key: cord-0690190-3ejychjf
authors: Ravi, Neeraja; Lee Cortade, Dana; Ng, Elaine; Wang, Shan X.
title: Diagnostics for SARS-CoV-2 detection: A comprehensive review of the FDA-EUA COVID-19 testing landscape
date: 2020-07-18
journal: Biosens Bioelectron
DOI: 10.1016/j.bios.2020.112454
sha: f55595c8a02ab130255ee3b18a0e013c718c3814
doc_id: 690190
cord_uid: 3ejychjf

The rapidly spreading outbreak of COVID-19 disease is caused by the SARS-CoV-2 virus, first reported in December 2019 in Wuhan, China. As of June 17, 2020, this virus has infected over 8.2 million people but ranges in symptom severity, making it difficult to assess its overall infection rate. There is a need for rapid and accurate diagnostics to better monitor and prevent the spread of COVID-19. In this review, we present and evaluate two main types of diagnostics with FDA-EUA status for COVID-19: nucleic acid testing for detection of SARS-CoV-2 RNA, and serological assays for detection of SARS-CoV-2 specific IgG and IgM patient antibodies, along with the necessary sample preparation for accurate diagnoses. In particular, we cover and compare tests such as the CDC 2019-nCoV RT-PCR Diagnostic Panel, Cellex's qSARS-CoV-2 IgG/IgM Rapid Test, and point-of-care tests such as Abbott's ID NOW COVID-19 Test. Antibody testing is especially important in understanding the prevalence of the virus in the community and to identify those who have gained immunity. We conclude by highlighting the future of COVID-19 diagnostics, which include the need for quantitative testing and the development of emerging biosensors as point-of-care tests.

A With the quickly changing landscape of available diagnostic tests for COVID-19, it is necessary for a holistic review and evaluation of diagnostic resources to be assembled. The goal of this review is to present an analysis of the current FDA-EUA diagnostic landscape for COVID-19, from patient specimen collection to commercially available diagnostic tests and future directions. We will begin our review by highlighting the structure of SARS-CoV-2 and the suspected roles and diagnostic interest of its proteins. We then cover relevant patient specimen collection techniques and sample preparation necessary prior to diagnostic testing, specifically focusing on existing viral RNA isolation methods and commercially available kits. Finally, we will move into our analysis of the current FDA-EUA diagnostic landscape, covering commercially available COVID-19 diagnostic platforms, and discussing COVID-19 immunity and how it will shape retrospective diagnostic development as well as epidemiological studies.

We conclude with a discussion on necessary future works and important avenues of research. Table 1 . Previous studies looking at SARS-CoV have demonstrated that the strong antibody responses against the spike and the nucleocapsid proteins are of high diagnostic utility (Chang et al., 2020) .

The CDC has mandated that testing for SARS-CoV-2 be conducted only in consultation with a licensed healthcare provider and on persons demonstrating symptoms of COVID-19

(CDC, "Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons for Coronavirus Disease 2019 (COVID-19)", 2020). The CDC has recommended nucleic acid testing of upper respiratory specimens collected by swabs. Nasopharyngeal (NP) specimens are the preferred choice for swab-based SARS-CoV-2 testing, followed by oropharyngeal (OP) specimens. As of June 2020, the CDC has allowed nasal swabs to be taken by the patient (self-swab) or health worker and used as a valid specimen for testing when NP swabs are not available. SARS-CoV-2 and its relevant biomarkers can be found in multiple specimen types besides NP, OP and nasal swabs, such as lower respiratory and blood-based specimens. Testing on alternative specimen types may be necessary depending on the goal of the test, variability of patient condition (i.e. intubation), or need for re-testing after a negative result.

Here, we will cover upper respiratory, lower respiratory, and blood/serum/plasma specimens. Currently, NP swabs are the recommended specimen for COVID-19 diagnostics, but they must be collected by a trained healthcare worker. The current surge in the number of patients displaying COVID-19 symptoms reduces the availability of healthcare workers and the appropriate personal protective equipment necessary to perform NP or OP swabs. Considering these shortages, the CDC has approved onsite patient self-swab collection for nasal mid-turbinate swabs and anterior nares (nasal) swabs. Self-collected nasal swabs are less invasive and more comfortable compared to NP swabs collected by a healthcare worker. As the pandemic escalates, many of the specimens collected will be nasal mid-turbinate or nasal swabs. Nasopharyngeal and nasal washes/aspirates are collected using saline filled syringes or mechanical suction to collect specimens; even though they are considered acceptable specimens for COVID-19 diagnostics, the resources and time needed to perform them make them lower priority specimens to collect.

