Wastewater Surveillance Testing Methods | CDC

Use this guide to perform wastewater-based disease surveillance. Sewage-based disease surveillance is a rapidly evolving science and the CDC will continue to update the guidance and information as it becomes available.

Overview of the test methods

Several test methods and laboratory procedures are used to quantify SARS-CoV-2 in wastewater in the USA. Laboratory controls can ensure that results are comparable by taking into account method performance and data quality. Based on the SARS-CoV-2 content in the wastewater, the methods can be adapted to higher or lower detection limits as required. If, for example, the SARS-CoV-2-RNA levels in the wastewater are sufficiently high, small amounts of wastewater (e.g. 1 ml) can be tested without additional concentration processes. Testing methods include sample processing steps, the use of laboratory controls, and the implementation of biosafety measures to ensure that public health data can be interpreted.

Overview of the processing and testing of wastewater samples for SARS-CoV-2

After sampling, the first step in SARS-CoV-2 wastewater testing is sample preparation. A matrix recovery control should be added to the sample during this step. The second step is the sample concentration. The third step is RNA extraction from the concentrated wastewater sample. The last step is the RNA measurement. In addition to measuring SARS-CoV-2 RNA at this step, several laboratory controls should also be measured, including matrix recovery controls, normalization of human stool, quantitative measurement controls, and controls to assess inhibition of molecular methods.

Sample processing

Sample processing for the measurement of SARS-CoV-2-RNA in wastewater includes sample preparation, sample concentration, RNA extraction and RNA measurement methods. The methods chosen at each step must be tailored for use with wastewater, which is a chemically and biologically complex and variable mixture. Evaluate the performance of these wastewater sample preparation methods using appropriate laboratory controls. The correct biosafety protocols for processing wastewater samples that may contain SARS-CoV-2 should be followed and are described later on this website.

Sample preparation

Proper storage and preparation of wastewater samples will ensure that the SARS-CoV-2-RNA wastewater measurements are accurate.

• camp: Immediately after collection, refrigerate the samples at 4 ° C and, if possible, process them within 24 hours in order to reduce the degradation of the SARS-CoV-2 RNA and increase the monitoring benefit. If you cannot process specimens within 24 hours of collection, you should add a Matrix Recovery Control to the specimen before refrigerating it at 4 ° C or freezing it at -20 ° C or -70 ° C.
• Homogenization: Both liquid wastewater and primary sludge samples should be mixed well before any portion of the collected wastewater is removed for downstream processing. Mix by inverting the samples several times (for liquid samples) or by mechanical mixing. Homogenizing samples may also include methods of breaking up wastewater solids and disaggregating virus particles, such as by ultrasound treatment.
• Sample clarification: Clarifying liquid wastewater samples by removing large solids can aid subsequent filtration-based concentration steps when used for sample concentration. However, removing solids also removes SARS-CoV-2 RNA that is attached to those solids. You can clarify samples with large pore size filters (5 µm or larger) or centrifugation.

Sample concentration

Concentrating wastewater samples can improve the detection of SARS-CoV-2 RNA. The concentration can be more important for untreated wastewater samples than for primary sludge samples. For more information on choosing a sample type, see "Sampling in" Developing a Wastewater Sampling Strategy.

So far evaluated concentration approaches that result in an adequate recovery for the SARS-CoV-2 detection in wastewater include:

• Ultrafiltration
• Filtration through an electronegative membrane with sample pretreatment by adding MgCl2 or acidification
• Precipitation of polyethylene glycol (PEG)
• Skimmed milk flocculation
• Ultracentrifugation

When choosing a virus concentration method, consider the following factors:

