RT-PCR is the workhorse in diagnosing viral diseases, and it has proven to be invaluable in confirming COVID-19 cases and guiding the act of isolation. Even though PCR is fast and sensitive, it suffers from inherent drawbacks that limit PCR to only being able to diagnose during the acute phase of the infection.
The two most significant methods for diagnosing viral infections are the polymerase chain reaction (PCR) and immunoassays.
What Is PCR?
PCR is a routine technique used in the laboratory to amplify small DNA samples into larger quantities that can be detected and analyzed. A viral RNA or DNA sample is taken from the patient with a swab or blood draw. Then, it is sent to a laboratory that specializes in viral infection analysis to determine if the patient has COVID-19.
RNA viruses, such as SARS-CoV-2 and COVID-19, have a PCR that is preceded by an extra step that produces a complementary DNA template (cDNA) from RNA. SARS-CoV-2 and COVID-19 do this by adding a reverse transcriptase enzyme.
Then PCR begins to add short DNA sequences called primers, which bind the viral DNA strands. The double-stranded sections of DNA are recognized and bound by a thermostable polymerase enzyme that acts as a molecular photocopier extending the sequences and producing a full complementary strand RT-PCR).
According to The Native Antigen Company’s website, “By controlling the annealing, extension and denaturation steps with changes in temperature, the initial sample of NA can be exponentially amplified, followed by the addition of specific DNA probes that produce a detectable signal (often fluorescent) to confirm the etiological agent.”
What Are Immunoassays?
Immunoassays detect the presence of immune proteins. These assays primarily consist of an antigen or antibody, immobilized on a surface (usually a titre dish or paper strip), which binds virus-specific antigens or antibodies from a patient sample. Lab technicians add another reporter protein to detect the virus-specific immune signal to confirm there is an ongoing or past viral infection.
PCR for COVID-19
Those who have COVID-19 can present with a wide variety of symptoms. Some patients may not have any while others may experience severe pneumonia and everything in between. T0 make things more complicated, COVID-19’s most common symptoms strongly overlap with those in other co-circulating respiratory illnesses. These symptoms include fever, dry cough, shortness of breath, headache, aches and pains, sore throat, fatigue, diarrhea, runny nose, and sneezing. These symptoms can also be found with the common cold, the flu, or allergies depending on the cluster of symptoms presented.
This is why diagnosing COVID-19 purely based on signs and symptoms alone is not accurate. COVID-19 must be confirmed by highly specific diagnostic testing. However, due to rapid sequencing and publication of the SARS-CoV-2 genome in January 2020, RT-PCR primers and open access protocols were quickly made available, and they are now being used by medical facilities worldwide to diagnose patients with COVID-19.
The primary advantage of RT-PCR is its speed and sensitivity. After a swab has been taken from the back of the patient’s throat, a sample is sent to a lab that provides results in hours.
Unfortunately, RT-PCR is a limited test. High-quality specimens are required with a sufficient amount of intact viral RNA. “Yet, SARS-CoV-2 loads in the respiratory tract have shown to vary considerably.” This has caused a high rate of false-negatives with probable cases remaining negative after multiple tests, further exposing healthcare workers to the risk of infection.
Additionally, processing COVID-19 samples require a specialized biocontainment laboratory that is operated by highly trained technicians who are usually found within medium to large hospital facilities, and COVID-19 is pushing these facilities beyond their limit.
In resource-limited countries, diagnostic infrastructure and training are hard to come by and many healthcare systems only have a handful of laboratories and technicians who are spread across vast geographical areas. Governments and private organizations are working hard to increase the capacity and speed of the PCR testing, however, as labs become strained by growing case numbers, “delays and complete lack of testing is becoming all-too-common.”
What Are Immunoassays and Where do They Fit In?
Immunoassays have some advantages over PCR. Antibodies and antigens are considerably more stable than RNA. This makes them less susceptible to spoiling when they are being transported and stored, reducing the opportunity for false-negatives. Accuracy is also improved because the antibodies and antigens are uniformly available in sputum and blood samples.
The biggest advantage of immunoassays is the ability to detect infections from the past.
Once a person has recovered from COVI-19 and it has left the body, viral RNA can no longer be detected in the respiratory tract. This leaves only a small window of opportunity during the acute phase of the infection in which COVID-19 can be detected. This works for diagnosing ongoing infections, however, there is no indication if the person has had COVID-19 previously, and if they are immune to COVID-19 or still susceptible to the infection.
Antibodies can remain in the bloodstream for years after the infection has left the body. Immunoassays allow technicians and researchers to identify patients who have had COVID-19, retrospectively. Additionally, the type of antibody found and its existing levels can indicate the stage of infection and estimate how much time has past since the person was exposed to COVID-19 for contact tracing.
Nevertheless, immunoassays have their limitations as well. As the data on antibody tests continue to emerge, it is becoming apparent that the body’s antibody response to COVID-19 is considerably slower than expected. Data is still limited but it appears that the initial lgM antibody response takes nine days to peak after the initial infection and the lgG antibody response takes 11 days.
Most viral infections elicit a primary lgM response within five days. This means the SARS-CoV-2 antibodies will not make good markers of acute COVID-19 infection. Early studies indicate that combined RT-PCR/antibody testing will reduce false negatives, the use of antibody kits for acute-phase diagnoses alone makes for a risky strategy.
Antibody testing may not be appropriate for acute-phase diagnoses, it still shows valuable applications for COVID-19. Possibly its most valuable use is that of a wide-scale antibody testing public health tool.
Worldwide, governments are using estimates of transmission rates, case numbers, and case-fatality rates to make the right decisions for public health. The problem is that 20-80 percent of estimated COVID-19 cases are asymptomatic, these figures have been difficult to accurately model.
On a population level, this means that the true size and scope of the pandemic is still undefined, leaving policymakers with little indication of how serious of a threat COVID-19 still is, and how long it can be expected to last.
By Jeanette Vietti
The Negative Antigen Company: Why We Need Antigen and Antibody Tests for COVID-19
The New York Times: Antibody Test, Seen as Key to Reopening Country, Does Not Yet Deliver
Featured Image Courtesy of NIH Image Gallery’s Flickr Page – Creative Commons License
Top Image Courtesy of Isabelle’s Flickr Page – Creative Commons License