- R & D
Various molecular tests can be used to detect RNA virus infections.
The RT-qPCR test is currently regarded as the gold-standard, but its sensitivity to residual viral RNA genome fragments can lead to false-positive results long after a patient has recovered from a viral infection.
We here modified the RT-qPCR protocol to make it less sensitive to residual viral RNA genome fragments, reducing the likelihood for false-positive results in recovered COVID-19 patients.
The method may improve test-to-release protocols, expand the tools available for clinical testing, and help reduce hospital encumbrance.
Introduction: Respiratory RNA viruses, such as influenza and coronaviruses, are important human pathogens that have substantial impact on our healthcare systems and economy. Currently, Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)-based tests are considered the gold-standard for the detection of viral RNA in clinical samples. But its sensitivity to residual viral RNA genome fragments can lead to false-positive results long after a patient has recovered from a viral infection.
Objective: To develop the RT-qPCR protocol to make it less sensitive to residual viral RNA genome fragments, reducing the likelihood for false-positive results in recovered COVID-19 patients.
Methods: To minimize the detection of viral RNA genome fragments, we here modified the RT-qPCR assay by replacing the routinely used random hexamers with an oligonucleotide [oligo(dT)20 primer] that binds to the 3′ end of the viral genome. We analyzed clinical samples obtained within the first 5 days of the onset of symptoms (i.e., when SARS-CoV-2 infected patients are typically infectious) and samples obtained at least 15 days after the onset of symptoms (i.e., when most patients have recovered). The qPCR was run and analyzed on a Quant Studio 5 qPCR machine according to the manufacturer’s instructions.
Results: We observed no significant difference between the two RT-qPCR methods (CoviPath COVID-19 RT-PCR Kit v/s Random Hexamers assay) for the COVID-19 samples taken within 5 days of the onset of symptoms, suggesting they are equally efficient at detecting infectious viral RNA levels in clinical samples. However, a significant difference was observed in samples obtained more than 15 days after the onset of symptoms, with most long-range RT-qPCR reactions producing no detectable signals.
Conclusion: Previous research has shown that COVID-19 patients can test positive for SARS-CoV-2 long after recovery. Our method can be used to reduce these false-positive RT-qPCR tests, and distinguish between incorrectly-positive cases and re-infections.
Reference: Reducing false-positive SARS-CoV-2 diagnoses using long-range RT-qPCR, Aartjan Velthuis, Dovile Juozapaite, CharlotteV. Rigby, Ingrida Olendraite, Pankaj Mathur, Kalyan Dhanorkar,
Vishalraj Hulle, Tejas Shah, Vijeta Jadhao, Shravan Mutha, Hamid Jalal, Vikram Gopal. MedRxiv 2021.11.11.21266219;
What is COVID-19?
COVID -19, also known as coronavirus disease 2019 is an illness caused by a novel coronavirus. It is also referred to as severe acute respiratory syndrome due to its ability to affect and cause damage to the respiratory tract. This was first identified amid an outbreak of respiratory illnesses in Wuhan City, China.
It was brought to the notice of the WHO towards the end of December 2019, a month after which, it was declared a global health emergency.Very soon thereafter, the WHO declared COVID-19 as global pandemic, the first of its kind since the H1N1 influenza outbreak in 2009.
When to perform COVID-19 test?
COVID-19 affects different people in varying ways.
Most infected people will develop mild to moderate symptoms which can be managed at home and will not need further medical intervention or hospitalization.
Some mild symptoms include
Some of the less common symptoms are
The serious symptoms which would require immediate medical assistance are
Type of tests & advantages
With the rampant spread of this disease across the globe, testing has become imperative. There are currently two types of tests available.
The RT-PCR (Reverse transcription polymerase chain reaction) test detects nucleic acid, such as RNA, belonging to the coronavirus. These tests are highly sensitive and extremely accurate.
On the other hand, the rapid antigen test (also known as the Rapid Test RAT) are generally quicker and relatively cheaper as compared to the RT-PCR test. These are also less sensitive and less accurate than its counterpart.
RT-PCR over Antigen
The RT-PCR diagnostic test is a more reliable way to get tested owing to its accuracy and high sensitivity to an active infection. This is very useful when testing is needed before resuming work or school/college. The test involves taking a sample of the mucus or saliva in the nasal or oral cavity using a specialized swab stick.
The Antigen test is quicker owing to the quick turnaround times of collected samples. The main drawback of this test is the possibility of missing an active infection. A negative result of the Antigen test, coupled with symptoms would generally be followed up with an RT-PCR test to confirm the result.
Advantages of RT-PCR test at Krsnaa Diagnostics
Our state-of-the-art facility has made it easy, accurate and timely to undergo a PCR at one of our centres and has several advantages:
Role of Radiology in COVID-19 diagnosis?
HRCT & advantages
Mild symptoms of COVID-19 are known to mimic upper respiratory viral infections, with advanced symptoms citing respiratory dysfunction as the principal cause of morbidity and (seems negative word). In a scenario such as this, chest imaging is crucial to evaluate the level to which the respiratory system may have been impacted.
The HRCT (High resolution computed tomography) provides a robust method of examination in the diagnosis and monitoring of diseases of the lung tissue and surrounding airways.
Non-invasiveness and avoidance of contrast solutions give this test an added benefit since it eliminates the possible allergic reactions that can be caused by contrast solutions.
X-Ray & advantages
The conventional chest X-ray is a strong and reliable method to evaluate problems related to the lungs, heart and chest wall.
It is also an effective diagnostic tool for shortness of breath, fever or a persistent cough. It uses a small dose of ionizing radiation to produce images of the inside of the chest cavity, making it very advantageous over alternate diagnostic methods.
Chest X-rays are very quick and easy. They require very little or no prior preparation, making them useful at times of emergency diagnoses.
What is COVID-19 Antibody test?
Antibodies are normally produced by the body’s immune system in response to an outside stimulus, namely a virus.
It is primarily a blood test and can detect whether a person has ever contracted the virus, albeit in the past. This test is particularly useful if you are showing COVID-19 symptoms but do not have access to the RT-PCR test.
A key factor to note is that antibodies are produced only about one or two weeks after the onset of symptoms. It is hence not advisable to take this test if you have been experiencing symptoms for less than a week.
Antibody tests are useful in determination of exactly how many people have been infected by and recovered from the virus. This would give a better understanding of the rate of the virus’ spread.