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Coronavirus Illness 2019 (COVID-19)

Use this steering to implement wastewater-based illness surveillance. Wastewater-based illness surveillance is a quickly growing science, and CDC will proceed to replace steering and knowledge because it turns into obtainable.

Testing strategies overview

A number of testing strategies and laboratory workflows are used to quantify SARS-CoV-2 in wastewater throughout the USA. Laboratory controls can make sure that outcomes are comparable by accounting for technique efficiency and information high quality. Primarily based on the degrees of SARS-CoV-2 in wastewater, strategies will be tailored to greater or decrease detection limits as wanted. For instance, if ranges of SARS-CoV-2 RNA are sufficiently excessive in wastewater, small volumes of wastewater (e.g., 1 ml) could also be examined with out extra focus processes. Testing strategies embrace pattern processing steps, use of laboratory controls, and implementation of biosafety measures to make sure that information will be interpreted for public well being use.

Overview of wastewater pattern processing and testing for SARS-CoV-2

After pattern assortment, step one in SARS-CoV-2 wastewater testing is pattern preparation. A matrix restoration management ought to be spiked into the pattern throughout this step. The second step is pattern focus. The third step is RNA extraction from the concentrated wastewater pattern. The ultimate step is RNA measurement. Together with measurement of SARS-CoV-2 RNA on this step, a number of laboratory controls must also be measured, together with matrix restoration controls, human fecal normalization, quantitative measurement controls, and controls to evaluate molecular technique inhibition.

Pattern processing

Pattern processing for measuring SARS-CoV-2 RNA in wastewater includes pattern preparation, pattern focus, RNA extraction, and RNA measurement strategies. Strategies chosen at every step should be tailor-made to be used with wastewater, which is a chemically and biologically advanced and variable combination. Consider the efficiency of those wastewater pattern processing procedures utilizing applicable laboratory controls. Correct biosafety protocols for processing wastewater samples which will include SARS-CoV-2 ought to be adopted and are described afterward this webpage.

Pattern preparation

Correctly storing and making ready wastewater samples helps make sure that SARS-CoV-2 RNA wastewater measurements are correct.

  • Storage: Refrigerate samples at 4°C instantly after assortment and, if potential, course of them inside 24 hours to cut back SARS-CoV-2 RNA degradation and improve surveillance utility. For those who can’t course of samples inside 24 hours after assortment, you must spike a matrix restoration management into the pattern previous to refrigerating it at 4°C or freezing it at -20°C or -70°C.
  • Homogenization: Each liquid wastewater and first sludge samples ought to be well-mixed previous to eradicating parts of collected wastewater for downstream processing. Combine by inverting samples a number of occasions (for liquid samples) or by mechanical mixing. Homogenizing samples may also embrace procedures to interrupt up wastewater solids and disaggregate virus particles, reminiscent of by sonication.
  • Pattern clarification: Clarifying liquid wastewater samples by eradicating massive solids can assist subsequent filtration-based focus steps if they’re used for pattern focus. Nevertheless, eradicating solids will even take away SARS-CoV-2 RNA adhered to these solids. You possibly can make clear samples utilizing massive pore dimension filters (5 µm or bigger) or centrifugation.

Pattern focus

Concentrating wastewater samples can enhance detection of SARS-CoV-2 RNA. Focus could also be extra vital for untreated wastewater samples than main sludge samples. See What to Pattern underneath ‘Growing a Wastewater Sampling Technique’ for extra info on choosing a pattern sort.

Focus approaches evaluated so far that yield satisfactory restoration for SARS-CoV-2 detection in wastewater embrace:

  • Ultrafiltration
  • Filtration via an electronegative membrane with pattern pre-treatment by addition of MgCl2 or acidification
  • Polyethylene glycol (PEG) precipitation
  • Skim milk flocculation
  • Ultracentrifugation

Contemplate the next elements when choosing a virus focus technique:

