My Experiments with WBE…

WBE which is a commonly used acronym for wastewater based epidemiology refers to an approach where community wastewater is analyzed to investigate the health risk factors associated with that community.

Traditionally this approach has been used by countries with properly devised sanitation systems for detection of illicit drugs in a community. The principle being -”If it's in your body it will be in your wastewater”.

During the first lockdown in Jaipur city, India, Dr Sudipti Arora came across this approach and wondered what if we can detect the COVID-19 virus in the community wastewater” It is this question which got me hooked onto this project.

Since then (May 5 2020) we have been regularly sampling the wastewater from communities all across the city to monitor the presence of this virus.

At the time there were rumors in which it was predicted that the virus might not survive harsh summers of Rajasthan and we might get rid of this whole virus in the blink of an eye. Therefore we started off with a very basic question- can we detect the virus at such high ambient temperatures from the wastewater samples which had a completely different organic composition than those being investigated in the aforementioned countries where the temperature remained quite low and the sewerage system was fully connected. Daily habits and food was also known to be different from the reported cases of Covid-19 detection by WBE. Our studies dispelled the might of virus degrading at high temperatures and proved that if sampling sites are chosen wisely the protocol that we could standardize was able to not only detect the viral presence through RT_PCR detection of viral genome but did so in about two weeks ahead for an area which reported a burst of case numbers making it a hotspot for the disease.

We could also show through this testing that the wastewater treatment plants were working adequately in virus removal. We also tested the hospital wastewater and found that the daily protocol of disinfection was working during the first lockdown. Since RT-PCR setups or any other sophisticated detection setups are not easily available everywhere we checked if the samples could be transported for the detection. We found that the samples could be processed for viral detection and quantification even after crossing nearly a thousand kilometers by road at 4 degree temperature. These were really great findings for several reasons: a) we found that WBE was useful even in the tier two cities of India. b) the results showed that early detection was really helpful in disease management c) the stigma of individual testing could be averted to a great extent. d)  since it could pin-point where to do testing in a city WBE could turn out to be a very cost effective measure and e) sample detections available at a distance can also be used f) since the patient testing only happened after the emergence of symptoms, WBE testing got a head start of this window (of 7-14 days which it takes to develop symptoms.) in prediction. Thus, even if there are no symptomatic patients in a community the presence of viral infection can be detected by infected feces dumped in the wastewater.

We further developed the sampling by increasing the sites and covering the dynamics of the whole second wave of the disease. WBE allowed our team to detect the rise two -three weeks ahead of the rise in cases in the city. After these findings we asked since the WBE approach can lead to detection of viral  genome and since that does happen way earlier than there is an actual rise in the case load, is it possible to use this advance detection window for sequencing and variant detection. This was not such a direct leap as the sample of genomic RNA of SARS-CoV-2 collected from patients is very different from that of the wastewater. We designed a setup where we  sequenced samples on a timeline of the whole second wave in jaipur. When we analyzed the results we were happy to find that data generated from such samples was meaningful and could detect the change in variant prevalence within the city during the second wave.

Currently based on these experiences we are trying to continually monitor the prevalence of different variants of SARS -CoV-2 virus in Jaipur city. We are grateful to the funding from DST-SERB which allowed us to do this monitoring quite regularly and also provided us access to various sites which made the resolution of our data even better.

Currently, we are monitoring 18 sites (Private and government regulated wastewater treatment plants) covering the city by collecting samples twice a week from each site. We also collect the treated wastewater to cross check the elimination of virus from treated wastewater. Our field team is doing tremendous work in collecting SOPs and taking records of onsite parameters.

The samples are transported back to our lab at 4 degrees (also called the cold chain transportation). Once in our lab the samples are tested for several more parameters , e.g. ph Temp, DO, conductivity etc. simultaneously the samples are processed for further steps or backup.

Further steps involve various sterilization methods and pre - processing steps such as centrifugation, filtration, etc. RNA is extracted from these preprocessed samples either by an automated RNA extraction machine or by KIT- Based method. The RNA samples can then be proceeded for qualitative or quantitative detection by RT-PCR technique. Samples with good viral load and RNA quality are sent for sequencing to our sequencing collaborators at NII, Delhi.

Once we have the final results relevant authorities are notified regularly. To sum up my view, wastewater based epidemiology is a very powerful approach and has great potential as an auxiliary sentinel system across the country.

-Aditi Nag, PhD

Assistant Professor

Dr. B. Lal Institute of Biotechnology