When possible, the CDC recommends healthcare providers also take lower respiratory specimens, which can be valuable samples for diagnosing COVID-19 in severe cases . We discuss three types of lower respiratory specimens: sputum, tracheal aspirate and bronchoalveolar lavage (BAL) fluid. Sputum specimens should be collected from patients with deeply productive coughs, but they should never be induced. A recent study has shown that when naturally produced, sputum is a more robust specimen for diagnosis compared to throat swabs (Lin et al., 2020) . Tracheal aspirates and BAL fluid specimen collection techniques involve flushing either the trachea or a small lung section with saline and aspirating it for analysis. These methods are quite invasive and should only be used if clinically indicated.

Whole blood samples are collected by a healthcare provider by inserting a needle into a vein and directly collecting whole blood into a sterile tube.

These samples can be stored as whole blood, serum, or plasma. In general, it is recommended that whole blood is processed into serum or plasma for storage if the sample will be analyzed at a later date. The CDC does not recommend that whole blood, serum or plasma be used as a specimen for an onsite diagnosis of COVID-19 at this time. However, whole blood is useful for conducting blood smears, looking at morphology and cell count, and examining blood cultures.

After whole blood collection in a sterile tube, serum is generated after leaving whole blood at room temperature to clot. The blood is then centrifuged and the liquid supernatant, or serum, is separated from the remaining clot. For plasma collection, whole blood is collected in a sterile tube containing an anticoagulant. The blood is centrifuged, and the plasma supernatant is separated from the red blood cells and buffy coat. Serum and plasma samples can be used in serological diagnostics for epidemiological studies and recovery analysis, which will be addressed later in the review. These samples can be further processed for the detection of viral RNA in molecular diagnostics. More recently, finger prick blood drop samples collected at point-of-care or drive-through sites are used in lateral flow assays (usually immunoassays); most of these products are currently pending FDA approval. A summary of the covered swabs and extraction methods can be seen in Table 2 In this section, we will cover the two main types of diagnostic tests with FDA-EUA approval: nucleic acid diagnostic testing to diagnose active COVID-19 infections, and serological testing to determine COVID-19 presence in a community. The practical diagnostic considerations of RT-PCR test and serological tests are summarized in Table 3 . Once a patient has SARS-CoV-2 specific antibodies circulating in his or her bloodstream, a natural next question is how long these antibodies remain, or how long a patient has protective

immunity. To answer this, researchers have analyzed immune responses to the coronaviruses that cause the common cold, finding that protection decreases by a year or two (Tyrrell et al., 1996) . to be conducted to determine how long this antibody-mediated immunity will last, these preliminary studies indicate that patients who have seemingly recovered should still exercise caution and maintain good practices such as social distancing. 

While there are many COVID-19 diagnostic products in the market with FDA-EUA approval, they can broadly be grouped into diagnostic tests and antibody tests. Diagnostic tests focus on nucleic acid or viral antigen detection, and are primarily utilized for active COVID-19

diagnoses. Antibody tests measure antibodies against SARS-CoV-2 in patients; these tests are utilized to glean who may still be at risk, and more broadly assess the prevalence of COVID-19 in a community. Some of the primary customers for diagnostic tests are hospitals, drive-through clinics, and academic institutions interested in providing COVID-19 diagnoses for the public; on the other hand, some of the main customers for antibody tests are healthcare providers, laboratories, public health staff, and community clinics interested in mass screening efforts to determine population prevalence.

One of the main reasons there are so many SARS-CoV-2 nucleic acid detection tests in the market is that there was never a standard protocol set in place (The Conversation US, Inc, 2020). After the CDC's initial test proved to be faulty, and test development restrictions were lifted at the end of February, many academic institutions and companies took the initiative to develop their own tests to help the country increase testing efforts. As a result, many of these There are many antibody tests circulating the market, including those that detect IgG and IgM, IgG only, and total antibody. Currently, the CDC reports that there is not a major advantage between serological assays that detect IgG specific to SARS-CoV-2, IgG and IgM specific to SARS-CoV-2, or total antibody (CDC. "Frequently Asked Questions about Coronavirus 

Many of the manufacturers selling FDA-EUA approved diagnostic products only have test prices available via inquiry. However, the major customers of these diagnostic tests, such as have presented with COVID-19 symptoms and have been referred by a medical provider (npr, 2020). In general, the clinical labs or hospitals conducting the tests will directly bill the test cost to an insurance provider; if uninsured, these costs go to a government program such as Medicare or Medicaid if the patient qualifies.

