Reverse Transcription Polymerase chain reaction (RT-PCR) and serologic testing for IgM-IgG antibodies are the dominant ways that global healthcare authorities are using to test for COVID-19.
What is RT-PCR testing?
RT-PCR is currently the main testing method used to detect COVID-19. It consists of, first, reverse transcription of RNA into DNA and then amplifying the complementary DNA (cDNA) segment to a large enough amount in order to study it in detail. By applying PCR amplification on collected samples (nasopharyngeal swabs in most cases), experts are able to detect the presence of SARS-CoV-2’s E gene, which codes for the envelope that surrounds the viral shell. Results with an E gene target detected will be reported as COVID-19 positive. If ambiguity arises with an E gene target result, RNA-dependent RNA polymerase (RdRp) gene detection or any other single gene target with a validated assay must be searched for to confirm COVID-19. If none of the aforementioned target genes is detected, laboratory testing is considered inconclusive.
By detecting viral RNA, which will be present in the body before antibodies form or symptoms of the disease are present, the tests can tell whether or not someone has contracted the virus.
Why sometimes testing is repeated?
If a sick patient suspected of having COVID-19, but with a negative first test, is worsening or not improving on standard care, RT-PCR should be repeated, even if previous tests were positive for another pathogen.
False-negative results are possible due to improper collecting methods and undetected low virus shedding.
What’s the correlation with viral shedding?
Viral shedding is the last phase of a virus’ life cycle, during which the expulsion of a virus’ progeny occurs after it has replicated and used all available resources of a host cell. A person is considered contagious if the virus that has infected them is shedding. The rate of shedding can be followed over time and is considered of interest by public health to establish the proper quarantine precautions needed to limit the spread of the virus. SARS-CoV-2 can cause asymptomatic shedding, actually spreading from person to person while clinically undetectable, and could be detected for up to 9 days in a completely asymptomatic patient.5 The quantification of viral RNA shedding is usually measured by quantitative RT-PCR (qRT-PCR), whereas right now, qualitative RT-PCR is used globally for the diagnosis of COVID-19.
It has been reported that some patients who were no longer symptomatic still had high viral titers and were able to infect their close contacts. Studies have been initiated to determine how long a patient can still be positive after a complete resolution of symptoms. A study carried out in Munich, Germany showed that viral shedding from the pharynx was at its highest during the first week of symptoms, with a peak at day 4. Symptoms started to decrease at the end of the first week, whereas viral RNA was still detectable in swabs during the second week. The clinical sensitivity of RT-PCR on swabs taken between days 1 and 5 of symptoms was 100%, supporting the hypothesis that viral shedding is at its highest in the first week of symptoms.3 Another study in China showed that total duration of viral shedding could range from 8 to 37 days, with a median duration of 20 days in survivors.6
For now, the estimation of SARS-CoV-2 viral shedding is still limited by a lack of quantitative viral RNA detection and a low positive rate of SARS-CoV-2 RNA detected in throat-swabs, but it appears that earlier positive RT-PCR results correspond to the timing of SARS-CoV-2’s highest viral shedding.
What is IgM-IgG antibodies serology testing?
Immunoglobulin (Ig) antibody production is an indication of the body’s immune response. IgM antibodies are the first immunoglobulins to be produced in the body in response to an infection. It disappears later as the virulence of the virus declines. IgG appears soon after infection and stays in the blood. By looking at these serologic markers, especially anti-SARS-CoV-2 IgG, it indicates who’s been infected and who should be immune to the virus.
Point-of-care lateral flow immunoassay tests that detect anti-SARS-CoV-2 IgM and IgG are gaining popularity to “replace” RT-PCR.1 It is simple and takes only 15 minutes to show a result.
While RT-PCR shows the status of an active infection. Serology tests will show the immune response.
| Throat swabs – Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) | |
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| Serological IgM/IgG antibodies testing | |
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What do studies say about testing?
