Covid-19, Heal
Testing For Covid-19 During Turbulent Times

Dr Shankhabrata Bagchi

Inspector General of Police in Andhra Pradesh & ISB Alumnus

7 min read

 

“Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2)” is the causative organism of “COVID-19,” the disease which has metamorphosed into a veritable pandemic. The name itself is a code – ‘CO’ stands for ‘corona,’ ‘VI’ for ‘virus’ and ‘D’ for disease, i.e. coronavirus disease 2019. COVID-19 is now a household name, and so are PCR and rapid antibody tests, the two major criteria for COVID-19. Yet questions regarding the reliability of these tests haunt our minds. Can we be certain of having contracted the infection if we test positive? Can we heave a sigh of relief if we test negative? 

Let us search for these answers today.

The validity of a test

How to know that a test is accurate? The answer lies in its validity. Efficacy is the ability of a test to separate those having disease from those who are disease-free correctly. It has two components: sensitivity and specificity. Sensitivity is the ability of a test to accurately identify everyone having the disease. In contrast, uniqueness is the ability of a test to correctly determine everyone who is disease-free (among those tested). We hardly find tests with one hundred per cent sensitivity as well as specificity. What becomes inevitable, then? False negatives who test negative despite having the disease and false positives who test positive despite being disease-free. A highly sensitive test will have less false negatives. On the contrary, there will be less number of false positives in a highly specific test.

Dangers of false negatives and positives

False-negative tests give a false sense of security. Such patients get delayed or no treatment, whereas they continue to spread infection. As governments across the world plan to relax restrictions and loosen lockdowns in a bid to reopen economies, there is a serious risk that people who are wrongly believed to be disease-free because of their false-negative test results will cause a re-infection in society. Hence, we must select tests with high sensitivity so that false-negative results are minimized. On the contrary, every false-positive result causes a healthy person to unnecessarily undergo isolation as well as retesting. People getting false-positive results in antibody tests may be misguided to believe that they are immune to COVID-19 as they have the protective antibody and they may negligently get exposed and infected. Therefore, false-positive results also need to be avoided.

RT-PCR test: false positives & false negatives

All validated RT-PCR tests have high specificity because they detect specific gene sequences of the RNA of SARS-CoV-2. Hence, false-positive results are rare here. But beware! RT-PCR test can sometimes produce false-negative results. An infected patient may even test negative several times before testing positive. Therefore, correlation with the history of travel and contacts, clinical observations, chest X-rays and chest CT scans is recommended in suspected COVID-19 patients who test negative. Retesting is also advised in such cases, 2 to 4 days after the first test.

RT-PCR test: causes of false-negative results 

RNA samples for RT-PCR test are usually collected from swabs of nasopharynx or oro-pharynx, e.g. throat. However, RNA samples are relatively unstable because enzymes that can split RNA are found in many places, including the surface of the human body. Therefore, if the samples are transported to the laboratory within 72 hours after collection, the swabs are placed in tubes containing viral transport medium (VTM) and stored at 2 to 8°C (cold chain). If testing is delayed further, specimens must be stored at -70°C or below or transported on dry ice. Meticulous maintenance of these conditions for storage and transportation of samples will prevent RNA-splitting enzymes from degrading viral RNA in those samples. Failure to ensure the prescribed environmental conditions may cause false-negative results. 

Besides, swab must be taken from a spot sufficiently back inside the throat to get a viable sample for this test. Otherwise, sample quality may be poor, leading to a false-negative result. False-negative results also occur due to improper reagents, poor technique in conducting the test in the laboratory because of lack of trained laboratory personnel and in very early or late stages of the disease when low levels of the virus are present. Besides, the failure to amplify the target sequences of viral RNA during the processing of the sample in the laboratory may cause false-negative results. False-negative results may also happen during the later stage of infection as the virus may no longer be present in the upper respiratory tract, i.e. nasopharynx or oropharynx at that time. Best way to avoid false adverse reports, specimens can then be collected from the lower respiratory tract, i.e. trachea/windpipe and below where the virus will still be present. 

Rapid antibody test: dilemma faced by governments/regulators

Amidst the frenzy of launching antibody tests on a massive scale at the earliest, we must not forget the crucial issue of reliability of those testing kits. Given the current public health emergency, the Food & Drug Administration (FDA) in the USA have issued emergency use authorization (EUA) to private companies for using their medical devices and kits despite the lack of adequate data to prove their accuracy. Many other countries have relaxed criteria for validating antibody testing kits or imported them in a hurry, only to find later that the results they yield are highly inaccurate. It is the need of the hour for the government and national regulators to do a proper balancing act so that the need for accuracy of any new diagnostic test is not sacrificed at the altar of public health urgency. 

