Insights Of Covid-19 Tests

Soumyadip Biswas

1st November, 2020

Abstract

On 21st Dec 2019, the outbreak was identified in Wuhan, China and the World Health Organisation (WHO) declared a Public Health Emergency of International concern on 30th January and a Pandemic on 11th March of 2020 respectively.  People of Wuhan who were experiencing pneumonia-like symptoms were diagnosed with Covid19 (CoronaVirus Disease 2019) caused by a zoonotic (human-infecting) virus of the CoronaVirus family - SARSCoV-2 (Severe Acute Respiratory Syndrome CoronaVirus 2). On 11th Jan, scientists from China published the genome of the virus SarsCoV-2-Hu-1(29983bp). Studies are further done and results were made free to understand the transmission, behavior, and prevention of the virus. Tests were necessary for tracking the spread of the virus, the development of a vaccine, and corresponding control measures.

1. Introduction

Every individual in society is susceptible to the disease and also shows symptoms differently during a long incubation period of 14 days. We need to ramp up testing dramatically to slow the spread of the virus. From past experiences, humanity had learned to prepare for unprepared challenges. Mathematical modeling tools and simulations with Tests had proved to be lifesaving. Tests are needed for tracking the spread of the virus, indicate and isolate infected individuals, development of vaccine, basis for designing suitable measures to control the pandemic; making it from a short duration pandemic with a huge number of infective with more deaths - to a longer pandemic with a slowing rate of infection. So, limited medical services are available to the most drastically improving recovery rates.

2. Sample collection

Recommended by CDC (Center for Disease Control and Prevention) are samples from the upper respiratory tract: nasopharyngeal specimen - a swab from the back of the nasal cavity, oropharyngeal swab – swab from the back of the throat. Both of these connected areas come in direct contact with inhaled air, and cell lining of the areas expresses the key receptor on their surface ACE-2 (Angiotensin Converting Enzyme 2) which is a friend-in-disguise-aid to the viral entry. Hence, viral load is thought to be higher than anywhere else, which is essential to get detected by the test.

The virus is a respiratory one, another best place to look for it was the mucosal fluid. SalivaDirect was given Emergency Use Authorization on Aug 15th by FDA, it needs the test subject to spit into a tube, the process excludes the involvement of a worker & few steps needed for processing, making it resource-saving and safe and hence is also very cost-effective in a way.

 

3. Types of detection

Tests are designed in a way to directly detect the viral nucleic-acid: qRTPCR, LAMP during the initial course of the infection when the viral load is high. (Indirect) Detect antibodies against the virus: antibody (serology) test. Other tests: cell fee assay, aptamers, are out there, these are selected based on sensitivity, specificity, and ease of use.

Tests: outline: a mixture of reagents added with a sample to be analyzed> viral genome is copied many times (signal amplification) > detected by color change/ fluorescence.

3.1. qRT-PCR

Qualitative Reverse transcriptase Polymeric Chain Reaction is a modified version of a conventional PCR. Since the genome of SARS CoV2 is ssRNA (single-stranded Ribonucleic acid), it needs to be converted to ds DNA (double-stranded DeoxyRibonucleic acid) for getting copied by PCR protocol, this job is done by Reverse Transcriptase enzyme. Qualitative/real-time is added because the result of the test could be visualized immediately after the reaction is over. This is achieved by using an additional fluorescent-labeled probe to the standard PCR protocol, which is a short complementary DNA fragment targeted to bind somewhere in the middle of the viral genome. In presence of the viral genome, the probe binds to its complementary part, and while being copied by a DNA polymerase enzyme, the fluorescent tag is detached from the probe released into the solution where it fluoresces. A fluorescent signal could be detected by the eye/ detector. To increase specificity and sensitivity CDC regulated tests check for: a. human sample - needs to be positive, b. two viral surface proteins N1 and N2 interpreted as if for anyone N protein is positive the result is inconclusive and accepted only when both are positive.

3.2. LAMP

Loop-mediated Isothermal amplification does not match with its acronym. This method has a much complex reaction mechanism, and forms long concatemers of DNA at one set temperature (isothermal), then small fragments of DNA in PCR in a thermocycler. The protocol involves 6 primers, which makes it highly specific, is also cheaper than a fluorescent-labeled probe for qPCR. This test could be done with raw samples. A high amount of hydronium ions are released into the solution while the reaction proceeds decreasing the pH of the solution, results could be visualized in real-time by pH indicators like cresol red, phenol red.

3.3. Antibody 

Our body fights disease by raising antibodies in response to a specific antigen (antibody generator) detected by the immune system which is mass-produced by plasma cells. We produce 5 different types of (Ig) immunoglobulin proteins (antibodies). At the initial stage of infection, we have a peak in IgM antibodies after someday IgG takes over. These are the two main antibodies tested for.  The antibody test kit has 3 main sections: a. a sample port, from subject 1-2 drops of blood is added with a few drops of a buffer solution that will carry the antibody in the blood to > b. an antibody present in blood flow through the section containing Gold Rabbit IgG conjugate (control), Gold CoVid S protein conjugate (binds to an antibody against CoVid). > c. it gives three signals: C (control) – always positive for the proper kit it contains anti-rabbit IgG. G – Anti-human IgG, M – Anti-human IgM either showing a positive line shows a positive case.

