Mumbai’s first genome sequencing lab aims to break Covid code
Thirty-year-old research scientist Vidushi Chitalia loads a cartridge into the sequencing machine with a large display screen. The cartridge contains all that is required for carrying out genome sequencing — 20 microlitres of barcoded samples of genetic material from Covid positive persons pooled into a tiny chamber.
It’s taken three days for the team, also comprising research assistants Reem Nomani (26), and Sanika Shirsat (26), to prepare the samples that made it into the machine: a select bunch of swab samples from Covid positive persons in Mumbai was first treated to inactivate the virus in a Biosafety Level 2 area; the RNA or Ribonucleic Acid (it translates genetic material to protein) was extracted and converted to complementary Dioxyribonucleic Acid (cDNA); this in turn was amplified, and cut into smaller pieces (a process called fragmenting) since the sequencer is designed for a very specific size of DNA fragments; these were tagged with linker sequences (to help link the samples for further steps like barcoding), cleaned of any impurities and barcoded for the machine — a white box slightly bigger than a microwave.
These sequencing will help identify the variants of the Sars-CoV-2 virus. And great care has been taken to select which ones will make it into the sequencer. The samples were taken from travellers arriving at the city airport, patients who had moderate to severe infection after receiving both doses of the vaccine, people who had been re-infected with Covid and those who were admitted in an Intensive Care Unit for a prolonged period of time.
“We want to focus on certain categories of patients to understand how variants impact the nature of the infection,” said Dr Jayanthi Shastri, who is at the helm of the genome sequencing facility housed inside the Kasturba Hospital’s molecular diagnostic laboratory. The lab, which works under the civic-run BYL Nair Hospital, has been operational since 2007 to study pathogens causing H1N1, dengue, and leptospirosis, among other diseases.
Last week, a batch of 376 samples of Covid-19 patients was processed and the Brihanmumbai Municipal Corporation (BMC) announced that 304 samples tested positive for the Delta subtype of the Covid-causing Sars-Cov-2 virus. None tested positive for the Delta Plus variant, a variant of concern (VOC) in India. Two samples were of the 19A subtype, four were of the 20A subtype and the remaining 66 were original strain of the coronavirus.
This was the second batch to be studied in what is Mumbai’s first facility to study the Covid disease-causing virus. The work being done here is crucial: it will help address the gaps in our understanding of the pandemic. For instance, do variants of the virus, such as the Delta variant prevalent in Mumbai and the rest of the state, play a role in breakthrough infections, re-infections or long Covid? This in turn would help civic and state authorities to decide the best course of action.
“If the SARS-CoV-2 continues to thrive, it will keep mutating,” said infectious disease specialist Dr Om Shrivastav, who is also a member of Maharashtra’s Covid-19 task force. “Genome sequencing will be our key tool to track the mutations and prepare strategies to tackle them. The changes in the virus could be more vicious or milder, but having that knowledge is the only way to combat it and stop it from spreading rapidly,” he said.
The laboratory, which was inaugurated on August 4, is not part of the Indian SARS-CoV-2 Genomics Consortium (INSACOG) — a network of 28 laboratories across the country tasked with studying the genomes of the various variants that emerge through the course of the pandemic — but its work contributes to the larger knowledge base being created about the coronavirus and its variants.
The second batch mimicked the findings from the first: A few days after its inauguration, 188 barcoded samples were sequenced. The BMC announced that nearly 65% of these samples had the more contagious B.1.617.2 or the Delta variant. None of the 188 samples tested positive for the Delta Plus variant. Out of the 93 patients from Mumbai in the first batch, 54 had needed hospitalisation, while 40 patients showed no symptoms at all. Of the Mumbai cohort, 27 patients had taken both doses of their vaccine, and 20 had received their first dose. Only four patients out of those who had been fully vaccinated needed oxygen support.
India is currently sequencing only 0.7% of the positive samples, Shrivastav said. “Ideally, for a dense country like ours, we should be sequencing at least 33% samples. Europe, UK, parts of Australia and New Zealand have been doing a lot more sequencing,” he said adding that cities like Mumbai should have many more genome sequencing facilities.
