Break from Movies and Covid-19 lockout

Buddhist Tibetan Prayer Flag (My Thunderbird's) - I got this while opening old boxes.

It’s almost two weeks now; staying at home without going anywhere outside. Previously, all these kinds of occasions arise due to accidents and broken bones. This time, fortunately(?) its different – COVID-19 lockout. Don’t know how much longer it will last. Nowadays spending free time watching Amazon Prime, Netflix and YouTube. Weekdays are filled with tasks from office.

Today I did something different. A break from watching movies. Cleaned up my study room. There were a couple of carton boxes filled with hundreds of A4 print outs and hand-written notes. Some of them are more than a decade old. A huge chunk of those notes is about subjects that I tried to study in between and dropped. But I kept all those, assuming someday it will be of use. But no, I didn’t even open or read those notes for at least the past three years. So, I threw all those out and cleared the space. At least I did something different today, rather than spending all the time watching movies.

If you are stuck at home due to lockout, try to do one thing differently in every day.

Sajeev 

Society hardly care about funding for research projects

To fight with bacteria and viruses we need help. This help comes in the form of vaccines and antibiotics. We frequently take these tablets just like candy. We most often fail to comprehend the hours of work behind that. Thousands of hours invested by researchers and millions of dollars spent by universities and pharma companies. Let me remind you, most of these universities are not super-rich and don’t have the kind of money, like European football clubs, to spend.

It will take more than a decade for a vaccine to move from the ideation stage to the commercial stage. During this time a lot of possible candidates will be dropped out of the race. This track is hard and often not appreciated at well.

Penicillin saved tens of millions of lives; BCG saves millions of people from TB; Hepa vaccines saved millions from Hepatitis diseases so as many other vaccines. But how many of us remember the people who were behind these vaccines. Some of us study their names just to get scores in quiz programs and competitive exams. We hardly care about those people. 

In medical profession, society pays respect to doctors and nurses. But those who run after vaccines and antibiotics rarely come to the limelight. We hardly know what happens to them and the programs they worked on. When an epidemic disappears, they are also forgotten (so as their programs).

Societies which care about every moment of their cinema, pop/jazz/eastern/western/Korean music stars, football, cricket celebrity’s life hardly cares about vaccine research and its funding; even though it’s critical for the very survival of humanity. 

When the Soviet Union broke up NASA lost funding, so as particle accelerators. By the way, NASA started getting funds again when China emerged as a challenge. It’s like administration cares more about beating opponents rather than what NASA really does. This problem is not limited to the US, it’s there in other countries as well. The same happened with vaccine research funding for SARS once that outbreak was over some years back.

I have no doubt that, once SARS-COV-2 came under control everything will go back to normal (or new normal). Whatever funding allocated to universities and labs for medicine/vaccine research will disappear... till next outbreak.

I earnestly request you; make sure that funding will always be on for medical research. Convince your representatives to pass bills; allocate grants etc. Because viruses are not going to give up. They will come back… let’s be ready.

Sajeev

PS: Everyone is interested in weapons research; taps for funds are always on to build better machines for killing other human beings.

How SARS-CoV-2 (aka Corona Virus, aka COVID-19) Test Kit work

SARS-CoV-2 stands for Severe Acute Respiratory Syndrome-Coronavirus-2

How Testing is Done?

I. Collecting the sample
Healthcare worker collects material from a patient's nose or back of the throat. This sample is later shipped in a cold container to the testing lab.

II. Separate RNA from everything else in the sample (RNA Extraction)

This can be done either manually or automatically.

a. Manually: Add chemicals and centrifuge the sample. During this process, RNA will go to a different layer.

b. Automatic:
Here instead of mixing the chemicals manually, samples are put into a diagnostic machine. The robot arm in the machine will do the pipetting process and mixes the liquids on several tests at once. According to Seegene (a South Korean company which is a lead producer of these kits), "this method takes only four hours to test samples from 94 patients - four times faster than the manual method. It also reduces the risk of human error or contamination."

