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How 'Feluda' will detect COVID-19 in minutes

By SWARUPA DUTT
Last updated on: May 05, 2020 20:08 IST
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'This health emergency has brought a lot of people together with the common purpose of getting Feluda to play detective as quickly as possible.'
'As a scientist, if we can make a small difference in people's lives, we are happy'

IMAGE: A medic holds up a sample for COVID-19 rapid testing at the Rajiv Gandhi Government General Hospital in Chennai. Photograph: R Senthil Kumar/PTI Photo

Seven pairs of eyes watched the strip of paper.

60 seconds. 80 seconds. 100. Nothing. 120 seconds. Dr Debojyoti Chakraborty glanced at his mentor and senior Dr Souvik Maiti. 140 seconds.

A line appears on the paper strip.

A pronounced dark grey line.

Positive.

After two months of work, up to 20 hours some days, the team had finally hit the jackpot.

"Frustration is synonymous with a scientist's job. I think that's our state of being 90% of the time. It was a defining moment for us," Dr Chakraborty says. "We celebrated with pizza and cake."

Happenstance gave birth to Feluda.

The team, Dr Chakraborty says, was experimenting on a sickle cell anaemia test kit for the last two years.

In January, news of COVID-19, a novel coronavirus, filtered in and the team directed their focus on it to study how the virus mutated.

Despite being a government body weighed down by bureaucracy, the team did not wait for instructions.

"We didn't have patient samples at the time so we worked on synthetic DNA and RNA. Using a paper strip to get test results for a viral disease is completely new technology -- the chemistry, the biology involved -- has never been used in this manner. We had to file a patent immediately so that the Intellectual Property was protected," Dr Chakraborty says.

In early March, Professor K Vijay Raghavan, principal scientific adviser to the Government of India, stepped in and asked CSIR-IGIB to develop a cost-effective COVID test that gave error-free results quickly.

"When the PSA called us we already had a prototype for Feluda ready. Actually, it was almost 3/4th ready. We have conducted over 100 tests till now and Feluda has detected the virus every single time. But the invention had to be christened," says Dr Chakraborty.

"My wife, who is not a Bengali, suggested we call the test kit Feluda. She knows how obsessed Bengalis are about Satyajit Ray, so who better to name the kit after than his detective!"

He admits to being a huge Ray fan. "Jana Aranya is my favourite Bengali film, a Ray classic, the complexity of the characters makes me watch it several times, just as I have watched Rang De Basanti for the emotions it evokes."

Ray's detective Feluda has a Dr Watson, Jatayu -- and so as a tribute to the Master, the scientists have their own JATAYU, which is a Web server that assists Feluda by designing the experimental parameters.

Feluda is itching to get to work.

"We are excited at the possibilities Feluda offers. Apart from COVID-19 detection, it can be put to use to detect any diseases caused by genetic mutations like Duchene's Muscular Dystrophy, Sickle Cell anemia, Cystic Fibrosis and cancer causing mutations," says the soft-spoken scientist.

"The possibilities are endless. All we have to do is tweak the test kit a little," he explains.

In less than 30 days, Feluda, an inexpensive, robust and user-friendly COVID-19 test detection kit will be available for use in India.

It can transform the way we deal with the pandemic by enabling what the World Health Organisation and health experts world over have been asking of each government -- intensive and widespread testing.

A seven-member team of scientists headed by Dr Chakraborty and Dr Souvik Maiti of the Council of Scientific and Industrial Research's Institute of Genomics and Integrative Biology (CSIR-IGIB), took just two months to invent Feluda for use in COVID-19 detection; in normal circumstances it would take much longer.

Dr Chakraborty speaks to Swarupa Dutt/Rediff.com on the making of, and way ahead for, Feluda.

When can India use Feluda?

Feluda is ready.

But we are doing a large number of blind sample tests where results from Feluda are matched with the gold standard for COVID tests -- the Quantitative Real-Time Reverse Transcription Polymerase chain reaction (qRT-PCR) Test -- which is what is used in India and the world over.

Feluda, incidentally, is an acronym for FnCas9Editor Linked Uniform Detection Assay.

We are working with laboratories as well as hospitals and we are doing third party verification too.

The paperwork with the regulatory authorities is almost at an end.

CSIR-IGIB has discussed and signed technology transfer agreements with several interested pharmaceutical and biotechnology firms, who, as we speak, are scaling up production plans.

As far as we at the lab are concerned, there is a lot of R&D that is still going on to make it even better, even faster, more cost effective, more robust, error-free and a more accurate test.

You see, Feluda is a totally indigenous design that uses CRISPR-Cas9 technology and the model is very new in that it uses paper strips unlike most other testing kits in use elsewhere around the world, so we have to ensure it doesn't ever fail.

The virus is also mutating, it is changing its sequence, so our detection has to also incorporate these changes.

Will CSIR-IGIB make the test kits and market them?

We are an academic institution.

So production of the kit or scaling up of the kit will have to be done by biotechnology or pharmaceutical firms.

Over the last one month, a large number of firms have got in touch with us and discussions are at an advanced stage with several of them, for manufacturing the kits.