  • Pattern sort: For untreated wastewater samples, a number of filtration and precipitation strategies, listed above, can be found. For main sludge samples, centrifugation is the best method to focus solids.
  • Pattern quantity: Giant untreated wastewater pattern volumes might require dividing the pattern previous to membrane filtration (on account of gradual filtration fee) or PEG precipitation (on account of centrifuge quantity constraints). Pattern volumes better than 5 L might require pre-concentration by strategies designed to pay attention massive quantity, reminiscent of massive cartridge ultrafiltration.
  • Potential provide chain points: Strategies that require industrial filtration merchandise, reminiscent of membrane filters or ultrafiltration cartridges, could also be extra delicate to provide chain points than different strategies.
  • Pattern processing time: Focus technique choice will probably be constrained by technique processing time and availability of laboratory personnel. Membrane filtration of turbid wastewater samples might take a number of hours.
  • Availability of laboratory tools: Centrifuge volumes and pressure capability, in addition to availability of membrane filtration items, will even constrain technique choice.

RNA extraction

Nucleic acid extraction and purification is a necessary step in isolating SARS-CoV-2 RNA from the sewage combination. Sewage is a fancy combination with supplies identified to intrude with molecular viral quantification strategies, so contemplate the next when choosing an extraction technique:

  • Choose an extraction protocol designed to supply extremely purified nucleic acid extracts from environmental samples. Industrial kits can be found for environmental pattern extraction.
  • Use an extraction package or a protocol designed particularly to purify RNA and that features RNase denaturants previous to lysis.
  • Keep away from degradation of extracted RNA on account of a number of freeze-thaw cycles by aliquoting extracts into separate tubes and storing them at -70°C or under.

RNA measurement

Detection strategies: Quantify SARS-CoV-2 RNA in wastewater utilizing both RT-qPCR (reverse transcription-quantitative polymerase chain response) or RT-ddPCR (RT-droplet digital PCR; different types of digital PCR are additionally potential however much less widespread). Every technique will be carried out as both a 1-step response, during which RT and PCR happen in the identical response combination, or a 2-step response, during which RT and PCR are carried out in separate, sequential reactions. A 1-step RT-ddPCR protocol is advantageous for wastewater as a result of RT is carried out in particular person droplets, which might cut back RT inhibition in comparison with RT in bulk answer, as in a 2-step course of and in RT-qPCR.

Genetic targets: Primers and probes concentrating on areas of the SARS-COV-2 N (N1 and N2, printed by CDC) and E genes (E_sarbeco, Corman et al., 2020 EuroSurveillance) have been reported to be delicate and particular for quantifying SARS-CoV-2 RNA in wastewater. When potential, evaluate wastewater measurements utilizing the identical goal genes.

Laboratory controls

Laboratory controls are important for evaluating SARS-CoV-2 RNA wastewater concentrations over time and throughout wastewater sources, particularly if you use totally different testing strategies. CDC recommends the next sorts of measurement laboratory controls for SARS-CoV-2 wastewater surveillance:

  • Matrix restoration management
  • Human fecal normalization
  • Quantitative measurement controls
  • Inhibition evaluation
  • Unfavourable controls

Matrix restoration controls

Use a matrix restoration management (additionally known as a course of management) to know the quantity of virus misplaced throughout pattern processing. This management is vital for evaluating concentrations ensuing from totally different testing strategies and over time. It is very important quantitatively assess restoration as a result of wastewater is chemically and biologically advanced and variable, and sometimes accommodates constituents that may intrude with pattern focus, nucleic acid extraction, or molecular quantification strategies. You could embrace a matrix restoration management in technique validation and, if potential, embrace it with every pattern to account for sudden adjustments in wastewater composition. All the time embrace a matrix restoration management when wastewater circumstances (reminiscent of from rainwater inflows) or laboratory strategies change.

A matrix restoration management that’s extra biologically just like SARS-CoV-2 might extra precisely characterize the restoration of SARS-CoV-2 from a wastewater pattern. Candidates for matrix restoration controls are enveloped viruses with single-stranded RNA genomes, together with murine coronavirus (additionally known as murine hepatitis virus), bovine coronavirus, and bovine respiratory syncytial virus.

