People infected with the virus have been found to shed the virus in their stools. As most of the infected population is asymptomatic, sewage surveillance could be useful to detect the cases
In the recent past, the world has not witnessed a disastrous disease of this scale and magnitude. The coronavirus pandemic has taken a toll on human health and the economy world over.
The governments are responding to prevent the spread of coronavirus by ramping up testing, requesting people to isolate themselves, and by announcing nationwide lockdowns. However, the world has, so far, failed in containing the spread of this three-month-old virus even after putting in so much effort, probably due to the limitation of resources and a knowledge gap.
The identification and isolation of those suffering from coronavirus have been difficult due to the highly infectious nature of the virus. Coronavirus-affected people can either be symptomatic or asymptomatic. The symptomatic patients are screened based on the symptoms and their samples are sent for testing. But, in the case of asymptomatic cases, they will go unnoticed and, in turn, help the virus to spread rapidly. India, which is a populous country of 130 crore, is conducting 220 tests per million population – we have a very low testing rate, which is underestimating the actual number of infected people.
Hiroshi Nishiura and Tetsuro kobayashi from the Hokkaido University in Japan have found that there is a possibility of 33.3%-41.6% of the asymptomatic patient turning positive out of the total infected population.
The Indian Council of Medical Research (ICMR) also reported that out of the total number of samples tested, 69% cases are found to be asymptomatic.
The current method adopted for the screening of patients — based on symptoms — will leave a large number of asymptomatic cases undetected. By adopting this method of testing, we are currently seeing only the tip of the iceberg, as most countries are testing only those who are showing the symptoms of coronavirus.
Sewage is municipal wastewater produced by communities, which consists of human waste — stool and urine. This sewage is sent to the centralised treatment plants in order to remove the organic load, suspended solids, bacteria, and virus before discharging it into the surface waters.
This sewage essentially contains important health information of the community — such as intake of drugs, diseases prevalent in the area, etc. When a person is infected with a disease, like, for instance, the polio virus, he/she will shed the virus through faeces or urine. Similarly, when illicit drugs are consumed, traces of those drugs will be present in the urine or faecal samples. It is important to select the specific markers of a virus or drug which we need to identify. When we will test for the virus, the genetic sequence of the virus (RNA) will act as a marker as it is unique to that virus. That can be identified in a testing facility.
Previously, sewage analysis was used in the identification of the effectiveness of the polio vaccine. The National Polio Surveillance Programme began in India in 2001 in Mumbai. Under this Programme, environmental samplings were taken from sewers in Delhi. They found two strains of polio viruses in circulation based on the genetic sequence analysis from the sewage sample. The polio virus was infecting 1 out of 200 people affected.
The polio eradication team increased the vaccination in migrant workers and communities. This increased vaccination helped to mitigate the virus. Identifying specific strains also helped in developing a specific divalent vaccine, which was more effective compared to the previous trivalent vaccine.
Finally, India became a polio-free country in 2014. Now, many countries are polio-free by adopting this practice of sewage analysis to identify community outbreak.
Many governments are using sewage analysis as a tool for monitoring drug levels in the community. The Sewage Analysis Core Group (SCORE) has been involved in sewage analysis for the past 10 years. They are active in 86 cities in 29 countries. They analysed samples from sewage which are urinary biomarkers of the drug. Samples were also tested for secondary metabolites, which are found in urine, due to the breakdown of the parent drug. In the case of cocaine and cannabis, the secondary metabolites that were monitored were benzoylecgnonine (BE) and THC-COOH.
Raw sewage samples are collected from the sewage treatment plants. A virus contains a single strand of genetic material known as Ribonucleic acid (RNA). The RNA is isolated from the raw sewage sample using the standard RNA extraction kit. After isolating the RNA strand, the single RNA strand is made into a double-strand by using primers and polymerase enzymes, which bind the complimentary DNTPS to the RNA strand. This produces the double-stranded C-DNA.
Once the C-DNA is synthesised, it has to be kept in thermocyclers to generate multiple copies. In thermocycler, the CDNA will denature into two single- stranded ones at a high temperature. The temperature is then lowered for the binding of the primers. Further, the temperature is increased to generate the complementary strands for each of single-strand C-DNA. This cycle will be run 25-40 times to amplify the DNA. Later, TaqMan probe is used to measure the fluroscence. Taqman probe of a reporter dye will show fluorescence when an enzyme cleaves the reporter side of the dye during the PCR process. As more and more C-DNA is generated, more fluorescence is generated.
The controls, such as n-COVPC, NTC, HSC, are used for monitoring failure of analysis. The failure of performance in any of the controls invalidate the results.
The coronavirus loads from a patient’s stool have to be quantified. A study on the infected patient will arrive in the average viral load shed in faecal sample of the patient. The average viral shedding, multiplied with the population in the community, will give you a rough estimation of the total fecal shedding if the total population is infected. Comparing the viral load quantified at a Sewage Treatment Plant (STP) to the population and average viral shedding data will give an insight into the number of infected people in the community.
This research will help in assessing the number of infected people in the community. It will help the government to get real-time data on community spread, thus providing an early warning sign, deciding on future lockdowns and also relapse of the virus.
The researchers at the Massachusetts Institute of Technology (MIT) found a significantly higher number of cases as compared to the clinically-confirmed cases, based on an initial study.
They collected samples from major wastewater treatment facilities from March 18-25. They have used RT-qPCR for the estimation and quantification of viral loads per milliliter of the sewage sample. All the samples tested between March 18 and 25 showed positive results. They have taken samples from the northern and southern catchment area for all the dates. They identified the number of copies present per milliliter of the sewage. They have estimated, on an average, 100 viral particles per milliliter of sewage sample. It shows that 5% of the population was affected compared to the clinical estimation of 0.026% of the confirmed cases. This indicates the community spread has taken place in that area.
This information is valuable to identify the hotspots or to monitor the reduction or increase in the level of infection in the municipal limits of every city. The sewage screening will help to identify the virus in circulation. This will, in turn, help the government in containment and planning mitigating measures effectively. It will also help to identify the effectiveness of measures, such as lockdowns and seal down.
In India, every district has a 2-3 centralised Sewage Treatment plants (STPs). The ICMR should lay down guidelines for the quantification of RT-qPCR and population estimation to COVID analysis-approved laboratories.
The district administration, along with the Municipal City Corporations (MCCs) should take up the responsibility of analysing the STP samples from that Centralised Sewage Treatment (STPs) and also Hospital STPs to identify communities which are at risk.
These samples must be tested at the COVID- approved labs. It will provide a real time snapshot of COVID hotspots district/communities in the state. Initially, a pilot-scale has to be started from the already identified hotspots.
Adarsh Manjunath is a Process Engineer, water and waste water.
(Views are personal)