Once the kits are ready they have to be validated by ICMR and other regulatory agencies.

At our end the prototype is ready and we are satisfied with it, but like I said, we are still working to make it better.

Reports say your COVID-19 detection kit has an edge over existing tests for its low cost and quick, real-time results. How so?

Beginning with testing the RNA to the final detection takes less than an hour.

If you break that down, the RT-PCR step takes about 30 to 35 minutes, the incubation with the CRISPR Cas9 complex takes about 15 minutes at the most and the action on the strip takes just about two minutes.

That's less than an hour or an hour at the outside.

The regular qRT-PCR tests take between three to six to eight hours for results to show.

Feluda can also be tested in a multiplex format which is testing different samples together.

Another important feature of the test kit is the visualisation.

You can see the test result.

It is visual.

You do not need very complex instrumentation to read it.

The result is right there on the strip in the form of a line.

What is this line?

The result shows up on a paper strip like that which is used in a pregnancy kit.

There are two lines on the strip.

The strip is normally totally white.

When you dip it into a solution which contains CRISPR-bound DNA, a line will show up, which we call the control line.

This control line tells you that the strip is working fine.

Thereafter, if another line -- a grayish line -- called the test line develops, you know that the sample is positive for COVID.

If that line does not appear then you know the sample is negative.

It's that simple.

IMAGE: A medic collects samples for swab tests from a COVID-19 mobile testing van in New Delhi. Photograph: Manvender Vashist/PTI Photo

What are the components of Feluda?

The components include chemical reagents, buffers and plasticware required for pipetting solutions.

In addition, we currently need a very basic Reverse Transcription Polymerase Chain Reaction (RT-PCR) machine (which we are trying to eliminate in future versions of the test) and finally, the paper strips.

We do not use the qRT-PCR that is used in Covid tests worldwide.

You must remember, the end user is not the man on the street.

This is not a home testing kit.

The user is a technician working in a pathology lab or a diagnostic centre.

Yet, the test is designed in such a way that it does not require any complex instrumentation.

How do you take a sample for your test?

The sample collection is pretty much the same as in qRT-PCR tests -- through oral swabs or nasal pharyngeal swabs.

You extract the RNA from that sample and you subject it to an RT-PCR reaction, in which you convert that into DNA and then you amplify that.

What will Feluda cost?

Our estimates at the lab level works out to between Rs 500 and Rs 600 for a kit.

The whole aim was to reduce this cost as far as possible so that a patient can benefit from the economies of scale.

As compared to the test kits currently in use which cost between Rs 4,500 and Rs 5,000, this is way, way cheaper.

Will the price be marked up before it hits labs?

It is almost impossible for us to say at the lab level whether companies who are picking up Feluda for mass production will retain our price or not.

CSIR labs do not have the resources to make 1 million kits or 10 million kits, or whatever the number.

But it's unlikely there will be too much deviation from our price.

But you haven't answered the question. When will Feluda be available for use in India?

The time factor can only be answered by the biotech or pharma companies which will manufacture the kits.

But because the components of the kit are very easy to procure, it should take not more than maybe three or four weeks at the most, which means by the end of May.

Who has sanctioned this project?

The sanction came from the CSIR-IGIB where we work and the PMO.

IMAGE: Dr Debojyoti Chakraborty plays the sitar when he has the time. "My time for riyaz is at night, but it is strictly rationed due to work obligations." His favourite raag? Bilaskhani Todi, created, legend has it, by Tansen's son. Photograph: Ashish Gupta

How is the test different from existing COVID-19 tests?

So, there are two different ways by which you can detect a COVID virus presence.

One is that you try to detect the virus directly, which is the viral RNA and the second one is you try to measure the body's immune reaction, which is the antibodies that it produces.

The RNA test or the nucleic acid test is the methodology being used in India and the world over where you directly detect the presence of the viral DNA or viral RNA.

This is called the RT-PCR test.

Now, for our test we get RNA samples of the virus from the patient. We then convert this RNA into genetic material or complimentary DNA (cDNA).

Then we amplify it using a polymerase chain reaction (done on a simple PCR machine) and mix this with a complex CRISPR Cas9 protein.

The Cas9 protein specifically binds on to the DNA signature of the Covid-19 virus.

Finally, this mixture is applied on a paper strip. And as I mentioned earlier, a line appears.

The Massachusetts Institute of Technology, USA and the University of California, Berkeley use other CRISPR proteins (Cas12/13) for detection.

Now, the other test, called the Rapid Test Antibody test is a secondary reaction of the human body against an infection from the virus.

So it is not a first line test, it happens only if you have been infected with the virus.

You have tested close to 100 samples. Did you test on patients directly?

No.

When a patient or suspected patient comes to a hospital or a collection centre, a swab is taken by the doctor or the technician; it's put in a viral inactivation media which means it basically kills the virus.

Then this 'dead' sample comes to our lab where the RNA is extracted from that virus or that sample and then we start using Feluda from thereon.

How accurate is Feluda?

From the large number of samples we have tested I can say that our accuracy is extremely high.

