Understanding the Utility of RT-PCR and Antibody Testing for Diagnosis of CoVID-19 Infection

-Pradeep Neupane

Laboratory diagnosis of human coronavirus (COVID-19 virus/SARS-CoV-2) infection mainly relies on detection of viral RNA in clinical samples by “gold standard” reverse transcription polymerase chain reaction (RT-PCR) or detection of viral proteins or viral-specific antibodies (IgM/IgA/IgG) by serological immunoassays (rapid diagnostic tests (RDT)). None of these tests has 100% diagnostic accuracy.

Although RT-PCR is a highly sensitive and specific, it generates numerous false negative results meaning persons with active COVID-19 infection may test negative. Recent studies indicate that relying solely on RT-PCR to diagnose the virus infection is a risky strategy. CoVID-19 viral loads may vary considerably in various types of respiratory samples from the same patient. Given the variability of viral RNA loads in clinical specimens, RT-PCR detection may miss some true cases due to low viral loads or sampling variability. The accuracy of test results is dependent on the integrity of specimens. RT-PCR is dependent on procuring high-quality RNA from respiratory samples. Improper collection and handling of clinical specimens can affect the quality of the sample and hence the outcome of the test results, particularly RT-PCR results. Erroneous results as a result of specimen mismanagement compromise the care of patients and hamper the efforts to curb the global CoVID-19 pandemic.

On the other hand, antibody-detecting RDT show some advantages over RT-PCR. IgM/IgA/IgG serological tests are used to detect anti-viral antibodies in serum (blood) that are produced by host immune system in response to the viral infection. Antibodies are more uniformly distributed in blood. IgM/IgA/IgG are considerably more stable than RNA, which makes IgM/IgA/IgG less susceptible to degradation during sample collection, handling, storage, and transportation. One of the major advantages of serological assay is that it can detect past infection since IgM/IgA/IgG (primarily IgG) can persist in the bloodstream for several weeks/months/years after the infection. It is unclear whether recovered CoVID-19 patients are immune to reinfection from CoVID-19. In other word, there is no evidence that antibodies provide protection against the CoVID-19 virus. Many components of host innate immune system including T helper cells, NK and/or cytotoxic CD8 T cells may play important roles in protecting against CoVID-19. 

Serological tests have several limitations including the following: 1) Antibodies are produced over days to weeks after initial infection, suggesting positive serological test result might not be indicative of active CoVID-19 infection; 2) Virus specific IgM/IgG antibodies may not be detected at least 7 to 11 days after infection since IgM and IgG antibodies response against CoVID-19 virus does not peak until ~9 and ~11 days after infection, respectively; 3) Presence of cross-reacting antibodies in serum (blood) from previous infections with other non-CoVID-19 human coronaviruses may result in false-positive test.

In conclusion, RT-PCR and serological assays, commonly used tests to diagnose the CoVID-19 infection, are often unreliable, generating erroneous test results. Although RT-PCR is the most frequently used “gold-standard” method for diagnosis of CoVID-19 infection, results of RT-PCR must be cautiously interpreted. Therefore, antibody detecting RDT should also be taken into consideration and their results in conjunction with RT-PCR can be invaluable in providing rapid diagnosis of CoVID-19 infection. Also, antibody testing will be crucial for accurate determination of serological immunity levels amongst populations and for investigating optimal strategies to control the spread of CoVID-19.

References

Liu, L., Liu, W., Wang, S. and Zheng, S., 2020. A preliminary study on serological assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 238 admitted hospital patients. medRxiv.

Li, Z., Yi, Y., Luo, X., Xiong, N., Liu, Y., Li, S., Sun, R., Wang, Y., Hu, B., Chen, W. and Zhang, Y., 2020. Development and clinical application of a rapid IgM‐IgG combined antibody test for SARS‐CoV‐2 infection diagnosis. Journal of medical virology.