How to detect COVID-19 in wastewater

How to detect COVID-19 in wastewater

What is COVID-19?

The disease COVID-19 (“corona virus disease 2019”) is caused by an RNA virus, SARS-CoV-2. So far it is known that this virus emerged in Wuhan in China in December 2019. Fever, cough and difficulty breathing, are the most frequent symptoms, which can lead to the onset of pneumonia. More recent studies report other less common symptoms such as diarrhea, nausea, vomiting and abdominal discomfort.

Why should we monitor COVID-19 in wastewater?

Studies carried out demonstrated the presence of SARS-CoV-2 in stool samples from patients with COVID-19, evidencing the release of viral particles through the stool.

In some cases, fecal samples were positive for SARS-CoV-2 and negative for samples from the respiratory tract. This study, among others, shows that in feces it is possible to detect and diagnose infected patients early, leading to anticipation in isolation and preventing the transmission of the disease in the early stages of clinical manifestation.

Besides, we have cases of transmission by asymptomatic patients. Although they are asymptomatic, they continue to transmit the virus to other people and release the virus through the stool. Ultimately, the viral particles reach sewage treatment plants.

In this way, the detection of the virus in wastewater becomes relevant when we intend to monitor in advance an outbreak of COVID-19.

How can we do it?

1 - Sewage samples
The first step is to collect the sample. The collection must be made in the influent, preferably composed samples. They must be refrigerated during the entire process, from collection to transport to the laboratory.

2 - Processing of samples in the laboratory
In the laboratory, the samples undergo a concentration process that allows the concentration of viral particles (if present) and to allow their detection. In this step, it is intended that the RNA of the viral particles is isolated from the samples using the following steps:

  • Viral particle precipitation;
  • Extraction / Purification of viral RNA;

3 – Real Time RT-PCR
The detection of the virus is done through a diagnostic test, called Real Time PCR. This technique is relatively quick, sensitive and very specific. It combines amplification targeting small fragments of the virus genome for detection with specific probes.

Potential risks of analysis

Negative results do not rule out the possibility of infected individuals. There are several factors for the existence of false negatives in the analyzes carried out on wastewater, such as:

1. Sample collection: the moment of the collection was not adequate, taken too late or too early, containing few or no viral particles in the samples; This problem can be mitigated if several samples are collected over time;

2. The specimen collected was not handled and / or properly sent to the laboratory. This problem can be mitigated if the sample during collection and transport is refrigerated;

3. Technical reasons inherent to the test, including mutations in the viral genome or inhibition of PCR amplification reactions. This last point is taken care of by using the ViroReal® Kit SARS-CoV-2 & SARS, which detects a highly conserved region of the N gene present in all SARS coronaviruses. This approach allows for universal detection of all known strains of SARS-CoV, including SARS-CoV-2 and SARS without discrimination between strains. The discrimination can then be confirmed by using the ViroReal® Kit SARS-CoV-2;

WU, Yongjian; GUO, Cheng; TANG, Lantian; et al. Prolonged presence of SARS-CoV-2 viral RNA in faecal samples. The Lancet Gastroenterology and Hepatology, 2020.
KUMAR, Manish; PATEL, Arbind; SHAH, Anil et al. First proof of the capability of wastewater surveillance for COVID-19 in India through detection of genetic material of SARS-CoV-2. medRxiv, 2020.