SAM Pathogen Methods

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 Amelia McCall ([email protected]) and accommodations will be made.

Pathogen	Technique	Method
Bacillus anthracis	Sample Processing	Journal of Microbiological Methods (2016) 130: 6-13
		EPA/600/R-17/213
Bacillus anthracis	Real-Time PCR, RV PCR and Culture	EPA/600/R-17/213
Brucella spp. [Brucellosis]	Sample Processing
		EPA/600/R-16/109
		EPA Method 1682
Brucella spp. [Brucellosis]	Real-Time PCR	Journal of Microbiological Methods (2008) 75(2): 375-378
Brucella spp. [Brucellosis]	Culture	ASM Sentinel Level Clinical Microbiology Laboratory Guidelines: Brucella species
Burkholderia mallei [Glanders] and Burkholderia pseudomallei [Melioidosis]	Sample Processing	PLoS Neglected Tropical Diseases (2013) 7(3): e2105
		EPA/600/R-16/109
Burkholderia mallei [Glanders] and Burkholderia pseudomallei [Melioidosis]	Real-Time PCR	Clinical Chemistry (2006) 52(2): 307-310 (PDF)
		Journal of Clinical Microbiology (2006) 44(1): 85-90 (PDF)
Burkholderia mallei [Glanders] and Burkholderia pseudomallei [Melioidosis]	Culture	ASM Sentinel Level Clinical Laboratory Guidelines: Burkholderia mallei and B. pseudomallei (PDF)
Campylobacter jejuni [Campylobacteriosis]	Sample Processing	Methods in Molecular Biology (2017) 1512:1-8
		EPA/600/R-16/109
Campylobacter jejuni [Campylobacteriosis]	Real-Time PCR	Journal of Clinical Microbiology (2010) 48(8): 2929-2933 (PDF)
	Culture	ISO 17995
Chlamydophila psittaci [Psittacosis] (formerly known as Chlamydia psittaci)	Sample Processing	EPA Method 1682
		EPA/600/R-16/109
Chlamydophila psittaci [Psittacosis] (formerly known as Chlamydia psittaci)	Tissue Culture and PCR	Journal of Clinical Microbiology (2000) 38(3): 1085-1093 (PDF)
Coxiella burnetii [Q-fever]	Sample Processing	EPA/600/R-17/213
		Applied Environmental Microbiology (2011) 77(23): 8355-8359 (PDF)
		Journal of Microbiological Methods (2010) 81(2): 141-146
		EPA Method 1642
		EPA/600/R-21/280
		EPA Method 1682
Coxiella burnetii [Q-fever]	Real-Time PCR	BMC Microbiology (2008) 8:77
Coxiella burnetii [Q-fever]	Tissue Culture	Antimicrobial Agents and Chemotherapy (1991) 35(10): 2070-2077
Escherichia coli O157:H7	Sample Processing	EPA Method 1680
		EPA/600/R-10/056 (52 pp, 747 K, About PDF)
		EPA/600/R-16/109
Escherichia coli O157:H7	Real-Time PCR	Environmental Science and Technology (2011) 45(6): 2250-2256
Escherichia coli O157:H7	Culture	EPA/600/R-10/056 (52 pp, 747 K, About PDF)
Francisella tularensis [Tularemia]	Sample Processing	EPA/600/R-16/109
		EPA/600/R-19/110
		EPA Method 1682
Francisella tularensis [Tularemia]	Real-Time PCR	EPA/600/R-19/110
Francisella tularensis [Tularemia]	Culture	EPA/600/R-19/110
Legionella pneumophila [Legionellosis]	Sample Processing	Procedures for the Recovery of Legionella from the Environment
		Methods in Molecular Biology (2013) 954: 3-25
		EPA/600/R-16/109
Legionella pneumophila [Legionellosis]	Real-Time PCR	ISO/TS Method 12869:2019
Legionella pneumophila [Legionellosis]	Culture	Methods in Molecular Microbiology (2013) 954: 3-25
Leptospira interrogans [Leptospirosis]	Sample Processing	Standard Method 9260 I
		EPA/600/R-16/109
		EPA Method 1682
Leptospira interrogans [Leptospirosis]	Real-Time PCR	Molecular and Cellular Probes (2005) 19(2): 111-117
Leptospira interrogans [Leptospirosis]	Culture	Standard Method 9260 I
