Coronavirus Outbreak: What are the challenges and limitations of RT-PCR testing?

As the world segued into the year 2020, we were faced with a global challenge called COVID-19.

As of 24 April 2020, there are over 27 lakh individuals infected with SARS-CoV-2 and nearly two lakh people have died worldwide. Like many other respiratory diseases caused by viral pathogens, COVID-19 cannot be diagnosed entirely based on the presentation of symptoms as they closely resemble symptoms of the common flu and are therefore not unique. Hence, diagnostic testing is key to the identification of infected individuals. Moreover, there exist no known antiviral therapies which can treat COVID-19, therefore, isolation and quarantining of infected individuals is the best way of containing the spread of the virus. This further underscores the importance of accurate identification of infected individuals.

In response to WHO’s advice of “test, test, test”, many countries have now ramped up testing and tracing to identify and isolate infected individuals(link). Testing during the nascent stages of the outbreak involved lung CT scans which revealed lesions resembling pneumonia, however, such syndromic tests have now been replaced by molecular techniques.

Testing for a virus

Molecular techniques to test for viral pathogens are established based on the structure of the virus.

So what does a virus really look like?

Viruses, ironically, are submicroscopic organisms that have minimal elements that constitute its structure and resemble a spiky ball.

The structure is similar to that of a Ferrero Rocher chocolate wherein the outer layer constitutes spikes, which are proteins that are unique to the virus. Once the virus enters the nasopharyngeal tract of human hosts, it uses these spike proteins as keys which fit into their corresponding locks on cells of our body, allowing the viral contents to enter. The spiky structure is followed by a layer of fatty membrane underneath, akin to the chocolate wafer layer, which has more membrane proteins embedded in it. The viral genetic material is encased in this structure exactly like the hazelnut core of the chocolate.

Viruses can be broadly classified based on the nucleic acid composition of their genetic material as DNA (deoxyribonucleic acid, like most animal species) and RNA (ribonucleic acid, typically synthesized using DNA as a template) viruses.

SARS-CoV-2 is an RNA virus. Despite our knowledge of its structure, the virus itself happens to be submicroscopic, thereby precluding any possibility of its detection under a simple light microscope. This is where some of the established techniques of molecular biology come in handy. Molecular diagnostic techniques either test for protein and genetic elements of the virus or the changes in protein and genes of host cells (like the ammunition made by our immune cells called antibodies) in response to the virus. Since the host response to the virus is variable and signature manifestations of the infection are still being verified, the detection of protein and genomic elements of the virus is used for diagnostic testing.

What is RT-PCR?

Like other organisms, the information to make all elements that constitute the virus is stored as genes in the viral genetic material. These genes can be identified using a molecular biology technique called Real-Time Polymerase Chain Reaction or RT-PCR. Since genes on the viral genome are present as few copies, and cannot be identified directly, the number of copies of the entire gene or a part of the gene is amplified using PCR. The amplification regime used in a PCR resembles a growing family tree, where each existing DNA/gene segment is used as a template to generate more copies. Exactly like each new member of the family tree gets a unique name and an Aadhar card, in a real time PCR, each newly formed copy gets fluorescent labelling using fluorescently tagged nucleotides and the net fluorescence intensity accumulated is measured as the reaction progresses.

Each cycle of amplification generates a fixed number of new copies, therefore the original number of copies of the gene (or part of a gene) can be calculated by assessing the number of new copies (based on fluorescence intensity) made after a fixed number of amplification cycles. For detecting SARS-CoV-2, three genes of the virus are being assessed — RdRP gene (RNA dependent RNA polymerase), E gene (envelope protein, spiky coat) and the N gene (nucleocapsid protein, encases the genetic material), and the human RNase P (RP, marker for human samples).

RT PCR is highly sensitive and can detect as low as 6.25 copies of genetic material per microliter of the sample.

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Challenges with RT PCR

Despite being one of the most sensitive assays available to detect viral pathogens, RT PCR is not devoid of challenges and limitations.

The efficiency of RT-PCR depends on the adequate collection of the viral RNA which is typically extracted from nasopharyngeal (throat) swabs of patients. This becomes a major challenge because the amount of viral RNA (viral load) changes drastically between patients and even within a single individual during the course of the infection. This will alter the amount of viral RNA that the reaction starts with, thereby increasing the risk of a false-negative result.

Additionally, the efficacy of collecting the swabs varies between people (individuals as well as health workers) thereby exacerbating variability in the amount of viral RNA and increasing the chances of skewed RT-PCR results. Some researchers are also debating whether samples should be restricted to nasopharyngeal swabs.

What other specimens could be collected which can maximize the efficiency of RT-PCR based detection? It has been shown by a research group that sputum is an appropriate sample along with nasal swabs.

Additionally, bronchoalveolar lavage fluid (BALF) may also be tested and monitored for severely affected patients. However, BALF requires a specialized suction instrument which cannot be manoeuvred without help from an expert operator thereby making it an impractical alternative for testing large numbers of people.

In addition to viral load, false positives or negatives may also arise from improper handling of the sample leading to contamination with agents that prevent appropriate extraction of RNA or act as quenchers of the fluorescent signal during PCR. This can be avoided by increased caution during sample handling and minimum transit time to the testing centre after collection of the swabs. Minor alterations to the primers and probes used for the PCR, and modification of the samples used as reference controls, may also aid in minimizing false positives and negatives in RT-PCR results.

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Antibody testing

As the name appropriately indicates, RT-PCR conveys the current infection status of the individual tested. This is inadequate in a public health scenario as the history of infection is also an important factor that not only allows for efficient record-keeping but also keeps a check on recurring infections in the same individual. This sort of holistic assessment requires additional tests like antibody testing. Antibodies are the ammunition generated by immune cells of our body to combat diseases. These are highly stable in serological samples like blood and are therefore less susceptible to spoilage during collection, transport and storage.

Interestingly, antibodies also remain in the blood for a prolonged duration (weeks, in some cases) thereby allowing us to detect the history of infection. Since antibody testing requires specialized kits and detection efficiency peaks only after three days after the onset of symptoms, it may not be an appropriate replacement for RT-PCRs. However, a combinatorial diagnostic testing approach involving both these techniques can allow for efficient detection as well as management of the infected individuals during this pandemic.

The author is a research scholar with a doctorate in Developmental Biology from the Tata Institute of Fundamental Research, Mumbai

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