Rapid antibody tests: false positives & false negatives

These tests usually detect antibodies that are produced in the human body against the spike protein of SARS-CoV-2. Why is this protein selected? Because this is the main antigen that attaches to the host cell, facilitates entry of the virus into the host cell and elicits neutralizing antibodies. Unfortunately, some antibody test kits also detect antibodies produced in the human body against the spike protein of other coronaviruses because the antigenic structures of the spike proteins of SARS-CoV-2 and common cold-causing coronaviruses have probably about 50 to 60% identity. Such kits will yield some inaccurate results as someone infected with some other coronavirus may falsely test positive. To eliminate the potential for such cross-reactivity, new antibody tests must select sections of the spike protein of SARS-CoV-2 that are completely dissimilar from all other coronaviruses. Another problem arises when some antibody test kits are validated with samples from patients who are severely diseased and will develop a strong antibody response. Such antibody test kits may be reasonably accurate in severely ill patients. Still, they may give false-negative results in patients with mild or no symptoms who usually have less viral load and produce fewer antibodies in their blood. 

We must know that the antibody response can be low in the following individual who may be elderly, have poor nutritional habits, less severity of disease (low viral load) and in the presence of immunodeficiency disorders (e.g. AIDS) or with regular intake of immunosuppressive drugs (e.g. steroids in high dose). An antibody test may be harmful even if you have had an infection recently or in the past. 

Way forward

No country in the world has yet tested all its inhabitants for COVID-19 because every nation aims to ensure optimal utilization of its available resources. It is widely acknowledged now that a massive increase of COVID-19 testing is crucial to control this public health crisis, irrespective of the date we decide to relax lockdown measures. Meanwhile, there is a pressing need to ensure that all the tests are conducted accurately. Detailed analysis reports help doctors as well as policymakers, from being misguided while taking decisions. To cater to the burgeoning need for testing in the future, every nation must massively invest in the medical diagnostics industry, which will help boost our country’s medical technology and infrastructure. Doing so can improve the quality of indigenously manufactured kits, research development and innovation of unique testing methods that yield more accurate results in less time. This will ensure the availability of a sufficient number of trained workforce for proper sample collection and testing. As aptly said in Purusha Sukta of Rig Veda, “Nanya pantha vidyate ayanaya” i.e. there is no other way on earth.

“Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2)” is the causative organism of “COVID-19,” the disease which has metamorphosed into a veritable pandemic. The name itself is a code – ‘CO’ stands for ‘corona,’ ‘VI’ for ‘virus’ and ‘D’ for disease, i.e. coronavirus disease 2019. COVID-19 is now a household name, and so are PCR and rapid antibody tests, the two major criteria for COVID-19. Yet questions regarding the reliability of these tests haunt our minds. Can we be sure of having contracted the infection if we test positive? Can we heave a sigh of relief if we test negative ? Let us search for these answers today.

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The validity Of A Test

One question which may linger our minds is, how do we know if your analysis is accurate? The answer lies in its validity. Efficacy is the ability of a test to separate those having disease from those who are disease-free correctly. It has two components: sensitivity and specificity. Sensitivity is the ability of a test to accurately identify everyone having the disease. In contrast, uniqueness is the ability of a test to correctly determine everyone who is disease-free (among those tested). We hardly find tests with one hundred per cent sensitivity as well as specificity. What becomes inevitable, then? False negatives who test negative despite having the disease and false positives who test positive despite being disease-free. A highly sensitive test will have less false negatives. On the contrary, there will be less number of false positives in a highly specific test.


Several experts opine that the virus may not be present in the upper respiratory tract, i.e. nasopharynx or oropharynx during the later stage of infection. Then specimens can be collected from the lower respiratory tract, i.e. trachea/windpipe and below where the virus will still be present. Otherwise, the test result may be falsely negative.


Downsides Of False Negative & Positive Results

False-negative tests give a false sense of security. Such patients get delayed or no treatment, whereas they continue to spread infection. As governments across the world plan to relax restrictions and loosen lockdowns in a bid to reopen economies, there is a serious risk that people who are wrongly believed to be disease-free because of their false-negative test results will cause a re-infection in society. Hence, we must select tests with high sensitivity so that false-negative results are minimized. On the contrary, every false-positive result causes a healthy person to undergo isolation as well as retesting unnecessarily. People getting false-positive results in antibody tests may be misguided to believe that they are immune to COVID-19 as they have the protective antibody and they may negligently get exposed and infected. Therefore, false-positive results also need to be avoided.

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RT-PCR Test: False Positives & False Negatives

All validated RT-PCR tests have high specificity because they detect specific gene sequences of the RNA of SARS-CoV-2. Hence, false-positive results are rare here. But beware! RT-PCR test can sometimes produce false-negative results. An infected patient may even test negative several times before testing positive. Therefore, correlation with the history of travel and contacts, clinical observations, chest X-rays and chest CT scans is recommended in suspected COVID-19 patients who test negative. Retesting is also advised in such cases, 2 to 4 days after the first test.

RT-PCR Test: Causes Of False-Negative Results 

RNA samples for RT-PCR test are usually collected from swabs of nasopharynx or oro-pharynx, e.g. throat. However, RNA samples are relatively unstable because enzymes that can split RNA are found in many places, including the surface of the human body. Therefore, if the samples are transported to the laboratory within 72 hours after collection, the swabs are placed in tubes containing viral transport medium (VTM) and stored at 2 to 8°C (cold chain). If testing is delayed further, specimens must be stored at -70°C or below or transported on dry ice. Meticulous maintenance of these conditions for storage and transportation of samples will prevent RNA-splitting enzymes from degrading viral RNA in those samples. Failure to ensure the prescribed environmental conditions may cause false-negative results. 