Listeria monocytogenes [Listeriosis]	Sample Processing	PLoS ONE 15(2):e0228956
		EPA/600/R-16/109
Listeria monocytogenes [Listeriosis]	Real-Time PCR	USDA Microbiology Laboratory Guidebook, Chapter MLG 8A.04 (2009)
Listeria monocytogenes [Listeriosis]	Culture	Bacteriological Analytical Manual Online, Chapter 10
Non-typhoidal Salmonella [Salmonellosis] (Not applicable to S. typhi)	Sample Processing	EPA Method 1682
		EPA Method 1200
		EPA/600/R-16/109
Non-typhoidal Salmonella [Salmonellosis] (Not applicable to S. typhi)	Real-Time PCR	Environmental Science and Technology (2011) 45(20): 8996-9002
Non-typhoidal Salmonella [Salmonellosis] (Not applicable to S. typhi)	Culture	EPA Method 1200
		EPA Method 1682
Salmonella enterica serovar Typhi [Typhoid fever]	Sample Processing	EPA Method 1682
		EPA 600/R-10/133
		EPA/600/R-16/109
Salmonella enterica serovar Typhi [Typhoid fever]	Real-Time PCR	CDC Laboratory Assay. Triplex PCR for Detection of S. Typhi Using Smart Cycler®
Salmonella enterica serovar Typhi [Typhoid fever]	Culture	EPA 600/R-10/133
Shigella spp. [Shigellosis]	Sample Processing	EPA Method 1682
		EPA/600/R-16/109
		SM Method 9260
Shigella spp. [Shigellosis]	Real-Time PCR	Journal of Clinical Microbiology (2010) 48(8): 2929-2933 (PDF)
Shigella spp. [Shigellosis]	Culture	SM Method 9260
Staphylococcus aureus	Sample Processing	EPA Method 1682
		EPA/600/R-16/109
		Environmental Science & Technology 49(24):14249-14256
Staphylococcus aureus	Real-Time PCR	Journal of Food Protection (2007) 70(12): 2855-2859
Staphylococcus aureus	Culture	SM 9213 B
Vibrio cholerae [Cholera]	Sample Processing	EPA Method 1682
		EPA/600/R-16/109
		EPA 600/R-10/139 (56 pp, 1000 K, About PDF)
Vibrio cholerae [Cholera]	Real-Time PCR	Journal of Microbiological Methods (2007) 68(2): 254-259
Vibrio cholerae [Cholera]	Culture and Real-Time PCR	EPA 600/R-10/139 (56 pp, 1000 K, About PDF)
Yersinia pestis [Plague]	Sample Processing	EPA Method 1682
		EPA/600/R-16/109
Yersinia pestis [Plague]	Real-Time PCR, RV-PCR and Culture	EPA/600/R-16/109
Adenoviruses: Enteric and non-enteric (A-F)	Sample Processing	PLoS ONE 16(1): e0244977
		Applied and Environmental Microbiology (2015) 81(17): 5987-5992 (PDF)
		Journal of Virological Methods 213: 65-67
		EPA Method 1642
		EPA/600/R-21/280
Adenoviruses: Enteric and non-enteric (A-F)	Real-Time PCR	Applied and Environmental Microbiology (2005) 71(6): 3131-3136 (PDF)
Adenoviruses: Enteric and non-enteric (A-F)	Tissue Culture	Journal of Microbiological Methods (2016) 122: 43-49
		Current Protocols in Microbiology. 00:C:14C.1.1-14C.1.19
Astroviruses	Sample Processing	PLoS ONE 16(1): e0244977
		Applied and Environmental Microbiology (2015) 81(17): 5987-5992 (PDF)
		Journal of Virological Methods 213: 65-67
		EPA Method 1642
		EPA/600/R-21/280
Astroviruses	Real-Time Reverse Transcription- PCR	Canadian Journal of Microbiology (2004) 50(4): 269-278
Astroviruses	Integrated Cell Culture	Canadian Journal of Microbiology (2004) 50(4): 269-278
Caliciviruses: Noroviruses	Sample Processing	PLoS ONE 16(1): e0244977
		Applied and Environmental Microbiology (2015) 81(17): 5987-5992 (PDF)
		Journal of Virological Methods 213: 65-67
		EPA Method 1642
		EPA/600/R-21/280
Caliciviruses: Noroviruses	Real-Time Reverse Transcription-PCR	EPA Method 1615
Caliciviruses: Saporovirus	Sample Processing	PLoS ONE 16(1): e0244977
		Applied and Environmental Microbiology (2015) 81(17): 5987-5992 (PDF)