Human fecal normalization

Normalizing SARS-CoV-2 wastewater concentrations previous to calculating developments is performed to account for adjustments in wastewater dilution and variations in relative human waste enter over time. If the variety of folks contributing to the sewershed is anticipated to vary over the surveillance interval (on account of tourism, weekday commuters, non permanent employees, and many others.), normalizing SARS-CoV-2 concentrations by the quantity of human feces in wastewater will be vital for decoding SARS-CoV-2 concentrations and evaluating concentrations between sewage samples over time. Human fecal normalization controls are organisms or compounds particular to human feces that may be measured in wastewater to estimate its human fecal content material.

Human normalization controls embrace, however are usually not restricted to:

  • Fecal indicator viral molecular targets: Pepper Gentle Mottle virus, crAssphage
  • Fecal indicator bacterial molecular targets: Bacteroides HF183, Lachnospiraceae Lachno3

Normalizing SARS-CoV-2 concentrations utilizing human fecal controls (e.g., the ratio of SARS-CoV-2 to human fecal management concentrations) may also account for viral losses that happen anyplace between fecal enter into the wastewater system and quantification on the laboratory. Nevertheless, human fecal normalization can’t substitute matrix restoration controls for technique efficiency analysis.

Quantitative measurement controls

You could embrace quantitative measurement controls for all SARS-CoV-2 RNA quantification strategies. For RT-qPCR, derive a calibration curve from a management of identified focus. For RT-ddPCR, embrace a management of identified amount with every instrument run. RNA controls are preferable to DNA controls for correct RNA goal quantification. Aliquot quantitative measurement controls to keep away from freeze-thaw cycles and retailer them at -70°C or under.

Inhibition evaluation

Use inhibition testing to find out whether or not RNA quantification processes (RT and PCR) are performing as anticipated. Wastewater is a fancy and variable combination, and sometimes accommodates compounds that may impede correct measurement by interfering with RNA quantification strategies.

Inhibition will be assessed utilizing a number of approaches:

  • When SARS-CoV-2 RNA concentrations are excessive, assess inhibition by evaluating whether or not the concentrations measured within the extracted RNA diluted to totally different ranges scale with the dilution as anticipated. This technique is most well-liked as a result of it permits direct analysis of inhibition in the identical response used to quantify SARS-CoV-2 within the pattern.
  • When SARS-CoV-2 RNA concentrations are too low to be quantified after dilution, assess inhibition by spiking viral RNA (for instance, artificial SARS-CoV-2 RNA or purified RNA from a non-human coronavirus, as described in Matrix Restoration Controls) into wastewater extracts, and evaluating the measured focus to both viral RNA spiked into molecular negatives (no template controls) or to a dilution of the spiked extract.

For those who encounter inhibition, it may usually be eradicated by diluting extracts. For those who incessantly encounter inhibition, additional optimize pattern processing or quantification strategies.

Unfavourable controls

Extraction blanks are made by extracting RNA with out the addition of a wastewater pattern. These controls are used to detect extraction reagent contamination. Embrace them with every set of samples extracted.

“No template controls” are molecular response reagents with out added wastewater pattern nucleic acid extract. Use these controls to detect molecular reagent contamination and embrace them with all PCR instrument runs.


Focus of SARS-CoV-2 from wastewater requires bioaerosol-generating processes. CDC recommends conducting these processes in a Biosafety Stage 2 (BSL2) facility with unidirectional airflow and BSL-3 precautions, together with respiratory safety and a delegated space to don and doff private protecting tools. Laboratory waste from wastewater samples which will include SARS-CoV-2 ought to be autoclaved and managed in accordance with BSL2 biosafety tips.


Warmth pasteurization of wastewater samples has been performed to cut back biosafety threat from bioaerosol-generating procedures throughout wastewater pattern processing. Contemplate the next when deciding whether or not to incorporate pasteurization:

  • The extent to which warmth pasteurization will harm the brief RNA fragments focused by PCR is unknown in wastewater.
  • Peer-reviewed stories have discovered that warmth treating respiratory specimens at 56ºC for half-hour causes a negligible change to the RNA measurement.
  • Some researchers have reported that heat-treating wastewater at 60ºC can enhance SARS-CoV-2 RNA measurement, however extra information are wanted to substantiate this impact.

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