A study done in China demonstrated these following results when comparing RT-PCR and antibodies for testing2:
Antibodies
- The cumulative seroconversion rate of total antibodies, IgG and IgM specific antibodies against SARS-CoV-2 reached 100% by day 39 of illness, with a median time to seroconversion of 11 days for total antibodies.
- From day 8 after the onset of symptoms, antibody assay showed a marked increase in sensitivity, reaching over a 90% sensitivity on day 12 for total antibodies detection.2
RT-PCR
- Within 7 days of the onset of disease, RT-PCR testing had the highest sensitivity compared to serological testing.2
Therefore, the study demonstrated that in later phase assays (days 15-39), the sensitivity of antibody assays was more than double that of molecular RT-PCR testing. Also, patients who have an undetectable dosage of viral RNA in their respiratory tracts, antibodies assay could come up positive depending on the number of days since symptom onset. Further results have suggested that a higher titer of total antibodies could be considered a risk factor for critical illness, thus raising a question pertaining to its usage in establishing a clinical prognosis.2
Finally, although IgM-IgG combined assay can have many limitations, it can be used along with RT-PCR tests and clinical presentations for better management and follow up on a large scale.
This data comes from multiple clinical studies to validate the clinical efficiency of the IgM-IgG combined immunoassay test. The first study analyzed the serological assay used on 397 PCR confirmed COVID-19 patients and 128 negative patients at 8 different clinical sites at 6 different provinces all over China. The second study used was conducted in Shenzhen Third People’s Hospital between January 11 and February 9, 2020, where 173 patients diagnosed with COVID-19 with RT-PCR were enrolled and agreed to donate their plasma samples. Patients had either acute respiratory syndromes and/or chest CT imagery with SARS-CoV-RNA that was detectable in their respiratory specimens. Assays using enzyme-linked immunosorbent assay (ELISA) kits to detect total antibody (Ab), IgM antibody and IgG antibody counts against SARS-CoV-2 were done. The third study was done in Munich, Germany, and provided a detailed virological analysis of the oro- or nasopharyngeal swab specimens of 9 hospitalized cases. The fourth study was carried out in Wuhan, China, in January of 2020, where epidemiological, clinical, laboratory and radiological data was collected on 41 patients hospitalized with a coronavirus diagnosis. Treatment options and clinical outcomes were also part of the study. The fifth study was done in Vietnam during January and February 2020, where 2 groups of 12 patients with SARS-CoV-2 infection were monitored with 30 throat swab specimens and viral RNA was analyzed with RT-PCR testing. The sixth study was completed at Jinyintan Hospital and Wuhan Pulmonary Hospital in Wuhan, China, with 191 patients. Their demographic, clinical, treatment and laboratory data were collected, as well as serial samples for viral RNA detection, to further understand the clinical course of COVID-19, including its viral shedding.
Sources:
- Li, Z., Yi, Y., Luo, X., Xiong, N., Liu, Y. (January 27, 2020). Development and Clinical Application of A Rapid IgM‐IgG Combined Antibody Test for SARS‐CoV‐2 Infection Diagnosis. Journal of Medical Virology. https://doi.org/10.1002/jmv.25727
- Zhao J., Yuan Q., Wang H., et al (March 28, 2020). Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019. Clinical infectious diseases. https://doi.org/10.1093/cid/ciaa344
- Wölfel, R., Corman, V.M., Guggemos, W. et al. (April 1, 2020). Virological assessment of hospitalized patients with COVID-2019. Nature. https://doi.org/10.1038/s41586-020-2196-x
- Huang C, Wang Y, Li X, et al. (January 24, 2020). Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet. https://doi.org/10.1016/S0140-6736(20)30183-5
- Le, T.Q.M., Takemura, T., Moi M.L., et al. (April 2, 2020). Severe acute respiratory syndrome coronavirus 2 shedding by travelers. Emerging Infectious Diseases. https://doi.org/10.3201/eid2607.200591
- Zhou, F., Yu, T., Ronghui, D., et al. (March 11, 2020). Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. The Lancet. https://doi.org/10.1016/S0140-6736(20)30566-3