NUS Community COVID-19 Wastewater Surveillance Programme
COVID-19 is an ongoing global pandemic caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2). Singapore has deployed numerous management controls to tackle the spread of COVID-19 pandemic, namely contact tracing, circuit breaker and travel restrictions.
Early warning through the detection of SARS-CoV-2 viral fragments shed by infected or recovering individuals, symptomatic as well as asymptomatic, in the stool and sputum would aid in putting in place prompt control measures to prevent COVID-19 spread in the community.
In November 2020, National University of Singapore (NUS) Environmental Research Institute (NERI) initiated the COVID-19 wastewater surveillance programme in NUS campus hostels and residential colleges. The programme was conducted in collaboration with Singapore’s National Environmental Agency (NEA) as a part of the national effort in the monitoring of SARS-CoV-2 in the community through wastewater surveillance testing.
Wastewater sample collection
A total of 118 auto-samplers has been deployed around NUS Kent Ridge campus for the wastewater surveillance programme. The auto-samplers are pre-set to collect wastewater samples at selected times during the day. The samples are sent daily to a Biosafety Level (BSL) 2+ laboratory at NUS Environmental Research Institute (NERI) for analysis.
Samples are collected at NUS halls and colleges including Prince George’s Park Residences, Utown Residences and Yale NUS College. Picture 1 shows an auto-sampler installed at a manhole or Inspection Chamber (IC) for the wastewater sample collection. Daily wastewater sample collection would usually commence around 9am to 10am. Samples would then be transported to the laboratory in cooler boxes by noon for analysis (Picture 2).
Picture 1: Auto-sampler at the Inspection Chamber
Picture 2: Sample bottles prepared for daily wastewater collection
Wastewater sample laboratory testing
The success of wastewater surveillance testing depends upon sampling as well as laboratory analysis. The BSL2+ laboratory has been scaled up to test a maximum of 80 samples per day, with turnaround time of less than 24 hours upon receipt of the samples. Experimental protocols have been validated for sample processing. These include sample preparation, sample concentration, ribonucleic acid (RNA) extraction and RNA measurement.
In the laboratory, wastewater samples are first heat inactivated in a water bath before diagnostic testing. Organic compounds and solid debris present in the collected wastewater samples that may interfere with the test are removed by centrifugation. Subsequently, viral fragments present in the wastewater sample are concentrated via ultracentrifugation (using centrifugal filters) or Polyethylene glycol (PEG) precipitation, followed by automated RNA extraction and quantitative polymerase chain reaction (qPCR) analysis. This type of surveillance testing requires that the RNA analysis be sensitive enough to detect low levels of RNA present in wastewater samples and hence, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is conducted. For our analysis, the N gene (nucleocapsid) of SARS-CoV-2 genome is used as the target sequence for the detection of the SARS-CoV-2 virus. The Pepper Mild Mottle Virus (PMMoV), an abundant virus commonly found in wastewater, is used as the process control for the analysis.
Picture 3: A researcher in Personal Protective Equipment (PPE) placing the wastewater samples in a water bath prior to the diagnostic test
Picture 4: A researcher dispensing aliquots solutions in a Biosafety Cabinet (BSC)