		Journal of Virological Methods 213: 65-67
		EPA Method 1642
		EPA/600/R-21/280
Caliciviruses: Saporovirus	Real-Time Reverse Transcription-PCR	Journal of Medical Virology (2006) 78(10): 1347-1353
Caliciviruses: Saporovirus	Tissue Culture	Archives of Virology (1991) 120(1-2): 115-122
Coronaviruses: Severe Acute Respiratory Syndrome (SARS) -associated Human Coronavirus (SARS-CoV-2, SARS-CoV and MERS-CoV)	Sample Processing	PLoS ONE 16(1): e0244977
		Journal of Virological Methods 297. 114251
		Journal of Virological Methods 213: 65-67
		EPA Method 1642
		EPA/600/R-21/280
Coronaviruses: Severe Acute Respiratory Syndrome (SARS) -associated Human Coronavirus (SARS-CoV-2, SARS-CoV and MERS-CoV)	Reverse Transcription-PCR	Science of the Total Environment 774: 145727
Coronaviruses: Severe Acute Respiratory Syndrome (SARS) -associated Human Coronavirus (SARS-CoV-2, SARS-CoV and MERS-CoV)	Tissue Culture	Applied Biosafety (2007) 12(2): 100-108 (PDF)
Coronaviruses: Severe Acute Respiratory Syndrome (SARS) -associated Human Coronavirus (SARS-CoV-2, SARS-CoV and MERS-CoV)	Rapid-viability reverse transcription-PCR	Journal of Virological Methods 297. 114251
Hepatitis E virus [HEV]	Sample Processing	PLoS ONE 16(1): e0244977
		Applied and Environmental Microbiology (2015) 81(17): 5987-5992 (PDF)
		Journal of Virological Methods 213: 65-67
		EPA Method 1642
		EPA/600/R-21/280
Hepatitis E virus [HEV]	Real-Time Reverse Transcription-PCR	Journal of Virological Methods (2006) 131(1): 65-71
Hepatitis E virus [HEV]	Tissue Culture	Pathogens and Disease 56(1): 73-79
Influenza H5N1 virus	Sample Processing	PLoS ONE 16(1): e0244977
		Applied and Environmental Microbiology (2015) 81(17): 5987-5992 (PDF)
		Journal of Virological Methods 213: 65-67
		EPA Method 1642
		EPA/600/R-21/280
Influenza H5N1 virus	Real-Time Reverse Transcription-PCR	Emerging Infectious Diseases (2005) 11(8): 1303-1305
Influenza H5N1 virus	Tissue Culture	Methods in Molecular Biology (2012) 865: 11-24
Picornaviruses: Enteroviruses	Sample Preocessing	PLoS ONE 16(1): e0244977
		Applied and Environmental Microbiology (2015) 81(17): 5987-5992 (PDF)
		Journal of Virological Methods 213: 65-67
		EPA Method 1642
		EPA/600/R-21/280
Picornaviruses: Enteroviruses	Real-Time Reverse Transcription-PCR and Tissue Culture	EPA Method 1615
Picornaviruses: Hepatitis A virus [HAV]	Sample Processing	PLoS ONE 16(1): e0244977
		Applied and Environmental Microbiology (2015) 81(17): 5987-5992 (PDF)
		Journal of Virological Methods 213: 65-67
		EPA Method 1642
		EPA/600/R-21/280
Picornaviruses: Hepatitis A virus [HAV]	Real-Time Reverse Transcription-PCR and Integrated Cell Culture	Journal of Food Protection (2011) 74(10): 1756-1761
Reoviruses: Rotavirus (Group A)	Sample Processing	PLoS ONE 16(1): e0244977
		Applied and Environmental Microbiology (2015) 81(17): 5987-5992 (PDF)
		Journal of Virological Methods 213: 65-67
		EPA Method 1642
		EPA/600/R-21/280
Reoviruses: Rotavirus (Group A)	Real-Time Reverse Transcription-PCR	Journal of Virological Methods (2009) 155(2): 126-131
Reoviruses: Rotavirus (Group A)	Tissue Culture	EPA Method 1615
Cryptosporidium spp. [Cryptosporidiosis]	Sample Processing	EPA/600/R-17/213
		Journal of Microbiological Methods (2010) 81(2): 141-146
		Applied and Environmental Microbiology (2011) 77(23): 8355-8359 (PDF)
		EPA Method 1622
