Selected Analytical Methods for Environmental Remediation and Recovery (SAM) provides a list of methods or procedures to be used in analyzing environmental samples for pathogens. According to the ICLN-Federal Responsible Agency Matrix, EPA is the lead agency for environmental and drinking water remediation during a response to any bioterrorism event. It is assumed that the CDC and the FBI have completed the identification, confirmation, and strain-level characterization of the bioagent/pathogen involved in the event, prior to EPA's remediation. The first phase of EPA's actions includes site characterization, to determine the extent and magnitude of contamination and guide remediation planning. Based on the results of sample analyses for site characterization, EPA will determine the approach for site decontamination. During the post decontamination (clearance) phase of remediation, samples are collected and analyzed to determine the efficacy of the decontamination treatment.

Based on lessons learned and experience gained through various interagency bioterrorism counter-measures programs, including the Interagency Biological Restoration Demonstration (IBRD), decision making during remediation includes scientific, public health, social, economic, and political concerns. As part of this process, the analytical methods selected for use should aid in decision making in an effective and time-sensitive manner. The purpose of this section is to provide guidance to stakeholders in determining the appropriate methods for each remedial phase (site characterization and/or post decontamination) of a response to a biological event. Emphasis is given to environmental sample types that are most prevalently used to fulfill EPA's homeland security responsibilities (e.g., aerosols, surface wipes or swabs, drinking water, and post decontamination waste water).

Selection of pathogen methods from Selected Analytical Methods for Environmental Remediation and Recovery (SAM) should be based on specific data and information needs, including consideration of the remediation phase and whether there is a need to determine either the presence of a pathogen, the viability of a pathogen, or both. The flow chart in Figure 7-1 presents a summary of the sample types, overall steps in sample analysis, and analytical techniques that should be used to address pathogens during EPA site remediation activities following a contamination event. As depicted in Figure 7-1, for SAM Pathogens, site characterization refers to the assessment phase and post decontamination refers to the clearance phase. See: Figure 7-1.

Methods for Site Characterization Phase: Since decontamination of the affected site has to quickly follow the site characterization phase, rapid analytical methods should be selected to determine the extent and magnitude of contamination. It is assumed here that, prior to site characterization, the identity and viability of the pathogen have been determined by the CDC and FBI. Therefore, in most cases, the analytical methods selected for this phase may not have to determine whether the pathogen is viable. The methods should also provide a high through-put analytical capability, so that a large number of samples can be rapidly analyzed to determine the presence or absence of the pathogen and allow for site decontamination planning in a time-efficient manner. For most pathogens, such methods routinely include PCR, ELISA, or other immunoassay-based methods. Depending on the pathogen, type of event, and response, culture methods may be appropriate for use during site characterization. In certain cases, the viability determination of the pathogen within this phase may drive decontamination planning.

Methods for Post Decontamination Phase: It is extremely critical that the analytical methods used for sample analysis during the post decontamination phase be highly sensitive, specific, rapid, and able to determine pathogen viability. For post decontamination phase samples, neutralization or removal of the decontamination agent may be required prior to analysis. Traditional microbiological culture methods typically include plating on selective medium to determine the viability of the pathogen and minimize or eliminate non-target growth. The absence of growth on the medium generally indicates the absence of live pathogen in the sample [with the exception of some pathogens which may become viable but non-culturable (VBNC)]. To minimize the analytical time needed to obtain results, typical colonies should be quickly analyzed to confirm the presence of the pathogen using reliable and rapid methods such as PCR, ELISA, or other immunoassay-based methods, as opposed to time and labor intensive traditional biochemical and serological procedures. For Bacillus anthracis, the recently published Rapid Viability-PCR method may be used because it provides rapid and high throughput results in addition to viability determination (Letant et al. 2011). However, if pathogen viability and concentration determinations are required for any samples, the culture method followed by rapid identification techniques as stated above should be used.

Please note: SAM pathogen information provides guidance for selecting pathogen methods that have a high likelihood of assuring analytical consistency when laboratories are faced with the analysis of a large number of samples during remediation. Not all methods have been verified for the pathogen/sample type combination listed in SAM. Please refer to the specified method to identify analyte/sample type combinations that have been verified. Any questions regarding information discussed in this section should be addressed to the appropriate contact(s) listed in the Technical Contacts. See: SAM Technical Contacts.

Pathogens that are categorized as biosafety level BSL-4, such as hemorrhagic fever viruses and smallpox, will be handled only by reference laboratories with BSL-4 capability and are not included in this document. All other pathogens are to be handled using BSL-2 or BSL-3 containment and practices, as appropriate. If known, the BSL classification for each pathogen is provided in the SAM pathogen method summaries. Pathogens that are considered to be solely of agricultural concern (i.e., animal and plant pathogens) are not currently included. Such pathogens may be considered for possible inclusion in future revisions of SAM.

In addition to analytical methods, SAM lists commercially available spore strips, which may be used as general indicators that a decontamination process (e.g., fumigation) has been successful. Spore strip results, however, cannot replace negative-culture results as an indicator of decontamination efficacy. Culture-based methods have been selected for many of the pathogens; however, due to technical difficulty and time constraints, molecular techniques such as PCR will likely be used for viruses.

Some of the methods in SAM include multiple analytical techniques by inference. The analytical techniques listed in SAM for each pathogen is intended to be a description of the predominant technique that is required to provide the data quality parameter (viability or detection and identification). This description does not preclude the use of other techniques that are within or referenced by the method. For example, a viability method or procedure listed as "culture" may include immunochemical or PCR - based assays for the identification of isolates. Several of the pathogen methods listed in SAM also include options such as the potential for use of multiple cell culture media for primary isolation and allowance for selection of a defined subset of a larger number of biochemical tests for biochemical confirmation. To expedite time-to-results, however, isolates should be confirmed using rapid techniques (e.g., PCR, ELISA).

Sample Processing: It is widely recognized in the scientific community that the processing of biologically contaminated environmental samples is one of the most challenging issues prior to sample analysis. Although details regarding sample processing is not within the scope of SAM, it is extremely important that end users and stakeholders select the most appropriate procedure for a given sample type and analytical method. It is highly unlikely that a single procedure will be applicable to all sample types and analytical methods. Inadequate sample processing not only limits the recovery of biological targets (e.g., pathogen, deoxiribonucleic acid/ribonucleic acid [DNA/RNA], antigen/protein) from the samples, but may also prevent accurate quantitation and high throughput. Note: For post decontamination samples it may be necessary to neutralize the decontamination agent.

The methods listed in SAM attempt to address multiple environmental sample types, each with different physical, chemical, and biological properties (e.g., pH, inhibitory substances, and background microorganisms). In this edition, major emphasis is given to the environmental sample types that are most prevalently used to fulfill EPA's homeland security responsibilities following an event involving pathogen contaminants (e.g., aerosols, surface wipes or swabs, drinking water, and post decontamination waste water). Other sample types may have to be analyzed, and for those sample types, specific requests should be sent to the SAM Pathogen Methods Lead and Alternate Lead provided in the Technical Contacts. See: SAM Technical Contacts.

Below is a list of all selected pathogen methods with a link to their method abstract. Due to the complexity of some tables and graphics, some of our information is not amenable to a screen reader. If you have trouble accessing information contact Kathleen Nickel (nickel.kathy@epa.gov) and accommodations will be made.

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