Definition

The SARS-CoV 2 virus concentration is the number of occurrences of the reference sequence identifying SARS-CoV 2 RNA in one milliliter.

The copy number is measured using qRT-PCR.

The SARS-CoV 2 virus concentration indicator is expressed as copy number per milliliter.

Method

Samples :

Monitoring of the overall carriage rate: 2 samples of 500 ml of wastewater are provided daily. These samples, averaged over 24 hours, come respectively from and according to the global constitution of the networks: from the segregated network (RS) and/or the combined network (RU)

Spatial distribution: Instantaneous samples of 500mL of wastewater are taken (within a 3-hour period) at identified sampling points. These allow the network to be divided into collection sectors and to identify local trends.

Analyses

The BMPM performs the analysis of the samples according to the following process

- extraction of the virus,

- amplification by RT PCR,

- quantification by comparison to an inactivated SARS-CoV-2 standard.

The calibration line used is as follows:

*The 2 x 105cg/ml inactivated SARS-CoV-2 control used is as follows:

EDX SARS-COV-2 Standard (lot 273020, expiration 31-01-2020) Exact Diagnostics, FORTWORTH.TX, USA

In our restitutions, 4 zones of concentration have been defined:

**Green:** Area where the carriage rate remains low (less than 0.4% of the population) corresponding to concentrations less than or equal to 160 copies/ml

**Yellow:** high carriage rate area (at least 0.4% to 1.2% of the population) corresponding to concentrations between 160 and 480 copies/ml

**Orange:** high carriage area (at least 1.2% to 4% of the population) corresponding to concentrations between 480 and 1600 copies/ml

**Red: **Very high carriage zone (at least 4% of the population) corresponding to concentrations greater than or equal to 1600 copies/ml

Definition of carriage rate

The WBE is a potentially robust tool for epidemiological monitoring of SARS-CoV-2 / COVID-19. Indeed, epidemiological monitoring of WBE can provide:

an anticipation of COVID19 outbreaks by 5 to 6 days,

an estimation of the population impacted by the epidemic by counting even asymptomatic cases,

a possibility to locate geographically the foci of infection

The COVID19 viral cycle admits an enteric phase that results in the release of virus in the feces (Wölfel et al., 2020).

The clearance of SARS-CoV-2 was studied in a group of 9 cases, and was 107 RNA copies/g of feces one week after the onset of symptoms and decreased to 103 RNA copies/g three weeks after the onset of symptoms.

For the interpretation of the data provided we will take a value of 107 RNA copies/g since we are monitoring a resumption of the epidemic.

The French National Society of Colo-Proctology (SNFCP) evaluates the average weight of seal discharged by an individual at 150 g/day.

We are therefore on an abscissa of 1000 for the table provided.

For example for Marseille :

The separate network implies a production of 70 x 106 liters of water per day for a population estimated at about 640,000 inhabitants. The wastewater production is therefore 175 l / inhabitant / day.

The combined sewerage system implies a production of 120 x 106 liters of water per day for an estimated population of about 360,000 inhabitants. The wastewater production is therefore 200 l / inhabitant / day.

Under these conditions, the detection threshold of our method (50 copies/ml) is therefore 0.125% of the population affected by SARS COV2 even in an asymptomatic way.

Corollary

We can also estimate a percentage of concentration in variants thanks to the following formula: