COVID-19 testing: How antibody, antigen, RT-PCR, TrueNat tests differ, their strengths and limitations

Limitations of antigen testing

An antigen test can only reveal whether a person is currently infected with SARS-CoV-2. Before or after the infection has passed, antigens won’t be present.

Since antigen testing doesn’t involve any processes of amplifying the virus or its genetic material, a swab sample may have too little antigen to be detected. This could produce a false negative result. As a precaution, a negative test should be followed up by the more accurate RT-PCR test, to confirm a true negative for COVID-19.

Accuracy is the single largest problem with antigen tests, which are much less sensitive than RT-PCR as a diagnostic tool.

How a PCR test works

A PCR test is a widely-used, highly sensitive test that seeks out traces of genetic material from a specific pathogen, if the pathogen is present in the body. The technique is a powerful diagnostic tool that can identify both DNA or RNA from specific microorganisms, irrespective of whether they are bacteria or viruses.

A PCR can capture a specific gene from genetic material (DNA or RNA) in a swab sample, and multiply it through a series of chemical processes so it can be detected using fluorescent dyes.

RT-PCR, a variation of the PCR test

The version of PCR testing used to detect viruses like the COVID-19-causing SARS-CoV-2 is called RT-PCR (reverse-transcription PCR).

While some viruses have only DNA, others like SARS-CoV-2, only contain RNA. Viruses infect a healthy cell and hack into the natural machinery that cells use to process our own RNA, so the virus particle can multiply and survive. Once inside a cell, the virus uses its RNA to take control of the cell’s machinery and ‘reprogramme’ the cell into a virus-making factory.

To detect an RNA virus like SARS-CoV-2, scientists use an enzyme (reverse transcriptase) to convert the virus’s RNA into DNA, in a simple and widely-used one-step process called ‘reverse transcription’. This allows a single molecule of DNA to be amplified exponentially (millions of times), which is the main goal of the PCR process, so even virus particles in single digits can be detected in the final result.

An RT-PCR test is considered very reliable because it can detect even a single virus particle in swabs taken from inside the mouth or nose, where the virus particles are most prevalent.

Advantages of RT-PCR testing

Swift, sensitive and specific: There are real-time variations of the RT–PCR technique that are quick, super sensitive, and specific. They can deliver a reliable diagnosis in three hours. That said, the process on average takes around eight hours to produce a conclusive result.

More accurate, less prone to contamination: Compared to other methods to isolate and detect viruses, RT–PCR is much faster and less likely to be contaminated or cause errors during the testing process. This is because the test – start to finish – is carried out in an enclosed tube, inside a specialised, automated machine.

Real-time RT-PCR continues to be the most accurate method we have to detect the SARS-CoV-2 virus.

Severity of infection can be estimated: A standard real-time RT–PCR set-up usually goes through 35 cycles, which means that, by the end of the process, around 35 billion new copies of the sections of viral DNA are created from each strand of the virus present in the sample.

Limitations of RT-PCR testing

Only detects ongoing infection: The main limitation of RT-PCR testing is that it cannot be used to detect a past infection with SARS-CoV-2, which is important for understanding the development and spread of the virus, as viruses are only present in the body for a specific window of time.

Other methods are necessary to detect, track and study past infections, particularly those which may have developed and spread without symptoms. Once you have recovered, the virus is eliminated and these tests can no longer tell if you’ve been infected. This creates significant uncertainty, especially if someone has self-isolated due to mild and unclear symptoms.

Requires specialised equipment, training: Unlike antibody testing, which requires a kit and medical/paramedical personnel trained in using the kit, RT-PCR testing needs specific instruments in a laboratory, which makes it more time-consuming. Even if the test itself takes just a few hours, the process of collecting and labeling samples, transporting them to a lab, and processing the samples in bulk ensure that it is days before the results from a single sample are known.

Portable, rapid RT-PCR machines are at the cutting edge of diagnostic technology, and often require specialised training to ensure the machine isn’t misused, which requires some level of expertise and training. Tests are only now being developed and made available for these advanced instruments.

Quality control is not a guarantee: In a pandemic, the use of even very accurate diagnostic tools is more error-prone than daily diagnostic tests. RT-PCR, while a revolutionary tool in diagnosis, can also be easily misused or misinterpreted. It depends on a reliable existing DNA sample of the virus, as a reference, to find the pathogen in a test. It is also sensitive enough to pick up on contamination as a false positive.

Back when the outbreak first began to spread in India, the National Institute of Virology in Pune was the only laboratory testing for COVID-19. Today, there are 1,000. The validity and accuracy of a test depend entirely on the laboratory’s quality. The Hindu reports multiple instances of private and public testing labs (including quality assurance labs) returning false positives at an alarming rate. Experts have called for internal quality control and external quality assessment as an urgent requirement if PCR testing is to be considered reliable.

Quantity meets quality with TrueNat testing 

Most modern PCR tests can detect a wide range of pathogens in real-time, with results visible as the chain reaction is underway. TrueNat is a chip-based, portable RT-PCR machine, originally developed as a portable diagnostic tool for tuberculosis by Goa-based startup  Molbio Diagnostics.

The device is on its way to becoming one of the main diagnostic tools for COVID-19 as India looks to expand its testing capacity. It is the fastest available PCR-based technique recommended by ICMR till date.