Results:

Where true Positive and negative we are looking for, false positive and negative are a major issue. There had been cases of faulty probes and contamination of samples. Hence now companies producing test kits are FDA (Food and Drug Administration of US ) approved and standardized by the CDC. There are cases of false positives with LAMP tests due to contamination, and false-negatives associated with qRTPCR, antibody tests.

 Failing of tests:  Viral load may not satisfy the calibration level of the test, vary with time, and area of sample collection, a time window of 4 – 10 days is good for the nucleic acid test, antibody test after that period.

4. Ways of testing

4.1 Drive Through testing

Health care professional take sample using appropriate wearing PPE (Personal Protective Equipment) - Hong Kong

 

4.2 Pooled testing

Sample from 64 patients is simultaneously run, proceeded only if a positive result is found, this is further optimized, and the infected could be detected in a population with fewer tests. Origami assays test 1122 patient samples by 93 assays only – West Bengal, Punjab, Germany, South Korea. 

5. Contact tracing:

It’s exactly tracing all individuals who came in contact with the infected person before it got detected, being a step ahead of the virus. Such individuals are instructed to self-quarantine and are tested. Smartphone apps come in handy for the process and are effective with only 60% of the population considered for it.

 

6. Mathematical Models:

SIR model, Travelling wave model and Simulations

Devices built on available data, to predict the future had changed the way we fight the pandemic. Most abstract mathematics had allowed making the most sense, answering questions: 1. Will disease spread? 2. Total number of infected in a population at a given time. 3. Maximum infective in a population. 4. How fat disease will spread?

The SIR model split the population into susceptible, Infected, and Recovered. Assuming the total population, infections, and recovery rate to remain constant differential equations are constructed with respect to time.  The traveling wave predicts the spread of the disease in a population like a wave traveling in a medium, here the equations are adjusted to space variables. These models are highly dynamic and are modified to fit real-life scenarios. Stimulations/computer models are the visual representation of this dynamic interplay. The basic reproduction number (R0) is a constant of the SIR model, its value tells us if one person in a population has the disease how many susceptible people will he infect, it’s about 3-4 for SARS CoV2.

7. Prevention

The most effective preventions are those we already know of, the math behind wearing a mask is more powerful than our intuitive sense. Because a mask works both ways, protecting others from you also the other way around. Practicing social distancing outside, washing hands directly reduces the R0 value. Making the pandemic without a miserable number of deaths. Those measures act as a social vaccine until the real vaccine is available.  

8. References:

  1. Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, co - Nucleotide - NCBI. (n.d.). Retrieved October 15, 2020, from https://www.ncbi.nlm.nih.gov/nuccore/1798174254

  2. Covid Trends. (n.d.). Retrieved October 15, 2020, from https://aatishb.com/covidtrends/

  3. (n.d.). Retrieved October 15, 2020, from https://nextstrain.org/ncov/global?animate=2019-12-02%2C2020-09-16%2C0%2C0%2C30000

  4. (n.d.). Retrieved October 15, 2020, from https://bit.ly/3aJnqUv

  5. MSc, E., MSc, S., BigCommerce, S., & MSc, P. (n.d.). COVID-19 Workplace Testing. Retrieved October 15, 2020, from https://bit.ly/2CIXT0S

  6. Jakhar, M., Ahluwalia, P., & Kumar, A. (2020, May 14). COVID-19 Epidemic Forecast in Different States of India using SIR Model (Rep.). Retrieved October 15, 2020, from https://www.medrxiv.org/content/10.1101/2020.05.14.20101725v1.full.pdf

  7. Appleby, J. (2020, March 30). What Takes So Long? A Behind-The-Scenes Look At The Steps Involved In COVID-19 Testing. Retrieved October 15, 2020, from https://khn.org/news/what-takes-so-long-a-behind-the-scenes-look-at-the-steps-involved-in-covid-19-testing/

  8. Prevalence of Asymptomatic SARS-CoV-2 Infection. (n.d.). Retrieved October 15, 2020, from https://bit.ly/2YgiOju

  9. Satyanarayana, M. (2020, June 23). Faulty probes are to blame for CDC coronavirus testing woes. Retrieved October 15, 2020, from https://bit.ly/3aIPlDJ

  10. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel (Rep.). (2020, July 13). Retrieved October 15, 2020, from https://www.fda.gov/media/134922/download

About the Author

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Soumyadip is completing his B.Sc in microbiology from St. Xavier's College, Kolkata. He is genuinely curious about science; from astro physics to the quantum world phenomenon, phycology to psychology. Besides these, he loves solving logical puzzles. You'll often find his hands full with experimental stuffs.