Is RT-PCR a reliable COVID test?

Types of Testing for COVID

1.Diagnostic tests are of two categories – Molecular [RT-PCR] and rapid tests [Antigen test].

2. Antibody tests [ELISA test].

About RT-PCR test

RT PCR means Reverse-Transcriptase-Polymerase Chain Reaction. RT–PCR is one of the most widely used laboratory methods for detecting the COVID-19 virus.
A virus has an outer molecular envelope like a capsule and inside is the genetic material. This genetic material can be either DNA or RNA.

DNA is double stranded and found in all organisms and which holds the genetic code for how these organisms are made and develop.
RNA is single strand molecule that copies, transcribes and transmits parts of the genetic code to proteins so that they can synthesize and carry out functions that keep organisms alive and developing.

SARS-CoV-2 coronavirus, which causes COVID-19, only contain RNA. They enter the cells to multiply and survive like a parasite. The viruses use their RNA to take control of the host cell and to make copies.
For the detection of the Virus, the RNA has to be converted into DNA by the enzyme reverse transcriptase. This is done since the DNA alone can be copied in the test.

Sample collection and basic principle of the RT-PCR test

• Sample is collected from the nose and throat where the virus harbors. The sample is treated to remove other than RNA and only the RNA is extracted.
• The extracted RNA contains both the RNA of the Virus and as well as the individual own genetic material.
• RNA is then reverse transcribed to DNA by the specific enzyme. Additional short fragments of DNA that are specific parts of the transcribed viral DNA are added. They are used for building and also for marker [For identification]
• If the virus is present in the sample, then the fragments attach themselves to the viral DNA.
• This mixture is placed in an RT-PCR machine with temperature for both heating and cooling.
• They now make copies of DNA. 1 becomes 2, that becomes 4 and to 8 and so on.
• At the end of the many cycles, millions of DNA is formed.
• The marker labels that attach to the DNA strands release a fluorescent dye which is detected by the computer. The amount of the dye is tracked after each cycle. The severity of the infection is determined by the amount of dye in each cycle. If dye is more in fewer cycles, then the infection is more severe.

Types of RT-PCR protocols

RT-PCR has two protocols, a one-step protocol and a two-step protocol

In one-step protocol the RT and PCR components are mixed in a single tube simultaneously. The one-step protocol works well for amplifying targets that are reasonably abundant. Also, there is less variation as both reactions take place in the same tube.

The two-step protocol includes – first reverse transcription and then the PCR. In a two-step protocol a stable DNA pool is generated which can be stored for longer periods and can be used for multiple reactions. The two-step method is more sensitive than the one-step method and yields better results of the rare targets and is a flexible priming option.

Reliability

RT PCR is a highly reliable, faster, sensitive test to detect the Virus and takes an average time between 4-8 hrs. Has a lower potential for contamination or errors. RT-PCR test is the gold standard for diagnosis or confirmation of COVID-19.

Limitations of RT-PCR test

False negative results- The patient has virus [Positive] but the test results shows negative and the reasons are:

• Inappropriate sample collection.
• Storage condition of the samples.
• Transfer procedure of the samples to the testing lab.
• Processing during the test.
• Genomic mutations.
• Inappropriate testing time at an early stage of infection. The highest shedding of SARS-CoV-2 is in the first few days of symptoms and peaks on day 4.

False positive results- The patient do not have virus [Negative] but the test results shows positive.

• Cross contamination of the sample. Fumigation of the sampling area to be done at frequent intervals.
• Testing errors like pipetting and processing.

Notwithstanding the probability of the above errors, RT PCR is currently the most accurate and the most sensitive available solution for the earliest and large-scale detection of SARS-CoV-2.

Rapid tests

Rapid antigen tests detect viral proteins [Known as antigens]. Samples are collected from the nose and/or throat with a swab. These tests are cheaper than PCR and will offer results more quickly, although they are generally less accurate. These tests perform best when there are more viruses circulating in the community and when sampled from an individual during the time they are most infectious.

Antibody tests

Antibody tests can tell us whether someone has had an infection in the past, even if they have not had symptoms. Also known as serological tests and usually done on a blood sample, these tests detect antibodies produced in response to an infection.
In most people, antibodies start to develop after days to weeks and can indicate if a person has had past infection. Antibody tests cannot be used to diagnose COVID-19 in the early stages of infection or disease but can indicate whether or not someone has had the disease in the past.

CT Test and report

Use of CT [Computed Tomography] for the diagnosis of COVID-19 are used for patients who may have classical symptoms of the illness but have a negative RT-PCR test result and testing is decided by the Physician. High-Resolution Computed Tomography (HRCT) tests is a diagnostic tool that involves imaging of the lungs. It showcases the presence of glass-like ground, opaque opacities in the lung cavities, which are a crucial marker of COVID severity. 

Interpretation of CT report

CORADS [COVID-19 Reporting and Data System] score determines the level of infection and involvement because of the virus. CORAD scoring is done on the basis of 1-6, wherein 1 signifies a ‘negative COVID’ or normal lung function, scores 2-4 signify suspected viral involvement and a reading of 5 means classic COVID-19.

A person who has detectable ground opacities in the lungs is given a score of 6 and said to be at a higher risk of COVID severity. Based on that, additional respiratory tests and blood work could also be done and treatment followed. 

Note-The images given for representation in this blog are taken from Google Images. Many thanks for Google.