I won't be able to disclose the figure because it is confidential, but it's better than the gold standard or the RT-PCR tests, that are being used widely.

I would always refrain from giving numbers because there is no test in the world which can give you 100 per cent results.

Even the gold standard test that is being used now, it is gold standard because they are supposed to give 100 per cent accuracy, but even in a lab setting where variables are controlled, one day it can give 60 per cent accuracy, another day 90 per cent accuracy.

So as a responsible scientist it is wrong to give out a number.

The issue is whether it is scientifically accurate. It is.

No test can say with 100 per cent confidence that every time it will give you 100 per cent results even if it has been validated at the lab.

Is a sample base of 100 good to go?

Yes it is, but that is also why I said there is continuous R &D involved to make Feluda absolutely perfect and what you get is the best possible level of detection.

Is there peer review done for your test?

Yes, absolutely.

The Feluda manuscript is on a preprint server (Biorxiv) and very soon an updated version will be available online.

The manuscript and its review in a journal is being done in parallel.

This is an emergency situation so all the processes are working in tandem.

We won't wait for one to finish and another to start.

In normal times, testing, validation, approvals and industry talk would take months and sometimes years to complete.

You say Feluda will be available for use by end May. Could it possibly have been done faster?

Most of us have been working day and night, without exaggeration, over the last two months.

Most of the work is being carried out by exceptional PhD students who have voluntarily stayed back at the institute and not gone home during this lockdown.

They stay up and work till 3 or 4 in the morning.

They are the actual heroes.

This health emergency has bought a lot of people together with the common purpose of getting Feluda to play detective as quickly as possible.

There are logistics teams who deliver patients's samples.

There are authorities in government who are working on permissions.

CSIR-IGIB has been has been behind us right from the security guard to the person holding the highest office at the institute.

It would have been a bit quicker actually but the lockdown has delayed processes a bit.

If there was no health emergency, the entire process of getting Feluda out in the market would take months and months.

Will you also be doing antibody tests?

No.

Our total focus and domain is on the nucleic acid test, which is the COVID test.

IMAGE: Health workers conduct door-to-door thermal screening at a slum in Kolkata. Photograph: Swapan Mahapatra/PTI Photo

Is there a fear of false negatives in your testing?

It's not a fear, it's just that you have to refine your work and ensure the test is as good as it possibly can be.

False negatives can happen in three ways.

One is if the test kit itself is not sensitive enough to detect the virus quickly and correctly.

In that case, the kit has to be refined more.

The second instance where you get a false negative is if the sample itself is not of the best quality, then the test may not work very well.

The third factor is human interface -- whoever is doing the test should have the requisite skills.

As scientists we need to ensure these variables are factored into the test kits.

We are very confident that our Feluda will work well with all conditions factored in.

What are the other diseases that Feluda can detect?

Feluda can detect DNA/RNA sequences including all possible mutations (disease causing or otherwise) in them and can also infer how the disease presents itself in an individual.

The basic technology will remain the same but kits would be optimized for every application.

In other words, Feluda can detect mistakes in DNA or RNA that manifests as disease. So the list is endless.

Rare genetic disorders, cystic fibrosis, cancer causing mutations, diseases that have multiple mutations (like COVID), blood group sensitivity, Duchene Muscular Dystrophy, Sickle Cell anemia.

The possibility is immense whether it is identifying a disease, diagnosing it correctly, or offering a foolproof test.

We are not the only ones doing this kind of thing in the world.

But in India I have not heard of anyone else using a paper strip.

But tests for these diseases are already available. So why bother?

Tests for these conditions are available but they are expensive and inaccessible in rural India.

So, for example, we were using a Feluda prototype to detect and diagnose Sickle Cell Disease.

The current tests for the disease are not genetic tests (a DNA or RNA test) but antibody tests.

The antibody test detects the body's response to an infection.

So with the existing antibody test you can identify if the person has the disease but you can't identify the carrier.

It is more difficult to find the carrier instead of a patient because the patient is anyway showing the symptoms.

The antibody test will not tell with a great accuracy if you are a carrier. Our effort is to identify a carrier because then you can save generations from the disease.

In India, tests for sickle cell anemia are almost never carried out.

In the extensive research that we have done, we have seen more cases in the rural populations in West Bengal and Odisha for instance, where almost every family has got a sickle cell patient.

There is no diagnosis, no screening and they don’t understand genetic loading.

There is also a social stigma attached especially with young women that if they undergo tests and if they are found to be a carrier, nobody will marry them.

So it is very important that screening can be done at a very early age. We don’t even need a blood sample, we use saliva, so it’s non-invasive.

A young child can be easily asked to spit.

We have developed a technology which will tell whether the particular genetic mutation is present in the DNA of a person or not.

Now, without Feluda, you have to do DNA sequencing.

It is not a very easy process and is only available in a few institutes in the country. And it's expensive.

So the reason we are using Feluda to test sickle cell disease is because the technique is cost effective and simple to use.

It can be used at small primary health centre in a village.

As a scientist, if we can make a small difference in people's lives, we are happy.

We are foot soldiers. We have to do the legwork to make that difference.

 

 

 
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