Besides, swab must be taken from a spot sufficiently back inside the throat to get a viable sample for this test. Otherwise, sample quality may be poor, leading to a false-negative result. False-negative results also occur due to improper reagents, poor technique in conducting the test in the laboratory because of lack of trained laboratory personnel and in very early or late stages of the disease when low levels of the virus are present. Besides, the failure to amplify the target sequences of viral RNA during the processing of the sample in the laboratory may cause false-negative results. False-negative results may also happen during the later stage of infection as the virus may no longer be present in the upper respiratory tract, i.e. nasopharynx or oropharynx at that time. Best way to avoid false adverse reports, specimens can then be collected from the lower respiratory tract, i.e. trachea/windpipe and below where the virus will still be present. 

Rapid Antibody Test: Dilemma Faced By Governments/Regulators

Amidst the frenzy of launching antibody tests on a massive scale at the earliest, we must not forget the crucial issue of reliability of those testing kits. Given the current public health emergency, the Food & Drug Administration (FDA) in the USA have issued emergency use authorization (EUA) to private companies for using their medical devices and kits despite the lack of adequate data to prove their accuracy. Many other countries have relaxed criteria for validating antibody testing kits or imported them in a hurry, only to find later that the results they yield are highly inaccurate. It is the need of the hour for the government and national regulators to do a proper balancing act so that the need for accuracy of any new diagnostic test is not sacrificed at the altar of public health urgency. 

Future Of Testing
We must introduce better and faster tests if we desire to conduct large-scale testing. One such test uses rapid, portable RT-PCR machines/hand-held DNA analyzers to conduct RT-PCR test based on isothermal nucleic acid amplification (INAA) technology. Polymerase enzymes used here don’t require a series of alternating temperature cycles, and they can amplify viral genetic material at a single temperature. Consequently, results are displayed within minutes. Higher the level of viral RNA in the sample; more significant is the rate of amplification of the target sequences of the viral RNA during the test. Thus the results of such rRT-PCR tests can also be interpreted semi-quantitatively. One institute in Kerala claims to have manufactured RRT-PCR test kits that work on the principle of reverse transcriptase loop-mediated amplification of viral nucleic acid. Availability of such accurate and scalable point-of-care PCR tests can immensely boost our testing capability. Such tests will facilitate diagnosis at home or in the community. Hence, cases can be identified earlier and subjected to isolation, quarantine or treatment as required. Another upcoming test can be the viral antigen test, a ‘dipstick’ style test that can detect viral proteins in swab samples and can be conducted at the point-of-care level. Unfortunately, all these tests are not yet widely available for use, and some of them are not validated so far. However, the future for COVID-19 testing looks promising with so many options on the anvil. We also need to regularly sequence a percentage of positive specimens from clinical cases to ascertain if there is any mutation of the viral genome. Any such evidence of variation can have momentous implications for diagnostic tests, development of vaccines and treatment of infection.

Importance Of Increased Testing
No country in the world has yet tested all its inhabitants for COVID-19 because every nation aims to ensure optimal utilization of its available resources. Nevertheless, it is widely acknowledged today that massive ramping up of testing for COVID-19 is crucial to permanently control this public health crisis, irrespective of the date we decide to relax lockdown measures in our country. Meanwhile, we must remember the pitfalls of testing, the possibility of inaccurate test results and the importance of retesting in case of doubtful results. Only if all cases and carriers are identified through testing, we can get the real picture about the spread of this pandemic. Only then can the government correctly categorize different zones based on the probability of transmission of this infection and enforce containment measures according to the changing field situation. Only then can the local administration take the right decision as to whom to isolate or quarantine to prevent the spread of the virus and whom from admitting in hospitals for treatment.

The views expressed in this article are of the author and not of the government.

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Way Forward
Any country or region may feel safe once new COVID-19 cases cease getting reported for some time. But this sense of security may be short-lived because further waves of infection can’t be ruled out. Even if we win the battle soon, the war against this disease may be long-drawn, and a country’s ability to swiftly test as many people as possible may be the key to containing this outbreak. To cater to the burgeoning needs for testing in the foreseeable future, every nation must massively invest in the technology and infrastructure of the medical diagnostics industry, continuously improve the quality of indigenously manufactured kits, progressively increase the stockpile of validated testing kits, swabs and reagents, research and invent newer testing methods that yield more accurate results in less time, increase the number of Virus Research and Diagnostic Laboratories (VRDLs) and accredited private testing laboratories, upgrade the capacity of all existing laboratories and ensure the availability of a sufficient number of trained workforce for sample collection and testing. As aptly said in the Purusha Sukta of Rig Veda, “Nanya Pantha vidyate ayanaya,” i.e. there is no other way on earth.


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