The genome sequencing process is a lengthy one. Smaller portions of the Covid-19 samples known as aliquots collected from across the city arrive at the laboratory every day in a cold chain. These samples are stored at –80 degrees Celsius to ensure that the RNA does not degrade during the process. The sequencer — Kasturba facility has two such machines — has a capacity of 376 samples each. In order to utilise the machine optimally, the team waits till they have a pool of 376.
The team of three handles the samples in a transit room equipped with an exhaust system with a virus burning unit that is meant to trap and destroy the pathogen in case of a leak. This room and other biosafety cabinets, where the cDNA is created and amplified, are a Biosafety Level (BSL) 2A [equipped to handle pathogens with a little more than moderate risk]. So, all researchers must follow strict Personal Protective Equipment (PPE) donning and doffing protocol while working in the transit room.
After inactivating the virus, the RNA is extracted. The researchers wear a laboratory coat, head cover, mask and gloves instead of a full PPE kit to extract the RNA and conduct a Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test to confirm the presence of the virus. “Only the samples that have a cycle threshold (CT) value below 30 are selected,” said Chitalia. “The CT value helps measure the viral load,” she said.
The extracted RNA is converted into complementary DNA (cDNA), which is amplified in a biosafety cabinet, tagged and fragmented. Once the impurities are cleaned, each sample is barcoded and pooled before being loaded into the sequencer. The cycle in the sequencer takes another 48 hours after which the data is relayed to the computer.
The server has preloaded information on all the variants identified by the World Health Organisation (WHO). Besides looking for the existing variants such as Alpha, Beta, Gamma or Delta among others, the researchers also keep an eye for newer mutations, while they analyse the data.
The two sequencing machines and server cost ₹6.5 crore. They were donated to the hospital by American biotechnology company Illumina. The company also bore ₹1.38 crore GST and customs levied to get the machines to India from Singapore. Other pieces of equipment such as extraction system, thermal cyclers, deep freezer, ice-making machine, pipettes costing nearly ₹4 crore were funded by the ATE Chandra Foundation. An alumnus of BYL Nair, Dr Mehul Mehta who is based in the US, facilitated the donations, said Shastri explaining how the lab came to be kitted with the machines.
“We started the discussion in May and it all moved so quickly,” said Shastri, who believes that the genome sequencing laboratory has an important role to play in the city’s public health landscape.
“We can use the facility for any pathogen. For instance, dengue is not very conclusively identified by PCR and whole-genome sequencing can prove to be beneficial for us. Or let’s say that the H1N1 virus becomes resistant to the oseltamivir drug in the future, genome sequencing will come handy then too,” she said.
As Chitalia, Nomani, and Shirsat work in a high-risk environment, vaccination is mandatory. The three also monitor themselves closely for even the slightest of symptoms. None has developed any to date.
The researchers at the laboratory want to compare the 2021 variants with the 2020 virus identified as the Wuhan strain in the hope of answering several pressing questions. Why, for instance, do the variants behave differently? Last year, hospitals saw a few instances of mother-to-child transmission but the virus did not have a serious impact on babies.
“Are the 2021 variants affecting children more or causing increased mortality and morbidity in the pregnant women and the children? This is something that we need to check,” Shastri said. That’s why the samples for genome sequencing are selected carefully.
Soon after the Kasturba facility opened, a circular explaining the criteria for selecting the samples were sent to all the wards and hospitals: in addition to breakthrough infections and re-infections, the laboratory also wanted samples of patients who were immunocompromised, as their immunity does not kick in for the virus to be cleared out completely. In such cases, there is a possibility of intra-host variation. “Put simply, the virus undergoes mutation within the host,” said Shastri adding that medical experts are also interested in the host genomics of patients who suffer from longer hospitalisations due to Covid.
But host genomics is a long-term plan at the moment. The laboratory is now focused on utilising its maximum capacity to sequence the samples from across the city and turn around the reports within a short span. Until now, samples from Mumbai were sent to Pune or Delhi for genome sequencing and the results would take anywhere between several weeks to three months. “There is no point in carrying out contact tracing if the reports come so late,” said Shastri.