News & Insight
News & Insight

BLT InsightKnow your stuff. The technology in COVID-19 diagnostic kits

2 Dec 2021

Level four social distancing is in effect in the Greater Seoul Area. Level four is the highest level of social distancing under the revised social distancing rules. This has still failed to curb the rise in COVID-19 cases, with around 1,600 new cases daily. Cases outside the GSA are also rising, and some are saying restrictions on gatherings past 6PM can be expected for non-GSA regions as well.


Some news media cite “COVID-19 self-diagnosis kits” as one of the reasons for the rise in cases. Many individuals who were positive for COVID-19 insisted they were negative after self-testing using these kits. Not long ago, a celebrity posted a “negative” result from a self-diagnosis kit they used before going out on social media. However, the Central Disease Control Headquarters are warning that COVID-19 infected persons who test negative using these self-diagnosis kits may become silent spreaders of the disease. That is to say, one should be wary of being too confident in self-diagnosis kit results.


That said, the populace cannot be completely barred from using these kits. Compared to COVID-19 examinations at community health centers, self-diagnostic kits are clearly faster and more convenient. Rather than rule them out completely, it may be better to have the general public sufficiently appreciate their technical limitations, better understand the test results, and encourage use of such kits as a reasonable reference for those who suspect they have COVID-19 in judging whether or not they can safely go about their daily life and activities. Obviously, it would be unwise to let one’s personal guard down based on the result of a self-diagnostic kit alone. It's about knowing your stuff.


COVID-19 self-diagnostic kits can be categorized into antigen, antibody and genetic testing kits. COVID-19 diagnostic kits are in-vitro diagnostic devices, employing immunochemistry (antigen and antibody testing) and molecular diagnostic technology (genetic testing).



Antigen-based diagnostic kits


Examples of antigen-based diagnostic kits include the “Standard Q COVID-19 Ag” and the “Standard Q COVID-19 Ag Home Test” from SD Biosensor. The home test product can be found relatively easily at pharmacies or online shopping malls. SD Biosensor supplies global pharmaceuticals company Roche with COVID-19 diagnostic kits. Achieving KRW 1 trillion in revenues in 2020, the company was listed on the KOSPI (not KOSDAQ) index on the July 16, 2021.


The “Standard Q COVID-19 Ag” product for expert use requires a nasopharyngeal swab for specimen collection, whereas the self-diagnostic kit “Standard Q COVID-19 Ag Home Test” collects a specimen from a nasal swab.



(Specimen collection method for Standard Q COVID-19 Ag (for expert use), source: SD Biosensor)


(Specimen collection method for Standard Q COVID-19 Ag Home Test (for self-diagnosis), source: SD Biosensor)



Antigen-based diagnostic kits use the immunochromatography method. Immunochromatography combines the principles of immune response (antigen-antibody response) and chromatography. When a diluted specimen solution is dropped onto the antigen diagnostic kit, the capillary effect moves the solution to the other end. Any antigens in the specimen bind to labeled antibodies. The labeled antigen-antibody complexes continue moving along until they bind again to an antibody fixed to a test line. Antibodies bind to both sides of the antigen, and therefore the reaction is known as a sandwich reaction. Any remaining labeled antigen-antibody complexes bind to secondary antibodies fixed to a control line. This control line is used to judge whether or not a test is normal. The visible lines created by the antigen-antibody complexes fixed to the test line and control line are used to diagnose. A positive diagnosis requires lines to be apparent at both the test (T) line and control (C) line.


(Concept of the antigen inspection concept, source: Creative-diagnostics)          


Despite the aforementioned advantage of quick testing, it is frequently pointed out that these kits are lacking in accuracy.


Diagnostic accuracy is evaluated based on sensitivity and specificity. It is important to be able to distinguish positive and negative results.


A positive result diagnoses a test subject as infected, and a negative results diagnoses a test subject as not infected.


More specifically, a true positive (TP) result properly diagnoses an infected person as infected , while a false negative (FN) result wrongly diagnoses an infected person as non-infected. A false positive (FP) result wrongly diagnoses a non-infected person as infected, and a true negative (TN) result properly diagnoses a non-infected person as non-infected.



Sensitivity is the probability of true positives, and specificity is the kit's true negative probability. Sensitivity and probability are relatively easy concepts. The positive predictive value (PPV) is the likelihood a person diagnosed as positive is actually infected, and the negative predictive value (NPV) is the likelihood a person diagnosed as negative is actually not infected. It is indeed possible that a positively diagnosed person is actually not infected, or that a negatively diagnosed person is actually infected.


Here, probability refers to conditional probability. We all struggled with “P(A|B)” in high school math class. Probability is always a difficult concept. Sensitivity, specificity, PVV and NPV are calculated as follows.



As a side note, the same method is used to evaluate the performance of AI machine learning models.

Low sensitivity raises the risk of false negatives, while low specificity raises the risk of false positives. Individuals falsely diagnosed as negative are in fact infected, and may spread COVID-19 by infecting others. On the other hand, individuals who are falsely diagnosed as positive pose no infection risk, but the false positive may result in unnecessary lockdown of facilities or quarantining of people who the falsely diagnosed person comes into contact with.

Relating to this, the manual for the Standard Q COVID-19 Ag Home Test presents the results from a clinical trial performed using a certified RT-PCR product with 40 confirmed-positive specimens and 105 confirmed-negative specimens. The sensitivity and specificity as stated here are 82.5% and 100%, respectively.



