Not all superheroes wear capes. Some prefer the lab coat. While most of us are trying hard to stay away from the dreadful virus, many microbiologists spend a big part of their waking hours around the SARS-CoV-2.
The seven-member team manning the Viral Research and Diagnostic Laboratory (VRDL) at the Madurai Medical College is among them.
The first step to fighting the virus is detecting its presence accurately. This is why laboratories like VRDL are critical to the containment exercise. And the pressure the staff of such labs are facing is massive. VRDL is nearly 20 years old. It became the eight facilities in the State to get an authorisation from the National Institute of Virology (NIV) to conduct the one-step real-time RT-PCR tests, on March 25.
Obviously, life is not easy for the VRDL staff these days. The lab, which usually functions from 10am to 4.30 pm, now works round the clock.
The staff rotate between three shifts. What more: the project staff and lab technicians do not get to go home.
They stay at a guest house inside the college premises. The administrative and technical heads, however, take turns to go home for short breaks.
The focus is on delivering accurate reports. The lab gets busier at nights these days as nearly two-thirds of samples come late evening. The facility handles swab samples taken from suspected COVID-19 patients in Madurai, Sivaganga, Virudhunagar and Ramanathapuram districts.
Currently, the lab performs three batches of screening tests and one batch of the confirmatory test, one after another, say GRH officials. So far, over 5,000 screening tests and 2,000 confirmatory tests have been done at VRDL. "Experts at the well-established VRDL need no special training to test for COVID-19," explains a retired microbiologist.
"This is because the genetic sequence of the virus was shared by the Chinese scientists as soon as it was isolated. It can now be replicated in any laboratory. RT-PCR tests are customised based on the genetic sequence of a given virus. However, technicians in a newly-established lab would require a month-long hands-on training."
Is there a risk?
There is a risk of exposure from the samples brought in for testing, affirms the retired microbiologist.
"All samples are, indeed, considered infectious at the time of receipt." GRH officials say they are keeping this in mind, and disinfecting the laboratory premises at least three times a day.
After testing, the vials, viral transport media (VTM), and test tubes are soaked in sodium hypochlorite solution for three hours.
They are then autoclaved before disposal. The protective suits are also disinfected, separately, in the same solution for three hours before being disposed of. Handling the waste from the lab is as critical as testing itself.
Wrapped in bags, the disinfected waste is collected in a box, sealed, and then handed over to bio-medical waste treatment facilities.
Special attention is being taken to collect waste from VRDL separately. The waste should be incinerated, to make sure there is no risk of transmission, say experts.
In the real-time RT-PCR test, the viral RNA extraction is done manually in a bio-safety cabinet.
The entire process takes about two hours. Later, a PCR tube containing the extracted RNA, mixed with chemical reagents is loaded into an automated thermal cycler that carries out an amplification process for two hours. The documentation (using pre-programmed software) takes another 30 minutes. Data entry, another 30-45 minutes.
Every time the test results are ready, reports are compiled and sent to the offices of GRH Dean, COVID-19 control room at the GRH, Deputy Director of Health Services, Joint Director of Epidemics (Chennai), ICMR-NIE (Chennai), and to the NIV in Pune.
Precautions in place
The lab staff have to wear PPE kits throughout the two-hour extraction process. Subsequently, it's enough if they use N95 masks and gloves.
The retired microbiologist says a total of 12 reagents are used per test. The thermal cyclers require at least a one-hour break every day for smooth operations.
Equipped with two functional thermal cyclers (that are usually imported from countries like Germany and Japan), 144 samples can be tested at VRDL in one go, reveals a senior GRH official.
While screening takes about six hours, confirmatory tests take another six hours. Only samples that test positive during screening go to the confirmatory test stage.
The VRDL is now training lab staff at government hospitals in Sivaganga, Ramanathapuram and Virudhunagar, where COVID-19 diagnostic labs will soon be set up. Sources say these new labs will begin the works only after the VRDL and NIV give a go-ahead.
RT-PCR test explained
RT-PCR is a combination of two laboratory techniques namely reverse transcription (RT) of single-strain RNA into double-strain Complementary DNA (cDNA) and amplification of specific DNA targets using Polymerase Chain Reaction (PCR). Though PCR, lakhs of copies of the viral DNA are made.
Different segments of the Coronavirus RNA sequence is amplified in different countries during the RT-PCR test.
RT-PCR is performed to measure the amount of a specific RNA. It is done by monitoring the amplification reaction by measuring the amount of fluorescence emitted with the passage of time during the test.
The cycling begins when the PCR tube containing the master mix (a mixture of reagents measured in microlitres) and the template RNA is placed in the thermal cycler. The first cycle is the reverse transcription. The second cycle is initial denaturation (loss of structure of protein through the exertion of external factors). The next 40-50 cycles are amplification.
If amplification of the DNA takes place, fluorescence is emitted by the amplified target DNA. A spike in fluorescence indicates the presence of the Coronavirus. If there is no increment in fluorescence with time, it denotes the absence of the target segment of the viral RNA, giving away a negative result.