III. Convert to DNA

RNA, we got from above step will be mixed with reverse transcriptase enzyme. This enzyme converts it to DNA (changing it from one strand RNA to two stand DNA). Later this DNA will be mixed with nucleotides and short synthesized DNA fragments called primers in a test tube.[1]

What primers will do?

Their job is to find specific segments of the viral genome and bind to it. What these specific segments are? These are the genetic materials from the virus. These primers should not mix with anything other than virus's materials. In this way selecting a primer is very important for these tests to work fine. 

IV. Polymerase Chain Reaction (PCR)

Well, these are not done by humans. Generally, these are done by PCR machines. This is a lab equipment which is used to amplify segments of DNA via polymerase chain reaction (aka PCR). "device has a thermal block with holes where tubes holding the reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps" [4]

When equipment is heated, DNA's double helix will separate into two strands. Now these strands are exposed. Now the temperature will be lowered, this time our primers will start doing their job. Primers will be locked into their targeted segments of the exposed DNA. How to identify these target segments? We will discuss it later. 

Now our enzyme uses these primers and starts building a complementary stance of DNA according to the exposed sequence. After 30-50 cycles a single DNA will be multiplied to hundreds of crores. This way we have enough DNA to detect something. [1]

V. Fluorescent dye

Now the question is how to detect viruses from this mix. This is not something we can identify using a naked eye or microscope. Here we use the concept of dye and tracing. Apply a fluorescent dye to the solution which produce fluorescence in the presence of certain agents; in this case, affected DNA. Higher the count of DNAs higher will be fluorescence. PCR machine contains an instrument that measures these light patterns which in turn tell us whether the sample is infected or not. [1]

Protocols

As a matter of fact, this procedure this very generic. RT-PCR is used to identify the specific genetic segment. What changes here is the components we need to add to detect each virus,

 - RNA extraction kit

 - Which PCR machine

 - Which primers to use

This combination is known as protocols. 

Well, that means someone must standardize and publish the protocols for these tests to work. For formulating the protocols, we need genetic sequences of virus. 

Sequencing

The world rushed to sequence SARS-COV-2 virus and did it successfully. Chinese sequenced and published the first full sequence of SARS-COV-2 genome in January. Other major labs across the world also did sequencing. Once the sequence is available to major labs across the world began designing, testing and publishing the protocols for detecting virus via RT-PCR. 

An extract from one of the papers,

"Sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions determined by rapid amplification of cDNA ends... 

The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%)

2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting beta corona virus. Although our phylogenetic analysis suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans." [6]

Protocol By CDC-US

Their protocol has four sets of primers.

N1, N2: Target unique regions of SARS-CoV-2 genome (a protein) that encapsulates and protects the genetic material of the virus.

Third Primer: Targets common gene for whole family of SARS-like virus. Remember nCov-19 (SARS-COV-2) falls in to SATS group.

Fourth: Targets the human genome. 

CRISPER (Clustered Regularly Interspaced Short Palindromic Repeats)

Companies like Sherlock Biosciences and Mammoth Biosciences are also trying to use CRISPR based technique to identify the virus. If this works then testing will be as simple as a pregnancy test using strips. [1]

Serological Test

This test looks for antibodies produced by humans against the virus. This will also tell us if a person is infected and then got cured. Even though this is less accurate than molecular tests, SARS-COV-2 also show up in this test.

Please note that the above article draws heavily from below mentioned reference links, especially Wired. In case you have more doubts and want to know more about this procedure please go through reference links given below.

Sajeev

References

1. Wired

2. WHO - Laboratory Guidance

3. CNN

4. PCR Machines

5. Novel primers and probes for detection of novel coronavirus in coronavirus

6. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding - The Lancet

7. Whole genome of novel coronavirus, 2019-nCoV, sequenced - ScienceDaily

8. SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) Sequences - National Institute of Health

9. Coronavirus disease (COVID-19) technical guidance: Laboratory testing for 2019-nCoV in humans - WHO

Guidelines Issued for Lock down By Ministry of Home, Government of India

Complete list of guidelines issued by the Ministry of Home Affairs, GoI for 21 days lockdown which started from March 25.

