Articles

Why is RT-PCR not enough for COVID-19 diagnostic?

Dr. Natalia Gonçalves

To better understand the transmission dynamics of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and develop effective countermeasures against it, antigen- and antibody-based immunoassays will be essential. In this article I will explain the difference among techniques and the importance of antibody detection for a complete diagnostic assessment for COVID-19.

RT-PCR: A quick summary

The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology. By using PCR, specific sequences within a DNA or cDNA template can be copied, or “amplified”, many thousand- to a million-fold using sequence specific oligonucleotides, heat stable DNA polymerase, and thermal cycling. In real-time PCR, the amount of DNA is measured after each cycle via fluorescent dyes that yield increasing fluorescent signals in a direct proportion to the number of PCR product molecules (amplicons) generated.

The main advantage of RT-PCR has been its speed and sensitivity. After taking a pharyngeal swab from the back of a patient’s throat, a sample can be sent to the lab to provide results within hours. And as only a very small amount of viral RNA needs to be present for amplification, these tests are highly sensitive in detecting virus from a sample. By controlling the annealing, extension and denaturation steps with changes in temperature, the initial sample of viral DNA or RNA can be exponentially amplified, followed by the addition of specific probes that produce a detectable signal (often fluorescent) to confirm the etiological agent.

However, RT-PCR diagnosis of COVID-19 has its limitations. Detecting SARS-CoV-2 from pharyngeal swabs requires high-quality specimens that contain a sufficient amount of intact viral RNA. Yet, SARS-CoV-2 loads in the respiratory tract have shown to vary considerably. This has not only led to high false-negative rates, with probable cases remaining negative after multiple swabs, but is further exposing healthcare workers to risk of infection.

For more resource-limited countries, diagnostic infrastructure and training is even harder to come by, with many healthcare systems having only a handful of labs and technicians spread across vast geographical areas. Governments and private organisations are working fast to increase the capacity and speed of PCR testing, but as laboratories are strained by growing case numbers, delays and complete lack of testing is becoming all-too-common.

Immunoassays (Immunity test)

Can people feel “free to go”?

Immunoassays are chemical tests used to detect or quantify a specific substance, in a blood or body fluid sample, using an immunological reaction highly sensitive and specific. Their high specificity results from the use of antibodies and purified antigens as reagents. An antibody is a protein (immunoglobulin) produced by B-lymphocytes (immune cells) in response to stimulation by an antigen. Therefore, immunoassays measure the formation of antibody-antigen complexes and detect them via an indicator reaction.

In other words, immunoassays are based on the principles that specific antigens will stimulate very specific immune responses; a antibody, which binds virus-specific from a patient, by adding a further reporter protein, it is then possible to detect a virus-specific immune signal to confirm the presence of ongoing (IgM or IgA) or past viral infection (IgG and immune status).

Unlike RNA, antibodies are long-lasting and can persist in the bloodstream for many years after infection. As such, immunoassays enable us to identify patients that have had COVID-19, retrospectively (positive IgG – immunity). The type of antibody and its relative levels could also be used to indicate the stage of infection and estimate time since exposure for contact tracing. 

However, antibody tests have their limitations too. With the studies we have so far, it seems that the initial IgM or IgA (acute infection) antibody response doesn’t peak until ~9 days after initial infection and the IgG antibody response doesn’t peak until day ~11, however this data is constantly changing, since we are talking about a new virus.

By conducting random antibody sampling of the general public (known as a serosurvey), public health bodies could better estimate the true levels of exposure and resulting population immunity. For COVID-19, this would be a game-changer, as true transmission could be calculated to forecast the intensity and longevity of the pandemic to direct decision-making. Furthermore, by identifying potential geographical ‘hot-spots’ of low population immunity, health systems could better allocate resources to prevent or manage transmission.

About the Author: 

Dra. Natalia Gonçalves is PhD in Science at the University of Sao Paulo and at University of North Carolina. She has a postdoctoral degree in Genetics and Molecular Biology at the University of Sao Paulo. She developed a project with CRISPR-Cas9 genetic editing for the correction of Duchenne Muscular Dystrophy.

References:

[1] Jiang, F., Deng, L., Zhang, L. et al. Review of the Clinical Characteristics of Coronavirus Disease 2019 (COVID-19). J GEN INTERN MED (2020). 

[2] Xia, N.; Wang, G.; Gong, W. Serological Test is an Efficient Supplement of RNA Detection for Confirmation of SARS-CoV-2 Infection.  Preprints 2020.

[3] A preliminary study on serological assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 238 admitted hospital patients. Lei Liu, Wanbing Liu, Shengdian Wang, Shangen Zheng. medRxiv 2020. 

[4] Li, Z, Yi, Y, Luo, X, et al. Development and clinical application of a rapid IgM‐IgG combined antibody test for SARS‐CoV‐2 infection diagnosis. J Med Virol. 2020; 1– 7. 

[5] Murray KO, Garcia MN, Yan C, Gorchakov R. Persistence of detectable immunoglobulin M antibodies up to 8 years after infection with West Nile virus. Am J Trop Med Hyg. 2013;89(5):996–1000. 

[6] Zhao, Juanjuan and Yuan, Quan and Wang, Haiyan and Liu, Wei and Liao, Xuejiao and Su, Yingying and Wang, Xin and Yuan, Jing and Li, Tingdong and Li, Jinxiu and Qian, Shen and Hong, Congming and Wang, Fuxiang and Yingxia, Liu and Zhaoqin, Wang and Qing, He and He, Bin and Zhang, Tianying and Ge, Shengxiang and Liu, Lei and Zhang, Jun and Xia, Ningshao and Zhang, Zheng, Antibody Responses to SARS-CoV-2 in Patients of Novel Coronavirus Disease 2019. Clinical Infectious Diseases, 2020.

[7] RUIYUN LI, SEN PEI, BIN CHEN, YIMENG SONG, TAO ZHANG, WAN YANG, JEFFREY SHAMAN. Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV2), Science, Mar 2020.

Leave a message