• Example type: Various filtration and precipitation methods listed above are available for untreated wastewater samples. For primary sludge samples, centrifugation is the most effective way to concentrate solids.
• Sample volume: For large untreated wastewater sample volumes, the sample may need to be split prior to membrane filtration (due to slow filtration rate) or PEG precipitation (due to centrifuge volume limitations). Sample volumes greater than 5 L may require pre-concentration by large volume concentration methods, such as. B. Ultrafiltration of Large Cartridges.
• Possible problems in the supply chain: Methods that require commercial filtration products, such as membrane filters or ultrafiltration cartridges, may be more sensitive to problems in the supply chain than other methods.
• Sample processing time: The selection of the concentration method is limited by the processing time of the method and the availability of laboratory staff. The membrane filtration of cloudy wastewater samples can take several hours.
• Availability of laboratory equipment: The centrifuge volume and power capacity as well as the availability of membrane filtration units will also limit the choice of method.

RNA extraction

The extraction and purification of nucleic acids is an essential step in the isolation of SARS-CoV-2-RNA from the waste water mixture. Wastewater is a complex mixture of materials that are known to interfere with methods used to quantify molecular viruses. Therefore, when choosing an extraction method, keep the following in mind:

• Select an extraction protocol for producing high-purity nucleic acid extracts from environmental samples. Commercial kits are available for environmental sampling.
• Before lysis, use an extraction kit or protocol specifically designed to purify RNA that contains RNase denaturants.
• Avoid the degradation of extracted RNA due to multiple freeze-thaw cycles by aliquoting the extracts into separate tubes and storing them at -70 ° C or below.

RNA measurement

Detection methods: Quantification of SARS-CoV-2 RNA in wastewater using RT-qPCR (reverse transcription-quantitative polymerase chain reaction) or RT-ddPCR (digital RT droplet PCR; other forms of digital PCR are also possible, but less common). Each method can either be carried out as a 1-step reaction in which RT and PCR take place in the same reaction mixture, or as a 2-step reaction in which RT and PCR are carried out in separate, sequential reactions. A 1-step RT-ddPCR protocol is beneficial for wastewater as RT is performed in single droplets, which can reduce RT inhibition compared to RT in bulk solution as in a 2-step procedure and in RT-qPCR.

Genetic goals: It has been reported that primers and probes targeting regions of the SARS-COV-2 N- (N1 and N2, published by CDC) and E-genes (E_sarbeco, Corman et al., 2020 EuroSurveillance) are useful for the quantification of SARS-CoV sensitive and specific. 2 RNA in wastewater. If possible, compare wastewater measurements with the same target genes.

Laboratory controls

Laboratory controls are important to compare SARS-CoV-2 RNA wastewater concentrations over time and across wastewater sources, especially when using different test methods. CDC recommends the following types of metrological laboratory controls for SARS-CoV-2 wastewater monitoring:

• Matrix recovery control
• Normalization of the human stool
• Quantitative measurement controls
• Assessment of the inhibition
• Negative controls

Matrix recovery controls

Use a matrix recovery control (also known as process control) to determine the amount of virus that is lost during sample processing. This control is important for comparing concentrations resulting from different test methods and over time. It is important to assess recovery quantitatively because wastewater is chemically and biologically complex and variable, and often contains components that can affect sample concentration, nucleic acid extraction, or molecular quantification methods. You need to include a matrix recovery control in the method validation and, where possible, add it to each sample to accommodate unexpected changes in wastewater composition. Always include a matrix recovery control whenever wastewater conditions (e.g. from stormwater inflows) or laboratory methods change.

A matrix recovery control that is more biologically similar to SARS-CoV-2 can more accurately depict the recovery of SARS-CoV-2 from a wastewater sample. Matrix recovery control candidates are enveloped viruses with single-stranded RNA genomes, including mouse coronavirus (also called mouse hepatitis virus), bovine coronavirus, and bovine respiratory syncytial virus.