		EPA Method 1623.1
		EPA Method 1642
		EPA/600/R-21/280
		Agriculture & Environmental Letters. 1(1):160031
Cryptosporidium spp. [Cryptosporidiosis]	Real-Time PCR	Applied and Environmental Microbiology (2011) 69(9): 5178-5185 (PDF)
		Applied and Environmental Microbiology (2011) 71(3): 1135-1141 (PDF)
Cryptosporidium spp. [Cryptosporidiosis]	Immunomagnetic Separation/Fluorescence Assay	EPA Method 1622
		EPA Method 1623.1
Cryptosporidium spp. [Cryptosporidiosis]	Cell Culture Immunofluorescence	Canadian Journal of Microbiology (2007) 53(5): 656-663
Entamoeba histolytica	Sample Processing	EPA/600/R-17/213
		Journal of Microbiological Methods (2010) 81(2): 141-146
		Applied and Environmental Microbiology (2011) 77(23): 8355-8359 (PDF)
		EPA/600/R-21/280
		EPA Method 1642
		African Journal of Medical Sciences. 40(4):321-5
Entamoeba histolytica	Real-Time PCR	The American Journal of Tropical Medicine and Hygiene. 88(6): 1041-1047
Entamoeba histolytica	Cell Culture	Journal of Parasitology (1972) 58(2): 306-310
Giardia spp. [Giardiasis]	Sample Processing	EPA/600/R-17/213
		Journal of Microbiological Methods (2010) 81(2): 141-146
		Applied and Environmental Microbiology (2011) 77(23): 8355-8359 (PDF)
		Applied and Environmental Microbiology (2011) 77(18): 6476-6485
		EPA Method 1642
		EPA Method 1623.1
		EPA/600/R-21/280
Giardia spp. [Giardiasis]	Real-Time PCR	Applied and Environmental Microbiology (2003) 69(9): 5178-5185 (PDF)
Giardia spp. [Giardiasis]	Immunomagnetic Separation/Fluorescence Assay	EPA Method 1623.1
Giardia spp. [Giardiasis]	Cell Culture	Transactions of the Royal Society of Tropical Medicine and Hygiene (1983) 77(4): 487-488
Naegleria fowleri [Naegleriasis] [Giardiasis]	Sample Processing	EPA/600/R-17/213
		Journal of Microbiological Methods (2010) 81(2): 141-146
		Applied and Environmental Microbiology (2011) 77(23): 8355-8359 (PDF)
		Journal of Parasitology Research (2013) ID 608367: 8 pages
		Clinical Infectious Diseases (2015) 60(8): e36-42
		EPA Method 1642
		EPA/600/R-21/280
Naegleria fowleri [Naegleriasis] [Giardiasis]	Real-Time PCR	Journal of Parasitology Research (2013) ID 608367: 8 pages
Naegleria fowleri [Naegleriasis] [Giardiasis]	Cell culture	Standard Method 9570
Toxoplasma gondii [Toxoplasmosis]	Sample Processing	EPA/600/R-17/213
		Journal of Microbiological Methods (2010) 81(2): 141-146
		Applied and Environmental Microbiology (2011) 77(23): 8355-8359 (PDF)
		Parasitology 144(13):1791-1801
		Journal of Microbiological Methods (2010) 81(3): 219-225
		Veterinary Parasitology 274: 108904
		EPA Method 1623.1
Toxoplasma gondii [Toxoplasmosis]	Real-Time PCR	Applied and Environmental Microbiology (2009) 75(11): 3477-3483 (PDF)
Toxoplasma gondii [Toxoplasmosis]	Cell Culture	Journal of Microbiological Methods (2010) 81(3): 219-225
Baylisascaris procyonis [Raccoon roundworm fever]	Sample Processing	EPA/600/R-17/213
		Journal of Microbiological Methods (2010) 81(2): 141-146
		Applied and Environmental Microbiology (2011) 77(23): 8355-8359 (PDF)
		American Journal of Veterinary Research. 44(5): 896-900
		EPA Method 1642
		EPA/600/R-21/280
		Parasitology Research (2010) 106: 499-504
Baylisascaris procyonis [Raccoon roundworm fever]	Real-Time PCR	Parasitology Research (2010) 106: 499-504
Baylisascaris procyonis [Raccoon roundworm fever]	Embryonation of Eggs and Microscopy	EPA/625/R-92/013

EPA/600/R-17/213