How to sample
There are two sample collection methods for wastewater surveillance
- Grab: Grab samples can be collected rapidly and do not require automated equipment. However, grab samples may be less representative of community fecal contributions than composite samples. For untreated wastewater and sludge, grab samples represent a single moment in time and are highly influenced by daily fluctuations in wastewater flow and composition. At the treatment plant level, grab samples may provide similar concentrations to composite samples if the proportion of the community that is infected is sufficiently high. However, at this time, the minimum proportion of the community that needs to be infected for grab and composite samples to be similar is unknown.
- Composite: Composite samples are collected by pooling multiple grab samples at a specified frequency over a set time period – typically 24 hours for wastewater surveillance. You can collect composite samples of untreated wastewater manually or using automated samplers with refrigeration capacity that collect flow-weighted samples (e.g., one sub-sample per 200,000 gallons of flow). Continuous composite samplers (versus flow-weighted) may improve how representative the sample is of the community contributing to the sewer. Composite samples are considered more representative of community fecal contributions than grab samples.
Selecting a sample volume
The volume of sample to collect will depend on the sample type (wastewater or sludge). A 1 liter (L) composite wastewater sample or 100 milliliter (ml) grab sludge sample volume should be adequate for testing. The maximum amount of sludge solids that may be directly extracted is typically around 2 grams. The remaining sample volume (if any) can be used for repeat measurement or to assess biological variability.
The volume of sample that is concentrated and quantified will determine the lowest amount of SARS-CoV-2 RNA that can be detected. Concentrating more than 1 L of wastewater may result in poor recovery or viral signal inhibition. If using grab samples, consult with wastewater treatment plant staff to collect representative samples that capture peak times of human fecal loading and to understand the solids residence time for sludge.
How to safely collect, store, and ship samples
Sampling safety: There is no evidence to date that anyone has become sick with COVID-19 because of exposure to wastewater. Standard practices associated with wastewater treatment plant operations should be sufficient to protect wastewater workers from SARS-CoV-2. These standard practices can include engineering and administrative controls, handwashing, specific safe work practices, and personal protective equipment normally required when handling untreated wastewater. Beyond CDC recommendations for how to protect against COVID-19, no additional COVID-19–specific protections are recommended for workers managing wastewater, including those at wastewater treatment facilities.
Storage: Never store samples at temperatures higher than refrigeration (4°C). Refrigerate samples during the collection process. If possible, process samples within 24 hours of collection, as effective actionable wastewater surveillance relies on rapid data collection. Remaining samples can be frozen at -70°C for archiving. Avoid more than one freeze-thaw cycle. Preliminary data have shown potential loss of signal following freezing.
Shipping: When sending samples to laboratories, CDC recommends packing samples with cold packs (4°C) and using same-day or overnight shipping. Package and ship samples as Category B infectious substance (UN 3373), in accordance with the U.S. Department of Transportation’s Hazardous Materials Regulations and the International Air Transport Association Dangerous Goods Regulations.