Sampling

At wastewater treatment plants, so-called flow-proportional composite samples of untreated wastewater are collected over a 24-hour period. This provides a representative picture of the water flow and virus levels across an entire day. In some cases, samples can be collected over longer periods, such as a week, to give an even more balanced view of virus occurrence. In our sampling programme, all treatment plants collect 24-hour composite samples, except Uppsala, which provides weekly composite samples. Samples are typically taken on Mondays.

During the sampling period, the total wastewater flow is also measured. This information is necessary to convert virus concentrations (genome copies per litre of wastewater) into the total amount of virus present during the period. After collection, samples are kept cold and transported to the laboratory in Uppsala for analysis.

Sample processing and analysis

In the laboratory, wastewater samples are processed to extract and concentrate the genetic material (DNA or RNA) of viruses and remove substances from the wastewater that could interfere with the analysis. A range of methods can be used for this step, including filtration, centrifugation, and chemical precipitation. At SEEC-SLU, we use a filtration-based method (Maxwell RSC Enviro TNA kit from Promega).

Once concentrated and prepared, the samples are analysed using reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). This technique enables us to determine the amount of genetic material from the different viruses present in each sample. At SEEC-SLU, we have RT-qPCR assays for influenza A and B virus, RSV, and SARS CoV-2.

Quality assurance

All analyses are subject to quality control, including the use of positive and negative controls and checks for PCR inhibition, to ensure reliable results. Samples that do not meet these quality criteria are marked as invalid and excluded from the figures presented in the dashboard. It is important to note that the methodology is still under development and is not accredited.

Handling values below detection and quantification limits

The limit of detection (LOD) is the lowest concentration of a target virus that can be reliably identified, while the limit of quantification (LOQ) is the lowest concentration that can be measured with acceptable precision. For each target virus, both the 95% LOD, i.e. the concentration at which detection is 95% probable, and the LOQ have been established for the RT-qPCR methods.

When results fall below these thresholds, they are treated in a standardised way. Samples with concentrations below the LOQ but above the 95% LOD are assigned a value equal to the average of the 95% LOD and the LOQ. Positive samples below the 95% LOD are assigned a value equal to half of the 95% LOD.

Population normalisation

To make viral levels from wastewater surveillance comparable over time and between treatment plants, it is important to estimate how many people the sample represents. The number of contributors can vary due to factors such as holiday periods, rainfall, snowmelt, or other circumstances that affect water flow.

A common approach is to first convert virus levels from genome copies per litre into genome copies per day, based on the measured daily wastewater flow. This value is then divided by the number of inhabitants connected to the treatment plant, giving an estimated number of viral genome copies per person per day.

An alternative, more dynamic method is to use a so-called population marker. This is a substance in wastewater that reflects how many people actually contributed to the sample, and ideally also mirrors how stable viruses are in the sewer system and during transport to the laboratory.

Although no perfect marker exists, the plant virus Pepper Mild Mottle Virus (PMMoV) is widely used in virus surveillance. Humans are not infected by PMMoV, but ingest it through food, particularly spices such as paprika powder and chilli, which are common in highly consumed processed foods. PMMoV is excreted in faeces and consistently found at high levels in human wastewater. By measuring PMMoV alongside the target virus, results can be normalised, which can improve comparability between different regions and over time.

In this dashboard, wastewater data are presented in three ways:

• Non-normalised: virus genome copies per litre wastewater
• Flow-normalised: virus genome copies per day per inhabitant
• PMMoV-normalised