The ongoing “coronavirus disease 2019” (COVID-19) pandemic is caused by the “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2), a positive-sense single-stranded RNA virus. Together with SARS-CoV, this virus belongs to the species “Severe acute respiratory syndrome-related coronavirus” from the subgenus Sarbecovirus of the family Coronaviridae 1. The symptoms of COVID-19 include cough, respiratory problems, fever, aches and pains, fatigue, diarrhea and taste and smell disorders 2. SARS-CoV-2 can also cause severe complications, including death, mostly in the elderly or in people suffering from comorbidities, such as diabetes mellitus, obesity, cardiovascular diseases, hypertension, cancer, chronic kidney disease or immunosuppression 3,4. Due to the virus’ capacity for human-to-human transmission and a lack of immunity in the population, many governments decided to implement a variety of sanitary restrictive measures 5, such as curfews, lockdowns and travel bans. Viral RNA and viable SARS-CoV-2 are shed in bodily excreta, including sputum, saliva and faeces, with respiratory droplets as primary viral transmission route. Hence, the measures meant to control the disease were aimed at diminishing close person-to-person contact and people’s movement 5,6. Additionally, many governments implemented intensive contact tracing, testing and isolation 7–10, allowing to monitor the spread of COVID-19 epidemic and to reduce transmission. This set of measures has been supplemented since the end of 2020 by mass vaccination campaigns.
The gold standard for the detection of SARS-CoV-2 is reverse transcription quantitative polymerase chain reaction (RT-qPCR) on extracted RNA from nasopharyngeal swabs for individual diagnostics. By using RT-qPCR for individual diagnostics, in May 2021 already more than 30 300 000 positive cases were detected in EU since the start of the pandemic in 2019 11. However, the number of confirmed positive cases is likely an underestimation because this depends on the willingness of the people to get tested. Additionally, testing such a large population results in a very high cost and during some periods of high virus prevalence, the number of COVID-19 cases exceeded the testing capacity of public health systems. Furthermore, some people are asymptomatic or pre-symptomatic while still being able to transmit the virus 12,13 and consequently are often not tested.
Therefore, in order to rationalize the monitoring of the virus spread at the level of a country or region, monitoring of wastewater was proposed for surveillance of SARS-CoV-2 14–16 based on previous experience for early surveillance of disease prevalence, such as poliomyelitis 17,18. Indeed, SARS-CoV-2 genomes can be detected also in faeces 19,20 with reported RNA loads ranging from 0.55–1.21x102 copies/µL 21 and consequently may be found back in wastewater. Additionally, it was shown that SARS-CoV-2 genomes in faeces can still be detected several weeks after respiratory samples tested no longer positive 22. This suggests that the viral excretion may last longer in faeces. The presence of SARS-CoV-2 has been reported in wastewater and an association was observed between an increase of the RNA concentration in raw wastewater 14–16 and an increase in reported COVID-19 cases 15. This renders wastewater-based epidemiology as an important early-warning tool to monitor the circulating viruses in a community. Wastewater-based epidemiology also provides opportunities to estimate the genetic diversity, geographic distribution and prevalence 23,24. Furthermore, wastewater surveillance could offer an unbiased method not limited by the asymptomatic nature of the viral infections leading to the under-diagnosis of positive cases compared to the clinical surveillance 25. Finally, this surveillance makes it possible to assess the spread of infection in different areas, even areas with limited resources for clinical diagnosis or delays in test reporting 26. However, there are several limitations to wastewater surveillance. The excretion rate during the course of the infection determines the viral load in the sample. Consequently, the correlation between the viral load and the specific number of positive SARS-CoV-2 cases may be challenging. Additionally, inconsistent capture of spatial variability makes the correlation with the number of positive SARS-CoV-2 cases difficult. This is the consequence of travel and use of multiple wastewater systems in time. It is also due to temporal delays, inactivation during the wastewater transport process and/or dilution due to rainfall. Additionally, infrequent or absent clinical testing of possible positive SARS-CoV-2 cases also complicates the correlation 27. Furthermore, the virus detection and quantification can be limited due to the instability of the genome in wastewater, low efficiency of virus concentration methods and the lack of sensitive detection assays 14.
Although RT-qPCR methods are the standard for clinical and consequently often used in wastewater samples due to the availability of these methods, many drawbacks were reported related to the use of this technology. First, the tests are expressed in cycle quantification (Cq). The Cq represents the PCR cycle at which the sample produced a fluorescent signal above the background. These Cq values are laboratory- and instrument-specific and a calibration to a quantitative standard is necessary to determine the absolute viral load. Furthermore, Cq values are not directly comparable across assays or technology platforms due to differences in nucleic acid extraction methods, viral targets and other parameters 28, thereby affecting inter-laboratory harmonization in interpretation of the test results. Finally, RT-qPCR is not adapted for wastewater samples that often contain inhibitors that might influence the Cq values. This could affect the accuracy of viral quantification 29.
Reverse-transcriptase droplet digital PCR (RT-ddPCR), may offer an interesting alternative for the detection and quantification of SARS-CoV-2 RNA 30,31. Similarly to RT-qPCR, a target-specific fluorescent probe coupled with primers are used, which makes adaptation of existing RT-qPCR assays straightforward. In a ddPCR, a reaction is emulsified into thousands of nanodroplets of which a proportion does not contain the template molecule 32. The nanodroplets are used as unique and small bioreactors to amplify the template 33–36. At end-point, the number of positive droplets are digitally counted relative to the total number of droplets. Furthermore, their known volume while flowing through microfluidic devices allows absolute target quantification using Poisson statistics 37,38, which enables an easier comparison between different laboratories and tests compared to RT-qPCR. To the best of our knowledge, eight RT-ddPCR methods designed to detect SARS-CoV-2 were published, of which two are commercial kits designed by BioRad 31,39−45. The performance of these methods was tested using reference standards, and four of the methods were tested on clinical samples of infected patient’s throat and nasopharyngeal samples. Three of these methods were tested on wastewater samples. Moreover, four of these RT-ddPCR methods were tested on respiratory samples, and in some cases were found positive compared to the negative RT-qPCR results 31,41. Additionally, the sensitivity of the RT-ddPCR methods for the detection of SARS-CoV-2 has been described previously as comparable or even higher compared to RT-qPCR methods 31,39−41. This makes this technology interesting in case of a low viral load. Furthermore, inhibition can be encountered in some matrices, like wastewater. RT-ddPCR separates DNA, inhibitors and reagents in droplets and is an end-point measurement, only measuring after the PCR amplification. Consequently, a reduction in the biases linked to the inhibitors are often observed in RT-ddPCR 46, which makes RT-ddPCR an interesting method for wastewater surveillance.
In this study, a new multiplex RT-ddPCR method specific for the detection of SARS-CoV-2 was developed. This method targets two different parts of the genome of the virus based on sequences used in the RT-qPCR methods developed by Institute Pasteur 47 and Lu et al. 48 In silico inclusivity of the target was verified using 154 489 whole genome sequences, including several circulating SARS-CoV-2 variants. This novel RT-ddPCR method was in-house validated, including specificity and sensitivity assessments. Additionally, the applicability of the proposed RT-ddPCR method was investigated using clinical and wastewater samples.