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. Following a microbial contamination incident, it is assumed that the identification, confirmation and strain-level characterization of the pathogen have been completed before the U.S. Environmental Protection Agency’s (EPA) remediation actions begin. The first phase of EPA’s actions includes site characterization, to determine the extent and magnitude of contamination and to 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.

Selection of methods 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 incident. For Pathogens, site characterization refers to the assessment phase, decontamination refers to the cleanup phase and post decontamination refers to the clearance phase.

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. Therefore, in most cases, the analytical methods selected for site characterization may not have to determine the viability of the pathogen. The methods should also provide a high throughput 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 polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) or other immunoassay-based methods. Depending on the pathogen, type of incident and response, culture methods could be appropriate for use during site characterization. In certain cases, the determination of the extent of pathogen contamination within this phase may drive decontamination planning.

Methods for Post Decontamination Phase: It is extremely critical that the analytical methods used during post decontamination 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 to minimize false negative results. Traditional microbiological culture methods typically include plating on selective medium to determine the viability of the pathogen and to 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.

Please note: SAM provides guidance for selecting pathogen methods that have a high likelihood of assuring analytical consistency when laboratories analyze a large number of samples during remediation. Not all methods have been verified for the pathogen/sample type combination. Please refer to the specified method to identify analyte/sample type combinations for which the method has been verified. Any questions regarding this information should be addressed to the appropriate Technical Contact(s).

Pathogens that require biosafety level (BSL)-4 containment and practices, such as hemorrhagic fever viruses and Variola major (smallpox) will be handled only by reference laboratories with BSL-4 capability and are not included in this document. All other pathogens should be handled using BSL-2 or BSL-3 containment and practices, as appropriate. Pathogens that are considered to be solely of agricultural concern (i.e., animal and plant pathogens) are not currently included. However, such pathogens may be considered for possible inclusion in future SAM updates.

Although culture-based methods have been selected for many of the pathogens, due to technical difficulty and time constraints, molecular techniques such as PCR will likely be used for viruses. Some of the selected methods include multiple analytical techniques by inference. The analytical technique listed 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” might include immunochemical or PCR- based assays for the identification and/or confirmation of isolates. Several of the methods also include options such as the use of multiple cell culture media for primary isolation and a selection of a defined subset of biochemical tests for 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 are not included, it is critical that end users and stakeholders select the most appropriate sample processing 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 may not only decrease recovery efficiency of biological targets (e.g., pathogen, deoxyribonucleic acid/ribonucleic acid [DNA/RNA], antigen/protein) from the samples, but also prevent accurate quantitation and high throughput. Samples should not be stored indefinitely, and should be processed and analyzed as soon as possible upon receipt. Note: For post decontamination samples it may be necessary to neutralize the decontamination agent.

The selected methods 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 of SAM, emphasis is given to the environmental sample types that are predominately used to fulfill EPA’s responsibilities following a contamination incident (e.g., aerosols, particulates [wipes or swabs], soils, drinking water, post decontamination waste water). Other sample types may have to be analyzed and, for those sample types, specific requests should be sent to the Pathogen Methods Lead and Alternate Lead. See: SAM Technical Contacts.

Below is a list of all selected pathogen methods with a link to their source. 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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