As the COVID-19 pandemic is rapidly spreading, one major focus of future work will be continuing development of POC and home tests that do not require extensive training for operation, are easily deployable to outpatient settings and clinics, and are low-cost while still preserving the accuracy of diagnosis. POC tests have a lower barrier to implementation than labbased tests: if FDA cleared, POC tests don't require a trained professional to operate, so users have the power to perform all the steps of the test on their own. In this way, users can know their results within minutes and seek professional help sooner, instead of waiting longer for results from a lab-based test. As we prepare for the possibility of a second wave, POC tests are more conducive to mass testing compared to lab-based tests, so we can rapidly provide accurate identification of not only symptomatic SARS-CoV-2 infected patients, but also provide detection of early infections or asymptomatic individuals. To increase the throughput and scalability of the number of tests that can be run, more POC tests should be combined with automated sample processing systems in the future, allowing more patients to get diagnoses in a timely manner. As the disease progresses through the population, it will be important to develop testing systems that can provide quantitative diagnoses, rather than merely qualitative 'yes or no' results regarding SARS-CoV-2 or IgG and IgM presence. It is imperative that we quantify the viral load to have a better sense of where a patient is in disease progression after symptom onset. Similarly, by quantifying the amount of SARS-CoV-2 specific IgG and IgM antibodies present, we can determine if a patient or a population has acquired immunity, and if so, exactly how much. This will be beneficial in identifying strong responders for providing convalescent sera.

Another future effort will focus on measuring SARS-CoV-2 infection with a host transcriptional response signature. In the past, these diagnostic profiles have been determined through a meta-analysis of publicly available data, resulting in a group of up to ten biomarker genes whose expression levels in the host are different at a specific disease state (Zhai et This could expedite vaccine trials, allowing a SARS-CoV-2 vaccine to reach the population sooner. One of the main challenges with creating such a signature is that a large population RNA-sequencing data needs to be curated first from patients with the COVID-19 disease, which will take time. Additionally, gene expression measurements involve isolating mRNA from peripheral blood mononuclear cells (PBMCs), reverse transcribing to cDNA, and amplifying the cDNA with the primers of interest. This will add on to the sample processing time, and requires a skilled technician to perform the task. Nevertheless, this is a promising quantitative approach to disease classification, and can hopefully improve the accuracy of future diagnoses.

In this review, we have covered diagnostics for measuring the presence of SARS-CoV-2, which can broadly be grouped into two categories: nucleic acid detection and antibody detection.

The standard of care for nucleic acid detection is RT-PCR, and this is currently being used to identify positive and negative cases of COVID-19 by testing for SARS-CoV-2 viral RNA in a patient swab sample. The most common swab types are nasopharyngeal swabs and oropharyngeal swabs. We have also covered antibody detection through serological assays, most commonly ELISA, in which SARS-CoV-2 specific IgG and IgM antibodies are detected to measure general immunity to SARS-CoV-2. This is important not only to examine the infection severity and chance for successful recovery in an individual, but also to determine if herd immunity has been reached for an overall population. Future work in this field will include quantitative testing approaches in nucleic acid and antibody/antigen assays and the development of a SARS-CoV-2 specific genetic signature. As the situation is rapidly evolving and we are learning more about this disease every day, we are more broadly advancing the field of infectious disease diagnostics.

E.N. would like to acknowledge support from the Cancer-Translational Nanotechnology

Training (Cancer-TNT) Program. D.L.K would like to acknowledge support from the Stanford Graduate Fellowship. This work is in part supported by National Institutes of Health grants R01 AI125197 and U54CA199075.

Authors have no competing interests to declare. Figure 1 : SARS-CoV-2 Virus structure, with five main proteins. Details of protein function can be found in Table 1 . 

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• Current standard-of-care for COVID-19 diagnosis is RT-PCR, used to detect SARS-CoV-2 viral RNA.• Serological assays for detection of SARS-CoV-2 specific IgG and IgM patient antibodies are useful to assess virus prevalence in the community.• Point-of-care tests for COVID-19 diagnosis can expedite a response to those who need it and alleviate the burden placed on healthcare providers.• Future testing strategies include developing quantitative COVID-19 diagnostic tools.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: