Viral infections are difficult to diagnose based on the patient’s clinical presentation alone. A reliable and accurate diagnosis requires laboratory-based testing for the pathogen of interest. Such laboratory testing involves either the direct detection of viral antigens, or detection of virus-specific antibodies in clinical specimens.
In the last few years, there has been a major evolution in lab-based detection of viral infections, leading to more accurate diagnosis and appropriate patient management. Traditional methods have been replaced by sensitive nucleic acid detection methods and measurements of virus-specific antibodies (1).
The COVID-19 era has revolutionized diagnostic virology and paved the way for large-scale implementation of real-time reverse transcriptase polymerase chain (RT-PCR) reaction technology to detect the virus’ genetic information (RNA) and solid-phase enzyme immunoassays (EIAs) to measure specific antibodies.
Merits of Molecular Testing
Most of these molecular technologies have major advantages such as high sensitivity and a wide dynamic range. They are easy to perform, and do not require biosafety level 3 (BSL-3) facilities. They generate reproducible and qualitative results and can be automated to yield high throughputs with quick turnaround times. Most infectious disease RT-PCRs, including all COVID-9 RT-PCR tests that have received emergency use authorization (EUAs) are qualitative assays.
These rapid advances in molecular assays resulted from developments in oligonucleotide synthesis, high-throughput automated processes for nucleic acid extraction, and real-time identification of PCR products (1). Some of the major advantages of viral detection by RT-PCR assays are high sensitivity and specificity, speed, applicability for all viruses and their variants, the ability to quantify the viral load, and the possibility of multiplexing.
Pitfalls of Molecular Testing
However, they do come with certain pitfalls—difficulties in sample collection, the need for sophisticated lab equipment and skilled personnel, high risk of contamination, false positives, and the need for appropriate quality controls (2). The shortcomings in existing lab-based systems for viral diagnostics were accentuated with the COVID-19 pandemic.
Due to the need for temperature cycling, RT-PCR is not adaptable for point-of-care (POC) detection. RT-PCR detection systems rely on adequate viral load at the sample collection site. When the viral replication time-window is missed, it leads to an increase in false-negatives. Approximately eleven days after the onset of COVID-19 infection, there is a rapid decline in positive RT-PCR rates as the infected patients start to produce anti-viral antibodies (3-7).
Alternatives
Despite RT-PCR being the gold standard for clinical COVID-19 diagnosis, other low-cost, rapid systems are emerging in the market such as reverse transcription loop mediated isothermal amplification (LAMP). LAMP-based testing solutions are becoming a quicker alternative to PCR since they do not need thermocyclers. LAMP is a simple, highly specific, rapid, and portable test that does not need viral extraction and hence, can be used in POC settings. Moreover, these systems can use saliva as an alternative matrix to other uncomfortable respiratory tract samples (8).
Other alternatives for high-throughput pathogen diagnosis are the CRISPR-Cas systems that sensitively detect nucleic acids. These systems are rapid and accurate with fast turn-around-times, low-cost, and scalability without the need for complex laboratory equipment and can be used in POC settings (9).
Other novel techniques such as Next Generation Sequencing (NGS) allow for sequencing of DNA directly from DNA fragments without the need for cloning in vectors, generating big data at high speed and low cost from a single run.
During COVID-19, NGS has allowed for an effective and unbiased way to identify new SARS-CoV-2 strains. NGS helps identify mutations rapidly to prevent the spread of new variants, detect mutations that can impact vaccine efficacy, and screen targets for COVID-19 therapeutics (10).
Immunodiagnostics
Direct detection of virus antigens in a patient sample has become popular for many infections, including COVID-19. Use of solid-phase enzyme immunoassays has allowed rapid detection of viral antigens with high sensitivity and specificity. In addition to laboratory-based testing, POC antigen assays based on the lateral-flow technology, like pregnancy tests, have been used to detect SARS-CoV‑2 antigens. These assays are rapid and provide results in about 15 minutes.
However, such assays are qualitative, might be difficult to interpret, are not as sensitive as the RT-PCR tests, and can provide false-positives (11). Other common immunoassays are radioimmunoassays, time-resolved fluorescence immunoassays, and immunoperoxidase stainings.
Serological tests measuring specific antibody response can detect past exposure to the virus in symptomatic and asymptomatic patients. Results are qualitative, semi-quantitative, or quantitative and useful for conducting epidemiological studies and for measuring herd immunity.
Measuring antibody responses to the spike protein is useful for monitoring vaccine response in clinical trials since most vaccines are based on this region of SARS-CoV-2. However, there are currently no guidelines on testing patients after vaccination to determine vaccine efficacy. Serological assays can identify individuals with antibody response against SARS-CoV-2 who can be donors of hyperimmune plasma to treat patients (12).
ELISAs can also be used to detect antibodies showing that a person has been exposed to the virus or to aid in diagnosis. Measuring IgG antibodies cannot detect active infections, but they can detect exposure to the virus retrospectively. For some viral infections (not including COVID-19), recent infections can be diagnosed by the presence of IgM antibodies. However, since these assays are never 100% sensitive or specific, chances of false-positives or negatives remain.
References
- Burrell CJ, Howard CR, Murphy FA. Laboratory Diagnosis of Virus Diseases. Fenner and White’s Medical Virology. 2017:135-154.
- Bustin SA, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech. 2004;15(3):155-166.
- Liu L, Liu W, Zheng Y, et al. A preliminary study on serological assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 238 admitted hospital patients. Microbes Infect. 2020;22(4-5):206-211.
- Cevik M, Kuppalli K, Kindrachuk J, et al. Virology, transmission, and pathogenesis of SARS-CoV-2. BMJ 2020;371:m3862.
- Cevik M, Tate M, Lloyd O, et al. SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and meta-analysis. The Lancet Microbe 2021;2:e13-e22.
- Hirotsu Y, Maejima M, Shibusawa M, et al. Comparison of automated SARS-CoV-2 antigen test for COVID-19 infection with quantitative RT-PCR using 313 nasopharyngeal swabs, including from seven serially followed patients. International Journal of Infectious Diseases 2020;99:397-402
- Ganguli A, Mostafa A, Berger J, et al. Rapid isothermal amplification and portable detection system for SARS-CoV-2. Proceedings of the National Academy of Sciences. 2020;117(37):22727.
- https://www.fda.gov/media/138249/download
- Palaz F, Kalkan AK, Tozluyurt A, Ozsoz M. CRISPR-based tools: Alternative methods for the diagnosis of COVID-19. Clinical Biochemistry. 2021;89:1-13.
- Chiara M, D’Erchia AM, Gissi C, et al. Next generation sequencing of SARS-CoV-2 genomes: challenges, applications, and opportunities. Brief Bioinform. 2021;22(2):616-630.
- Potential for False Positive Results with Antigen Tests for Rapid Detection of SARS-CoV-2 – Letter to Clinical Laboratory Staff and Health Care Providers. FDA. Published 2021. Updated 11/03/2020. Accessed 04/17/2021.
- Venkataraman I, Sabalza M, Naides S. The significance of serology antibody testing for SARS-CoV-2. 2020;52. Published 06/24/202.
Iswariya Venkataraman, PhD, is associate director of scientific affairs at EUROIMMUN US, a PerkinElmer company.
