been sufficiently decontaminated to allow re-occupancy of the area without the use of personal protective equipment (PPE).

  • Special Re-occupancy Considerations: There may be site-specific circumstances where additional sampling should be considered.

Sampling Approach
After the sampling objectives have been identified, a logical approach and schedule must be developed to carry out the sampling tasks. The approach and schedule will depend on the objectives.

For Assessment/Characterization:
  • Targeted Sampling Strategy: If a building or area becomes contaminated from a known source and the source is quickly isolated, the sampling approach will center on the source area and move outward away from the source to define the extent of contamination.

  • Statistically-Based Strategy: If contamination is likely to be present in a building or area and the source has not been identified, sampling will focus on areas that are statistically the most likely to be contaminated. The objective of statistical sampling is to maximize the probability of detecting contamination.

  • Air Movement and HVAC Considerations: Spore-bearing particles less than 10 microns in size, or spores themselves, may remain suspended in the air for long periods of time (such as hours or days). In such cases, spores may spread throughout an air space and into adjacent areas on localized air currents, such as those created by people walking by, and also through generalized airflow created by HVAC systems. To fully assess the extent of contamination, the investigator should extend beyond the targeted approach to sample areas on projected contaminant pathways, such as those associated with air movement, dust collection, or work process flow.
For Verification Sampling Prior to Occupancy:
  • Post-Decontamination Surface Sampling: The effectiveness of decontamination should be confirmed by post-decontamination environmental sampling in ambient air; and in areas and on surfaces that were previously contaminated. Verification sampling prior to occupancy should include surfaces and air in areas outside of the exclusion zone (for example, the area where decontamination activities were conducted) to ensure that the outside areas continue to remain free of spores.

  • Air Sampling: Aggressive air sampling techniques have been developed for recent anthrax responses that model EPA guidance for clearing facilities for re-occupancy after asbestos decontamination.  While the area is under negative pressure, all surfaces are aggressively agitated and air is continuously disturbed while samples are collected.  An air sampling method that maximizes the likelihood of detecting contamination should be used.
Sampling Quality Assurance and Quality Control (QA/QC) Considerations:

QA/QC measures must be incorporated into the sampling approach to insure the legitimacy of sampling results. The sampling objectives will dictate the rigor of the QA/QC program for a given site or task. QA/QC includes four key elements:
  • QC of field activities

  • Sample documentation and management

  • Sample handling and shipment

  • Data validation and management

Sampling Techniques
There are various sampling techniques available for determining the presence of anthrax in air and surface samples.

Surface and Bulk SamplingThe following techniques are available and have proven useful for surface and bulk sampling:
  • Wet wipe,

  • Wet swab,

  • High-volume vacuum with "HEPA" sock (Alsock), and

  • Bulk sampling.
Air SamplingThe following techniques are available and have proven useful for air sampling:
  • Gelatin filter (low volume),

  • Mixed cellulose ester (MCE) filter,

  • Anderson air sampler and single-stage impactors with seetle plates,

  • Open agar plate,

  • Dry filter unit (high-volume air sampler with polyester 1-micron filter), and

  • Liquid impingers.

Analytical Methods
The following analytical methods are available for the detection of Bacillus anthracis:
  • Immunoassay Tests: Hand-held assays, sometimes referred to as "smart tickets," are sold commercially for the rapid detection of Bacillus anthracis and other biological agents. These assays are intended only for screening environmental samples. Due to their low and questionable sensitivity, they cannot be relied on for a determination that anthrax is or is not present.

  • Polymerase Chain Reaction (PCR): PCR is a technique that amplifies DNA and compares sequences to known test probe standards for Bacillus anthracis. PCR can be used in the field or the laboratory; in either case, the samples must be cultured to confirm that the bacteria is Bacillus anthracis and that it is viable. Field PCR systems are very selective, but do not work well with heterogeneous environmental samples, and the probes are very expensive. PCR has been shown to work best as a final confirmation of positive samples taken from plated colonies.
  • Petri DishCulturing: Samples may be analyzed for Bacillus anthracis using a traditional lab culturing technique. The sample is appropriately prepared for elution and plating, after which it is inoculated onto plates containing sheep blood agar. The plates are allowed to incubate for several hours and are then examined for growth of suspicious colonies.
The laboratory selected for the sample analyses should be consulted by the team developing the sampling plan. The lab should be authorized for work with Bacillus anthracis and laboratory procedures must conform to guidance provided by CDC or the Association of Public Health Laboratories (APHL). The lab should also be a part of the Laboratory Response Network (LRN), which was developed by CDC, APHL, and the FBI to help public health labs prepare for and respond to acts of terrorism, including bioterrorism. 

The following links provide additional information regarding analytical methods:

Packaging and Transportation of Samples
Packaging and Transportation of SamplesThere are strict requirements for packaging and transporting anthrax samples to ensure that the general public and workers transporting the samples are protected from exposure.  These requirements include:
  • Rigorous packaging designed to withstand rough handling and prevent leakage,

  • Appropriate marking and labeling that identifies the contents of the package,

  • Documentation of the hazardous contents of the package and emergency point-of-contact, and

  • Training of transportation workers on how to handle the contents in the event of an emergency.
Packaging and transporting anthrax samples are subject to various regulations established by the DOT, CDC, USPS, OSHA, and the International Air Transport Association (IATA). It is also important to consult with the analytical laboratory receiving the samples to determine whether they have additional packaging or shipping requirements.

Additional details on packaging and shipping procedures for anthrax samples, according to CDC guidelines, can be found at:
  • Packing Critical Biological Agents. Centers for Disease Control and Prevention, Public Health Emergency Preparedness and Response. Protocols for packing, shipping, and transport of biological agents/diseases.

  • Interstate Shipment of Etiologic Agents. Centers for Disease Control and Prevention, Office of Health and Safety, Biosafety Branch.

Interpretation of Results
Interpretation of ResultsSince analytical methods are not fully validated for Bacillus anthracis, the following considerations are recommended for interpretation of analytical results:
  • Use a multi-disciplinary team of technical experts to interpret the analytical results.

  • Consult with field investigators and laboratory personnel during the interpretation process. This will provide the best insight into sample collection and recovery.

  • Consider the analytical method limitations and use professional judgment in interpreting any positive or negative findings as well as quantitative or semi-quantitative results.

  • In the case of preliminary assessment sampling, positive findings will usually require more extensive sampling in that particular area.
  • Laboratories will sometimes report "colony counts" with positive results. In some cases, colony counts can help the data interpretation teams decide how much more extensively to sample in that area, prior to establishing additional isolation and starting decontamination. However, quantitative methods have not been validated for a variety of sample collection methods and the use of "colony count" data is not yet accepted as a reliable indicator of large populations of colony forming units.

  • The multi-disciplinary team described above should establish a decision tree for evaluating the adequacy of the sampling and decontamination to coordinate its efforts in reaching a unified conclusion.

Coordination with Affected Parties
Before, during, and after a response, the sampling team should coordinate with the following affected parties:
  • Facility manager,

  • Union officials, and

  • Other individuals who might have site-specific knowledge of the contamination.
The sampling plan should indicate the following:
  • Employees working within the affected sampling area need to be informed of the sampling objectives and methods.

  • The method of providing results to employees working within the affected sampling area.

  • Before sampling begins, employees should be told when, where, and why sampling may occur and they should also be advised when plans are changed.

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