The Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technology is recognised by the World Health Organization (WHO), United States Centers for Disease Control and Prevention (CDC) and our Health Ministry for the confirmation of a diagnosis of Covid-19.
It permits the detection of minute quantities of the ribonucleic acid (RNA) of the SARS-CoV-2 virus which causes Covid-19. Its utility is helpful provided its accuracy and limitations are understood.
While the RT-PCR test detects RNA in minute quantities, it is important to remember that it
does not answer the key question of whether the virus detected is infectious.
The SARS-CoV-2 virus only contain RNA, which means it relies on infiltrating healthy cells to multiply and survive.
Upon entry into the body’s cells, the virus uses its RNA code to take over and reprogramme the cells, converting them into virus-producing factories.
In order to detect SARS-CoV-2, the viral RNA is converted into DNA in the test in a process called reverse transcription because only DNA can be copied (amplified).
A specific part of the transcribed viral DNA is amplified hundreds of thousands of times.
Instead of trying to detect a miniscule amount of virus among millions of genetic information, amplification permits sufficient amount of the target sections of the viral DNA to confirm that the virus is present.
A sample is treated with chemical solutions that extract only the RNA present in it. The extracted RNA contains the person’s own genetic material and the viral RNA, if present.
The RNA is reverse transcribed with a specific enzyme.
Additional short DNA fragments complementary to specific parts of the transcribed viral DNA are added.
These fragments attach to target sections of the viral DNA, if present.
The mixture is then placed in a RT-PCR machine which repeatedly cycles through temperatures that heat and cool the mixture to trigger specific chemical reactions that create new, identical copies of the target sections of viral DNA.
Each cycle doubles the previous number – two copies become four; four becomes eight and so on.
As new copies of viral DNA sections are produced, the marker labels attach to the DNA strands and release a fluorescent dye, which is measured and presented in real time on a screen.
The amount of fluorescence in the sample is tracked after each cycle.
Cycle threshold cut-off
The test is considered positive when the fluorescent signal is amplified sufficiently to be detectable.
The cycle threshold (termed Ct value) is the number of amplification cycles required for the fluorescent signal to cross a certain threshold.
The number of cycles it takes to reach this level provides an estimate of the viral load which is equated with the severity of the infection.
The lower the Ct value the greater the amount of RNA (genetic material) there is in the sample. The higher the cycle number, the less RNA there is in the sample.
A standard RT-PCR usually goes through 35 cycles, which means that by the end of the process, billions of new copies of sections of the viral DNA are created from each viral strand present in the sample.
Different test kits
There are different RT-PCR test kits available with different primers which target different sections of the virus genetic sequence – some target one section, others target more than one.
Both types of kits are used in Malaysian laboratories.
Therefore, there is a need to provide such information to the person undergoing the test.
Viral culture involves the injection of viruses in the laboratory cell lines to detect if they cause cell damage and death, thereby releasing a whole set of new viruses that infect other cells.
Like bacterial culture, viral culture is the gold standard or reference test against which any diagnostic index test for viruses is measured and calibrated, to determine the predictive properties of that test.
There is limited data on the relationship between RT-PCR and viral culture results.
There is some evidence of a relationship between the time from collection of a specimen to test, symptom severity and the likelihood that someone is infectious
When an individual is infected by the virus, the mean time for symptoms to develop (incubation period) is five to six days, with a range of between one to 14 days following exposure.
In general, about 90% of mild cases clear the virus within an average of 10 days after symptom onset.
Apart from symptom severity, the sampling location also determines when peak viral loads occur.
The virus may be detectable in the upper respiratory tract (URT) one to three days before the onset of symptoms.
Its concentration in the URT is highest from about the time of symptom onset to four to six days later, after which it gradually declines.
Peak viral loads in the lower respiratory tract appear to arise later.
In some patients, the viral RNA may only be detectable for several days and in others, it can be detected for weeks, possibly months.
Prolonged presence of the viral RNA does not necessarily mean prolonged infectiousness.
There is a correlation between reduced infectiousness and increased number of days elapsed since symptom onset and resolution, decrease in viral load in respiratory secretions and an increase in neutralising antibodies.
What does this mean?
A true positive test result means that the individual has or has had Covid-19 at the time of the test.
However, there are some individuals who get a false positive result i.e. they get a positive result but do not have or have had Covid-19.
Such individuals are unnecessarily quarantined.
The causes of such errors include the number and which gene sequences are measured, the length of the viral probes and technical errors especially due to contamination.
It is vital to remember that one or more negative results do not necessarily rule out infection.
A true negative test result means that the individual can go about their daily activities without the risk of infecting others.
The various factors that could lead to a false negative result include poor specimen quality containing too little patient material; the specimen was collected late in the course of the disease; the specimen was taken from a site that did not contain the virus at that particular time; the specimen was not handled and/or transported appropriately; or technical reasons inherent in the RT-PCR test.
Those with a false negative result are informed that quarantine is not required.
They go about their daily activities and may infect others.
No test is 100% accurate
Interpreting the result of a Covid-19 test depends on the accuracy of the test, and the probability of infection before testing.
A positive RT-PCR test carries more weight than a negative test because of the test’s high specificity but moderate sensitivity.
If the test comes back positive, it usually means that the person has or has had Covid-19 at the time of the test.
However, person(s) with Covid-19 can be missed by the RT-PCR tests because of false negatives.
If anyone has been in close contact with a Covid-19 positive person, it is safest to self-quarantine, even if the RT-PCR test is negative.
A single negative Covid-19 test should not be used to rule out those who have been in close contact with a Covid-19 positive person.
Anyone with suggestive symptoms of Covid-19 or is in doubt should seek medical attention.
Likewise, anyone undergoing RT-PCR tests should be informed about the accuracy and limitations of the tests.
Dr Milton Lum is a past president of the Federation of Private Medical Practitioners Associations and the Malaysian Medical Association. For more information, email email@example.com. The views expressed do not represent that of organisations that the writer is associated with. The information provided is for educational and communication purposes only and it should not be construed as personal medical advice. Information published in this article is not intended to replace, supplant or augment a consultation with a health professional regarding the reader’s own medical care. The Star disclaims all responsibility for any losses, damage to property or personal injury suffered directly or indirectly from reliance on such information.
Dr Milton Lum is a past president of the Federation of Private Medical Practitioners Associations and the Malaysian Medical Association.