Normalization of the human stool

The normalization of SARS-CoV-2 wastewater concentrations prior to calculating trends is performed to account for changes in wastewater dilution and differences in relative human waste input over time. If the number of people contributing to the sewer is expected to change during the monitoring period (due to tourism, weekday commuters, agency workers, etc.), it may be important to adjust SARS-CoV-2 levels by the amount of human Normalize feces in wastewater to interpret SARS-CoV-2 concentrations and to compare concentrations between wastewater samples over time. Human fecal normalization controls are organisms or compounds that are specific to human feces and can be measured in sewage to estimate human fecal levels.

Human normalization controls include, but are not limited to:

• Viral molecular targets of the stool indicator: Pepper Mild Mottle Virus, crAssphage
• Fecal indicator bacterial molecular targets: Bacteroides HF183, Lachnospiraceae Lachno3

Normalizing SARS-CoV-2 levels using human stool controls (e.g., the ratio of SARS-CoV-2 to human stool control levels) may also explain virus losses that occur somewhere between feces entering the sewage system and quantification in the laboratory . However, normalization of human stool cannot replace matrix recovery controls for evaluating method performance.

Quantitative measurement controls

They must include quantitative measurement controls for all SARS-CoV-2 RNA quantitation methods. For RT-qPCR, derive a calibration curve from a control of known concentration. For RT-ddPCR, include a known amount control with each instrument run. RNA controls are preferred over DNA controls for accurate quantitation of the RNA target. Aliquot quantitative measurement controls to avoid freeze-thaw cycles and store at -70 ° C or below.

Assessment of the inhibition

Use inhibition tests to determine if the RNA quantitation processes (RT and PCR) are going as expected. Wastewater is a complex and variable mixture and often contains compounds that can hinder accurate measurement by interfering with RNA quantitation methods.

The inhibition can be assessed using different approaches:

• If the SARS-CoV-2 RNA concentrations are high, evaluate the inhibition by assessing whether the concentrations measured in the extracted RNA, which have been diluted to different levels, scale with the expected dilution. This method is preferred because it allows a direct assessment of the inhibition in the same reaction that was used to quantify SARS-CoV-2 in the sample.
• If SARS-CoV-2 RNA concentrations are too low to be quantified after dilution, assess inhibition by spiking with viral RNA (e.g. synthetic SARS-CoV-2 RNA or purified RNA from a non-human coronavirus, as described in Matrix Recovery Controls)) in wastewater extracts and comparison of the measured concentration either with viral RNA to which molecular negatives were added (no template controls), or with a dilution of the spiked extract.

If you encounter an inhibition, it can often be removed by diluting the extracts. If you frequently encounter an inhibition, further optimize sample processing or quantification methods.

Negative controls

Extraction blanks are produced by extracting RNA without adding a waste water sample. These controls are used to determine contamination of the extraction reagent. Include them in each extracted set of samples.

"No template controls" are molecular reaction reagents with no added waste water sample nucleic acid extract. Use these controls to determine molecular reagent contamination and include it in all PCR instrument runs.

Biosecurity

The concentration of SARS-CoV-2 from wastewater requires processes to generate bioaerosol. CDC recommends performing these processes in a Biosafety Level 2 (BSL2) facility with unidirectional airflow and precautions for BSL-3, including breathing protection and a designated area to put on and take off personal protective equipment. Laboratory waste from wastewater samples that may contain SARS-CoV-2 should be autoclaved and treated in accordance with the BSL2 guidelines for biological safety.

pasteurization

Heat pasteurization of wastewater samples was performed to reduce the risk of biosafety from bioaerosol generation techniques during wastewater sample processing. When deciding if you want to include pasteurization, keep the following in mind:

• The extent to which heat pasteurization damages the short RNA fragments directed by RNA is not known in the wastewater.
• Peer-reviewed reports have found that heat treating airway samples at 56 ° C for 30 minutes resulted in negligible change in the RNA measurement.
• Some researchers have reported that heat treating wastewater at 60 ° C can improve SARS-CoV-2 RNA measurement. However, more data are needed to confirm this effect.