In these days of the COVID-19 contagion, Kerala's daily mood is invariably determined by sunset. It is at this time, by around 7pm that Chief Minister Pinarayi Vijayan meets the media after a review conference and provides an update on the outbreak.
A couple of new cases would have tested positive or, like in the last two days, there would be no new cases at all. Accordingly, there would be either anxiety or relief.
But for the Chief Minister to set the mood when the sun sets, a handful of virology labs across the state and the country and their specialists have to function in peak form round the clock.
Health Minister K K Shailaja said that at the moment seven labs in Kerala did this highly specialised virus detection function. The National Institute of Virology wing at Alappuzha Medical College was ready for testing almost at the start of the outbreak. On March 10, Kozhikode Medical College was ready. The very next day, on March 11, Thiruvananthapuram Medical College began testing at its Virus Research and Diagnostic Laboratory.
Barely a week later, Thrissur Medical College was ready to provide provide the first stage of tests. Indian Council of Medical Research (ICMR) then gave the green signal to Sree Chitra Tirunal Institute of Medical Sciences and Technology, Public Health Lab and Rajiv Gandhi Centre for Biotechnology.
Collection point
First step is to optimise the pressure on these labs. So the serum is collected from only those with symptoms. Sterile swabs are used to collect nose or throat serums and they are then placed inside what is called a 'viral transport medium', the serum's travel bag. These transport mediums are then protected within a triple layer packing. The name, age, sex, and ID number of the patient will be given in bold on top of this triple covering.
Along with the serum package should be provided a request form detailing the illness of the patient and his/her travel history and also the complete address.
Triple layer packing is done if the serum samples are sent to the nearest virology lab. If these samples are sent to a distant place, like the Pune Virology Institute, then the swabs will be kept inside thermocool boxes with dry ice pack facility. Once the samples reach the lab, the samples will be shifted to freezers. If they are to be kept for a longer time, then they would be placed inside minus 80 degree Celsius lab freezers.
Incidentally, only the samples of the first three medical students from Wuhan were sent to Pune.
First stage testing
Confirmation of the presence of the virus is done in two stages. First, a real-time reverse transcription polymerase chain reaction (RT-PCR) is done to detect the presence of E gene, which is a gene shared by all coronaviruses. It has also to be noted that coronaviruses are RNA viruses.
RT-PCR is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR). The PCR is a method widely used in molecular biology to rapidly make virtually infinite copies of a specific DNA sample allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail.
RT-PCR is done using reagents supplied by the Pune Virology Institute.
The reverse transcription, from RNA to DNA, takes about three to four hours. Then the DNA so produced is passed through a real-time PCR machine so that infinite clones of the DNA is produced. One of them is amplified and in about two hours it will be clear whether the sample has an E gene or not. If there is, the first test is recorded as positive. If not, the sample is negative.
A single lab can at the most do 40 such 'first stage' tests.
Confirmatory test
Now for confirmation, there is an additional test. This is a test to detect other types of genes, namely RdRP (RNA Dependent RNA Polymerase), ORF1b. It is said that two-thirds of the RNA of coronaviruses encodes RdRp, which are RNA synthesis materials, and two large nonstructural polyproteins that are not involved in host response modulation (ORF1a-ORF1b). Sars-CoV-2 has only the ORF1b polyprotein.
For this, the DNA sequence got through reverse transcription is once again passed through a real-time PCR machine. In about three hours the results will be clear. The presence of ORF1b will confirm COVID-19.
But this is not all. The test is repeated once more after 48 hours. The sample will be declared negative only if the first result is repeated. Then, the unused part of the serum samples will be destroyed using hypochlorite.
Each test will cost Rs 3000. But it is now done for free.