(Clinical sensitivity and specificity of Standard Q COVID-19 Ag Home Test, source: SD Biosensor)


Despite this, differences between actual test results and clinical trial results presented by the manufacturer (which indicate lower actual sensitivity), decreased accuracy when testing with trace amounts of virus, and lower virus concentration in nasal specimens compared to nasopharyngeal specimens remain problems.



Antibody-based diagnostic kits


SD Biosensor also supplies antibody-based diagnostic kits. The product name is “Standard Q COVID-19 IgM/IgG Plus.” Like Ag, the product uses immunochromatography to detect the immunoglobulin antibodies IgM and IgG in the subject’s blood. The IgM and IgG antibodies generated by immune response upon COVID-19 infection are made to bind with labeled antigens. A positive diagnosis is made by comparing the IgM (M) line, IgG (G) line, and control (C) line.


IgM is the first antibody created in the primary immune response, and activates complements. IgG is created in large amounts during the secondary immune response, and is reported to aid phagocytosis by macrophages and neutrophils. A positive antibody diagnosis kit result can indicate a current COVID-19 infection or a history of infection.


Use of antibody-based diagnostics kits must take the time at which IgM and IgG antibodies are created into account. Antibodies are not yet formed in the early stages of COVID-19 infection, and at this point antibody-based diagnostics kits have limited effect. According to research, IgM antibodies last a relatively short period of time, and IgG antibodies last around four months as well.


Antibody-based diagnostic kits are not sold to the general public.


(Testing methods according to time of COVID-19 infection, source: SD Biosensor)



Neutralizing antibody diagnostic kits


Individuals who have been vaccinated against COVID-19 will want to measure the immunity caused by the vaccine. This requires a neutralizing antibody test.


The spike protein of the COVID-19 virus attaches to ACE2 (Angiotensin-converting enzyme-2) receptors on the surface of human cells to infiltrate cells. ACE2 receptors bind to a specific site on the spike protein, called the RBD (receptor binding domain). Neutralizing antibodies bind to RBDs to inhibit the binding between spike proteins and ACE2 receptors, preventing intrusion of the virus and suppressing virus proliferation.


(Neutralizing antibodies, source: BioWorld)


Neutralizing antibodies make use of this principle wherein neutralizing antibodies inhibit binding between the RBDs of spike proteins and ACE2 receptors.


There are two methods of diagnosis using neutralizing antibodies. In the first, an ACE2 receptor protein is fixed to a plate and a recombinant RBD protein is labeled. In the second, a recombinant RBD protein is fixed to a plate, and an ACE2 receptor protein is labeled.


Neutralizing antibodies in the specimen (blood) bind to the recombinant RBD protein in competition with ACE2 receptor proteins. The degree to which binding between the recombinant RBD protein and ACE2 receptor protein is inhibited is measured. The presence of the neutralizing antibody is detected using the reduction in binding between the recombinant RBD protein and ACE2 receptor protein.


As the RBD of recombinant viruses are used instead of live viruses, the test is not dangerous, and a high level of biosafety is not required.


Neutralizing antibodies can be created through vaccination. If neutralizing antibodies are not detected in a vaccinated person, then the vaccine has failed to create immunity.


(Principle behind neutralizing antibody diagnostic kits, source: Creative Diagnostics)



Genetic testing


The most powerful and accurate means of testing uses genes. In this method, the existence of the COVID-19 gene in the subject’s body is detected. Individuals checked for COVID-19 at community health centers or screening centers are tested using the genetic testing method. Genetic testing makes use of molecular diagnostics technology, specifically real-time RT PCR (Real-time Reverse Transcription Polymerase Chain Reaction).


PCR tests are different from self-diagnosis kits, in that a medical professional collects a specimen from a nasopharyngeal swab of the subject. Even trace amounts of virus can be used for testing, as gene amplification technology is used.


First, RNA is extracted from the specimen. Single strand RNA is unstable and difficulty to amplify, and a reverse transcription process is used to synthesize a cDNA (complementary DNA) complementary to the extracted DNA. Using reverse transcription enzyme, primer and dNTP, a single strand cDNA based on the RNA template is synthesized.

 



(Overview of the RT-PCR process, source: ThermoFisher)



Then, the cDNA is amplified using PCR. Using the cDNA as a template, dual-strand DNA is synthesized using DNA polymerase, primer and dNTP. This process is repeated 30 to 40 times to amplify the DNA. The PCR process consists of denaturation, where double-strand DNA is separated into single-strand DNA; annealing, wherein a primer is bound to the single-strand DNA; and extension, where the DNA is replicated by synthesizing double-strand DNA by bonding complementary bases to the single-strand DNA. Reaction time and temperature must be controlled for each step.


(Example of DNA amplification, source: ThermoFisher)


For diagnosis, a fluorescent marker probe that binds specifically to the template DNA is used. The fluorescent marker probe is excited and gives off fluorescent light when exposed to light of a certain wavelength. If COVID-19 exists in a specimen, the amount of DNA synthesized using COVID-19 RNA is amplified, making the fluorescent signal more intense. A fluorometer is used to read the fluorescent signal. PCR devices diagnose positive subjects by reading the intensifying fluorescent signal in real-time.


Currently, only the genetic testing method is recognized as a COVID-19 diagnostic test.




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