Laboratory Testing Methods for Novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2)

D'Cruz, Roshan J.; Currier, Arthur W.; Sampson, Valerie B.

doi:10.3389/fcell.2020.00468

REVIEW article

Front. Cell Dev. Biol., 04 June 2020

Sec. Molecular and Cellular Pathology

Volume 8 - 2020 | https://doi.org/10.3389/fcell.2020.00468

This article is part of the Research Topic Coronavirus Disease (COVID-19): Pathophysiology, Epidemiology, Clinical Management and Public Health Response View all 400 articles

Laboratory Testing Methods for Novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2)

$\nRoshan J. D&#x;Cruz$ Roshan J. D'Cruz

Arthur W. Currier

Valerie B. Sampson^*

Nemours/Alfred I. duPont Hospital for Children, Wilmington, DE, United States

Following the first reports of coronavirus disease-19 (COVID-19) by China to the World Health Organization (WHO) on 31st December 2019, more than 4,302,774 novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cases have been reported by authorities in 212 countries and territories by 12th May 2020. The outbreak and spread of COVID-19 worldwide, highlights the critical need for developing rapid and accurate diagnostic testing methods for emerging human coronavirus (CoV) infections. Testing is crucial to track the spread of disease during a pandemic, and to swiftly permit public health interventions including isolation, quarantine, and appropriate clinical management of afflicted individuals. The key components of viral diagnostic tests are (1) collection of the appropriate sample (blood, nasal swab, and throat swab), (2) availability of the genetic and proteomic sequences of the novel virus for analysis, and (3) rapid and accurate laboratory testing methods. The current gold standard for the molecular diagnosis of SARS-CoV-2 infection is the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the qualitative and quantitative detection of viral nucleic acids. Other relevant laboratory methods include enzyme-linked immunoassays (EIA) for viral antibody and antigen detection, and serum viral neutralization (SVN) assays for antibody neutralization determination. The challenges faced in developing a diagnostic test for a novel pathogen are the ability to measure low viral loads for early detection, to provide low or no cross-reactivity with other viral strains and to deliver results rapidly. Several point-of-care molecular devices are currently being integrated for fast and accurate diagnosis of SARS-CoV-2 infections. This review discusses the current laboratory methods available to test for coronaviruses by focusing on the present COVID-19 outbreak.

Introduction

Coronavirus disease-19 (COVID-19) is caused by a novel coronavirus (CoV) that was originally reported in Wuhan, Hubei province, China in December 2019 (World Health Organization, 2020a). The International Committee on Taxonomy of Viruses named the virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection by SARS-CoV-2 causes a respiratory illness that varies in severity from mild upper respiratory symptoms akin to the seasonal flu, to severe progressive respiratory failure that requires intensive care and can lead to death. Asymptomatic carriers of the virus have also been reported and pose a significant public health threat due to their ability to unknowingly spread the virus (Chan et al., 2020a). SARS-CoV-2 represents the third CoV in this millennium to cross species from animals to humans and cause a severe respiratory disease after Middle-East respiratory syndrome coronavirus (MERS-CoV) in 2012 (Zaki et al., 2012), and SARS-CoV in 2003 (Drosten et al., 2003; Ksiazek et al., 2003). This novel CoV has now been identified as the seventh CoV that is transmissible between humans (including HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1) (Salata et al., 2019). On 30th January 2020, the World Health Organization (WHO) declared the SARS-CoV-2 epidemic a public health emergency of international concern and was upgraded to a pandemic on 11th March 2020. At least 4,302,774 confirmed cases and 289,561 deaths worldwide were reported as of 12th May 2020 (worldometers.info/coronavirus/). Diagnostic testing is critical during a pandemic as the ability to track the spread of SARS-CoV-2 is essential for effective disease management and control.

