Detection of SARS-CoV-2 in raw and treated wastewater in Germany - Suitability for COVID-19 surveillance and potential transmission risks

Abstract

Wastewater-based monitoring of the spread of the new SARS-CoV-2 virus, also referred to as wastewater-based epidemiology (WBE), has been suggested as a tool to support epidemiology. An extensive sampling campaign, including nine municipal wastewater treatment plants, has been conducted in different cities of the Federal State of North Rhine-Westphalia (Germany) on the same day in April 2020, close to the first peak of the corona crisis. Samples were processed and analysed for a set of SARS-CoV-2-specific genes, as well as pan-genotypic gene sequences also covering other coronavirus types, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, a comprehensive set of chemical reference parameters and bioindicators was analysed to characterize the wastewater quality and composition. Results of the RT-qPCR based gene analysis indicate the presence of SARS-CoV-2 genetic traces in different raw wastewaters. Furthermore, selected samples have been sequenced using Sanger technology to confirm the specificity of the RT-qPCR and the origin of the coronavirus. A comparison of the particle-bound and the dissolved portion of SARS-CoV-2 virus genes shows that quantifications must not neglect the solid-phase reservoir. The infectivity of the raw wastewater has also been assessed by viral outgrowth assay with a potential SARS-CoV-2 host cell line in vitro, which were not infected when exposed to the samples. This first evidence suggests that wastewater might be no major route for transmission to humans. Our findings draw attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater.

Keywords: COVID-19; SARS-CoV-2; SARS-CoV-2 replication in vitro; Wastewater treatment; Wastewater-based epidemiology (WBE).

Graphical abstract

Fig. 1

SARS-CoV-2 specific RNA fragment detected by (A) M-gene and (B) RdRP RT-qPCR in the aqueous phase of untreated and treated municipal sewage. Error bars indicate one standard deviation of duplicate samples. Results of a single PCR measurement are shown for M-gene. For M-gene RT-qPCR, C_T values for the standard ranged between 8.6 (standard 1 = 10⁸) and 38.4 (standard 6 = 10²). Values of tested wastewater above C_T 39 were considered negative for SARS-CoV-2. Standard curve was calculated using Bio-Rad CFX Manager software with E = 61.4%, R² = 0.952 and slope = −4.813. Results of two independent PCR measurements of the same samples are shown for RdRp. For RdRP RT-qPCR C_T values for standard range between 18 (standard 1 = 10⁸) and 37 (standard 6 = 10²). Values of tested wastewater above C_T 38 were considered negative for SARS-CoV-2. Standard curve was calculated using Bio-Rad CFX Manager software with E = 96.5%, R² = 0.990 and slope = −3.408.

Fig. 2

Specificity and sensitivity of viral (A) M-gene, (B) RdRP-gene, (C) E-gene and (D) N-gene RT-qPCR with respect to control C1 sampled in July 2019 before the known onset of the pandemics in Germany, untreated sewage P5, P11, P13 sampled during the pandemics, SARS-CoV-2 control, and two other human coronaviruses HCoV-229E and SARS-CoV. Results are from wastewater samples measured in duplicates in a single PCR. Standard curve for M-gene PCR was calculated using Bio-Rad CFX Manager software with E = 90.8%, R² = 0.996 and slope = −3.565. Efficiencies of other primer pairs were not calculated by serial dilution.

Fig. 3

Correlation of reciprocal C_T values between M-gene and RdRP-gene detection.

Fig. 4

Alignment of sequenced amplicons of P12, C2, and C4. Sequences of wastewater sample P12 and retrained samples C2 and C4 were analysed using Geneious - Bioinformatics Software for Sequence Data Analysis and compared to the SARS-CoV-2 synthetic construct sequence (GenBank: MT458696.1). In green primers binding sites are indicated. Dots in the sequence represent sequence mismatches to the SARS-CoV-2 synthetic construct sequence. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

SARS-CoV-2 removal detected by RdRP RT-qPCR measured by the sum of aqueous and solid-phase of (A) untreated (AC-Inflow) and (B) treated sewage after ozonation. Results are shown as mean + SD of two independent PCR measurements from same sample material. RdRP RT-qPCR C_T values for standard range between 18 (standard 1 = 10⁸) and 37 (standard 6 = 10²). Values of tested wastewater above C_T 38 were considered negative for SARS-CoV-2. Standard curve was calculated using Bio-Rad CFX Manager software with E = 96.5%, R² = 0.990 and slope = −3.408.

Fig. 6

Viral outgrowth assay setup. Caco-2 cells were inoculated with either wastewater samples or with SASR-CoV-2 containing cell culture supernatant (positive control) or culture medium only (negative control). Cells were investigated daily by light microscopy for the appearance of a cytopathic effect caused by replication-competent SARS-CoV-2 infection for up to 10 days. The experiment was performed in a single replicate for four different wastewater samples (P2, P5, P11, P12) from April 8th 2020.

Fig. 7

Bioindicator load for each WWTP: Creatinine [CREA μmol/day], urea [Urea mg/day], benzotriazole [BZT μg/day], clarithromycin [CLA μg/day], diclofenac [DCF μg/day], metoprolol [MET μg/day]. For regression lines and coefficients of determination, see Supplementary Table S5.

Fig. 8

Correlation between the measured SARS-CoV-2 M-gene copy loads and the nominal numbers of (A) cumulative and (B) acute COVID-19 cases on April 8th, 2020, in the catchment areas of the 9 WWTP studied. In comparison, creatinine-corrected numbers of (C) cumulative and (D) acute COVID-19 cases are displayed. For regressions and coefficients of determination, see Supplementary Table S5.