Ministry of Home Affairs, GoI

Corona (COVID-19): India needs to ramp up testing

SARS-COV-2 Testing (from OutWorldInData[5]). China is excluded from this list

Conducting more tests will help to identify all those who need care early. This will, in turn, reduce the mortality rate. For e.g. Germany (a country which do mass tests) has a mortality rate of just 0.3% whereas Italy stands at 7.9%. In Asia, South Korean test rate is 6150 tests/million people and the Indian one is just 6.8.

Tests/Day – An Overview [3]

South Korea:
South Korea has the capacity to test 15,000 people/day. As of last Friday, they tested over 3,16,600 people.
Test rate is more than 6,150 tests/million people. One of the highest in the world.

Spain:
Struggling with tests. Don't have enough resources to diagnose all cases. Just more than 39,000 tests conducted so far. This is going to create a lot of problems for them in the future.

France:
Has a capacity to carry out more than 2,500 tests a day. Priority is given for healthcare professionals and very sick people.

Germany:
Ability to conduct 12,000 tests/day

UK:
Currently 4,000 tests/day. Plan to increase that to 25,000/day.

Poland:
Currently 1,500 tests/day
China promised 10,000 test kits to Poland. Close to one week and there is no information on whether they got it or not.

Some of the numbers I gave above are a couple of days old.

Some companies which produces these test kits are, Thermofisher Scientific, Seegene, LabCoprp, Hologic, Danaher, Roche Diagnostics, Integrated DNA Technologies, Abbott Laboratories, Quidel, Qiagen, Sherlock Biosciences etc.

India

We are probably sitting blind. As per reports, India conducted just 17,000 tests till 22nd March. This in stark contrast to South Korea - whose population is much less than ours and a smaller country - which almost does that much in a day. We need to ramp up testing efforts rapidly. Otherwise, we won't even know how many people are affected. If the current number of affected people is correct, then we are very lucky. However, without doing proper coverage testing it is hard to say anything conclusively.

Test coverage is very much critical for containment and reduced mortality rate. There are examples that we can adopt. For e.g. South Korea came up with 'drive-through testing'. In this way, they are limiting the exposure of possibly affected people with the outside world[4] In South Korea, a company called Seegene has the capacity to produce 10,000 kits/week. Each of these kits can test 100 patients. This means, one million patients/week at cost of $20/test (1522 INR)

We need to accelerate testing. Government is taking some right steps in this direction. Last week union government gave its approval for private laboratories to begin testing samples and granted test licenses to a handful of companies to start trials. However, this is not enough. There are Indian companies that produced and gave testing kits to China and there are companies whose US arms already produce testing kits. We can't relay longer on the import of testing kits from Germany and other countries. If the virus spreads faster there, then they may not be able to export so much for us.

As per the latest updates, New Delhi approved a test kit developed by Mylab known as 'Mylab PathoDetect COVID-19 Qualitative PCR kit' for testing [1].

Till medicines are developed to stop SARS-COV-2, the one thing we should be doing is testing.

References:

1. Times of India
2. Bangladesh approves locally developed low cost Corona testing Kit - DD News
3. EuroObserver
4. Corona Virus South Korea Testing - CNN
5. Corona Virus Testing Data - Our World In Data

PS: By the way as per the reports, five days back 'Gono shasthaya Kendra' from Bangladesh also developed their own testing kit. Government of Bangladesh alreday gave the approval. Now its send for approval from WHO. They are employing serology-based technique. This kit will use 'Rapid Blot-Dot' technique and looks for anti- bodies created in the body in response to Corona virus infection. According to the Gano shasthaya Kendra, the kit will cost approximately 250-300 Taka (227-273 INR as per current rates).[2]