SARS-CoV-2 is a positive-sense, single-stranded RNA (ssRNA), group IV virus. The genome was sequenced from the bronchoalveolar lavage fluid of a patient (Genbank: MN908947) and shared through the Global Initiative on Sharing All Influenza Data (GISAID) platform on 12th January 2020 (Wu et al., 2020). The ~30 k base pair genome is highly similar to the human SARS-CoV and bat CoV-SARS-like genomes with 14 open reading frames (ORFs) that encode structural, replication and non-structural accessory proteins, as depicted in Figure 1. Molecular modeling studies demonstrate that like SARS-CoV, SARS-CoV-2 is surrounded by a lipid bilayer membrane, containing structural membrane (M) and envelope (E) proteins that interact to form the viral envelope (Durrant et al., 2020). This layer also contains spike glycoproteins (S) that give the characteristic “corona” appearance of this family of viruses. The spike proteins bind specific host cell receptors to facilitate host cell attachment and entry (Graham and Baric, 2010). The nucleic acid-associated protein binds the RNA genome and forms the nucleocapsid (N). Other proteins include replication and non-structural accessory proteins that are listed in Table 1. Reports of different strains of SARS-CoV-2 suggest an early split from the SARS-CoV-2 lineage and/or the virus is mutating. Ongoing research provides insight into the unique and conserved features of the genome and proteome of SARS-CoV-2 to track mutations and generates evidence about the evolution of the virus (Phan, 2020; Wang et al., 2020). This is important as these changes may affect key structural and non-structural components of SARS-CoV-2 that can render some diagnostic tests ineffective or less sensitive and can also impact the selection of epitopes for the development of new tests.

FIGURE 1

Figure 1. Schematic representation of SARS-CoV-2 genome. SARS-CoV-2 contains a positive-sense, positive-stranded mRNA genome with a 5′ capped mRNA sequence (C) and a 3′ poly-A tail. The coding genes are: ORF1a, ORF1b, Spike (S), ORF3a, ORF3b, Envelope (E), Membrane (M), ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF14, Nucleocapsid (N), and ORF10.

TABLE 1

Table 1. Proteins associated with the 14 ORFs of SARS-CoV.

The spread of SARS-CoV-2 is primarily by respiratory droplets that arise from individuals that harbor the virus. Symptomatic individuals with the disease are one source of virus, but a major public health concern is transmission by mildly ill or asymptomatic individuals during the incubation period. Rapid viral diagnostic testing for SARS-CoV-2 is critical to identify these individuals and facilitate the implementation of protective measures such as social distancing, quarantine and isolation that help to mitigate the spread of the virus in the community. The development of rapid and accurate tests that detect antibodies post-infection provide information about an individual's exposure to the virus and can be used to monitor the possibility of immunity, relapse or reinfection. This supports interventions to protect higher risk populations from developing more severe illness and can be used to investigate the efficacy of passive antibody therapies for COVID-19 infection. This review describes the available testing methods for SARS-CoV-2 and brings to light the importance of laboratory testing to control this disease and prepare for possible future disease threats.

Detection of SARS-CoV-2 by Real Time Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)

RT-PCR detects the genetic material of SARS-CoV-2 to identify the virus and/or quantify viral load. Comparisons of the ssRNA genetic sequences of this virus have shown similarities to SARS-CoV and several bat coronaviruses (Lu et al., 2020). This detailed knowledge has allowed the rapid development of RT-PCR assays for SARS-CoV-2 using SARS-CoV and known CoVs as references.

Sample Collection

Upper and lower respiratory samples are collected for detection of HCoV. Nasopharyngeal swabs are high priority specimens for SARS-CoV-2, and low priority specimens include oropharyngeal swabs, bronchoalveolar lavage, tracheal aspirates, and sputum (CDC, 2020a).

RNA Isolation

RNA is extracted from clinical specimens using approved viral isolation kits (Corman et al., 2020).

Real-Time RT-PCR

RNA is reverse transcribed to cDNA and subsequently amplified using a real-time quantitative PCR instrument. WHO announced various primer and probe sets for SARS-CoV-2 previously developed in China, Germany, Hong Kong, Japan, Thailand, and USA (World Health Organization, 2020b; Table 2). Primers targeting different sections of the virus genetic sequence including the envelope E gene, the RNA-dependent RNA polymerase (RdRp) gene, and the N gene (Chu et al., 2020; Corman et al., 2020; World Health Organization, 2020b). Targeting the E gene is reported for highest sensitivity, followed by the RdRp gene for confirmation (Corman et al., 2020). Some laboratories have multiplexed PCR tests consisting of multiple primer and probe sets located at different regions in the SARS-CoV-2 genome. These assays can be designed to contain primer sets targeting multiple genes simultaneously (RdRp/hel, S, N) (Chan et al., 2020b), or to detect different regions in a single target such as the N gene (U.F.A.D. Administration, 2020 Table 2). The use of multiplex assays is potentially beneficial as this can improve sensitivity in cases of loss or degradation of viral RNA during specimen collection and nucleic acid extraction, or in the event of mutation of the virus genome. These assays use in vitro synthesized RNA derived from transcripts (e.g., BetaCoV_Wuhan_WIV04_2019, GISAID Access number: EPI_ISL_402124) as positive controls and to generate standard curves. An internal control using RNAse P (RP) verifies the presence and quality of nucleic acid in samples and molecular grade nuclease-free water is used as a negative amplification control. A negative patient sample serves both as a negative extraction control to monitor cross contamination across samples and to validate test reagents.

TABLE 2

Table 2. Table of primer and probe sequences for detecting SARS-CoV-2 genes.

Advantages

RT-PCR is the frontline diagnostic test for COVID-19 that is capable of analyzing thousands of specimens in a single day and shows a testing sensitivity of 95% (Corman et al., 2020). The anticipated limit of detection of the SARS-CoV-2 RT-PCR test is <10 copies/reaction (Chu et al., 2020) which allows early detection of low viral titers. Gene amplification indicates a positive result for the presence of SARS-CoV-2 RNA and should correlate with clinical observations, patient history, and epidemiological information.

Disadvantages

False positive results could be generated by cross-reactivity of primers with nucleic acids arising from co-infection with other viruses or bacteria. In these cases, the agent detected may not be the definite cause of disease. Matching of the SARS-CoV-2 RT-PCR primers and probes using reliable libraries (e.g., BLAST) is necessary to ensure there is no homology with other CoVs like SARS-CoV from 2003 or other organisms such as Staphylococcus aureus and Candida albicans. False positives can also occur if reagents in a laboratory become contaminated, which is a major concern, particularly with the high volume of testing encountered during a pandemic. A negative patient sample is useful to identify this error in testing.

False-negative results could potentially arise from mutations occurring in the primer and probe target regions in the SARS-CoV-2 genome. Negative results do not preclude SARS-CoV-2 infection, and results should be validated with different primer sets against the same gene target and combined with patient history and other clinical data to accurately determine patient infection status.

Key Logistics

Provisions for testing laboratories, the use of approved tests and validation of results with governing authorities to develop master protocols for use by multiple investigators must be in place to achieve rapid testing capacity. The output for number of tests per day and number of individuals tested per day relies on the laboratory capacity, trained staff, reagents, supplies and equipment. Large quantities of specific high-grade reagents are needed to perform tests and supplies can be quickly depleted in a pandemic. This impacts the turnaround time for RT-PCR diagnostic testing that ranges between 2 and 5 days. Strategies to rapidly scale up testing for novel HCoVs must be considered for future diagnostic testing.

Detection of Antibodies Against SARS-CoV-2 Proteins by Enzyme Immunosorbent Assay (EIA)

EIA assays are diagnostic methods used to identify antibodies in patient blood sample or nasopharyngeal swabs. Enzyme-linked immunosorbent assays (ELISAs) for antibody detection against SARS-CoV-2 measure the host humoral response including IgM, IgG, and IgA to define previous exposure to the virus (Guo et al., 2020; Okba et al., 2020). IgM is the first immunoglobulin that is produced in response to an antigen and is primarily detected during the early onset of disease (3–7 days). IgG is the most abundant immunoglobulin that is produced in response to an antigen (7–25 days) and is maintained in the body after initial exposure and may have a protective role for acquired immunity. The IgA immunoglobulin plays a crucial role in the immune function of mucous membranes.

The SARS-CoV-2 S glycoprotein that mediates attachment and entry into cells is surface exposed and is a key target for the production of host neutralizing antibodies (Walls et al., 2016). This feature has made the S protein the focal target of antibody and vaccine development. The N protein in HCoVs functions as an antagonist of interferon (Kopecky-Bromberg et al., 2007; McBride et al., 2014) and viral encoded repressor (VSR) of RNA interference (RNAi) that facilitates viral replication, and is also a key target for antibody design (Leung et al., 2004). Recombinant antigens derived from the receptor binding domain of S protein (rS) as well as recombinant N protein (rN) are being developed as suitable diagnostic targets to detect IgM, IgG, and IgA antibodies. Dual detection of IgM/IgG and IgG/IgA immunoglobulins is under development for use in conjunction with nucleic acid detection for detecting active infection and to define previous exposure to SARS-CoV-2.