key: cord-341416-6bh08901
authors: Smithgall, Marie C.; Whittier, Susan; Fernandes, Helen
title: Laboratory Testing of SARS CoV-2: A New York Institutional Experience
date: 2020-07-19
journal: nan
DOI: 10.1016/j.yamp.2020.07.002
sha: 
doc_id: 341416
cord_uid: 6bh08901

nan

title, status or ethnicity.

• There are two main categories of tests used to detect current or past viral infection: molecular and serologic assays.

• Molecular assays aim to determine if a patient is actively infected with a pathogen of interest, while the purpose of serologic testing is to determine prior exposure.

• Serological assays determine a patient's exposure history. At this time, it is unknown whether antibody detection equate to immunity. (EUA). Perhaps the most noteworthy outcome is that this scenario has made laboratory professionals more visible and respected and induced a deeper sense of ownership of the profession. This brief report will provide an overview of the types of testing available for SARS-CoV-2 patient management, as well how testing has impacted the situation in New York City. 

Clinicians rely on laboratory testing to provide clinically relevant, actionable results, which can direct both inpatient and outpatient care. There are two main categories of tests used to detect current or past viral infection: molecular and serologic assays. Antigen-detection assays have also been used historically for diagnostic purposes. Molecular assays aim to determine if a patient is actively infected with a pathogen of interest, while the purpose of serologic testing is to determine prior exposure. The most widely used assays for detection of SARS-CoV-2 utilize reverse transcriptase polymerase chain reaction (RT-PCR). This technique is already commonly used in microbiology laboratories to detect RNA specific to respiratory viral pathogens, such as influenza, respiratory syncytial virus (RSV), and others [3] . The World Health Organization developed the first quantitative RT-PCR test for detecting SARS-CoV-2 and subsequently the U.S. Centers for Disease Control and Prevention (CDC) began shipping its own RT-PCR test kits after receiving Emergency Use Authorization (EUA) by the U.S. Food and Drug Administration (FDA) on February 4, 2020. However, there were complications that became apparent during the validation process that caused a setback in deploying the assay to the diagnostic community [4] .

On February 29, 2020 the Wadsworth Center of the New York State Department of Public Health's RT-PCR assay was the second test to receive EUA. This assay, though, was not designed for high-throughput testing, and analyzed approximately 50 -60 specimens per day per platform with a turn-around-time of 4-6 hours from sample to answer. Consequently, testing remained at a minimum until mid-March 2020 when commercially-available, fully automated SARS-CoV-2 real-time assays began receiving EUA. These high-throughput automated assays include, but are not limited to, the cobas® SARS-CoV-2 Test run on the Roche COBAS 6800/8800 platform and the Abbott RealTime SARS-CoV-2 assay with the m2000 platform.

Rapid point-of-care tests such as Xpert® Xpress SARS CoV2 (Cepheid) and ID NOW TM COVID-19 (Abbott), which test single specimens, also became available. These molecular assays detect various viral targets including SARS-CoV-2 specific targets such as ORF1 a/b, a non-structural region and N2, a nucleocapsid recombinant protein as well pan-Sarbecovirus targets such as the envelope E-gene.

The ability to batch samples greatly increased testing capabilities in New York City. However, due to significant shortages of testing reagents, positive controls, collection swabs, transport media and personal protective equipment, only the most critically-ill patients presenting to the hospital were being tested. As a result, the biased positive rate of patients tested in New York State was around 50% and New York City was upwards of 70%. This crucial shortage in testing capacity significantly impacted the public health response's ability to contain the virus. The number of SARS-CoV-2 positive cases increased exponentially in New York and adjoining states such as New Jersey, making this region the epicenter of the pandemic (Fig. 1 ).

With the increase in the number of assays that were verified in several hospitals and laboratories within New York, testing was gradually expanded in April 2020, beyond individuals with a very high pre-test probability, to include all symptomatic individuals and people with exposure to known SARS-CoV-2. With this increase in the overall number of tests performed, the overall positive test rate decreased to approximately 20%, a more accurate reflection of the incidence of patients suffering from COVID-19 ( Figure 2 ). With practicing of social distancing and contact precautions, in addition to expanded testing, the positive rate within the NY community has remained steady since early May 2020, at about 5-7%. The overall statistics for New York from early March until May 26 th , 2020 can be visualized in Figure 2 . Briefly, since the start of the pandemic, over 2 million tests have been performed with an overall positive rate of approximately 20%. In terms of demographics, not only was incidence higher in males, but they also had a much higher fatality rate (58.2%) compared to females (41.8%). Communities of color and lower socioeconomic status also were more seriously impacted with higher rates of infection and mortality [5] .

To date there are more than 80 commercial laboratories and/or test kit manufacturers that have received approval for emergency use by the Federal Drug Administration (FDA) for SARS-CoV-2 testing with molecular assays accounting for the vast majority [6] . Various reports document success with different specimen types ranging from nasopharyngeal, oropharyngeal, anterior nasal and mid-turbinate nasal swab, swabs to nasal washes and saliva, some of which have received EUA [7] . In addition, the FDA recently granted EUA for an RT-PCR lab developed test for qualitative detection of SARS-CoV-2 in saliva specimens and a test that uses a home collection kit with nasal swabs [6] for details see https://www.fda.gov/emergency-preparednessand-response/mcm-legal-regulatory-and-policy-framework/emergency-use-authorization]. A recent report showed comparable detection of respiratory viruses by RT-PCR with saliva and NP specimens [8] . Saliva as a specimen type, is appealing for its reduced risk posed at the time of collection; however, larger studies comparing saliva with other validated specimen types are essential for documenting the reliability of this specimen type. In an effort to expand testing capabilities, manufacturers and laboratories have adopted self-collection devices using predominantly anterior nares and mid-turbinate for sample collection [9] . The wide range of specimen types and their varied collection times during the course of COVID-19 infection however, could contribute to the false negative rates seen in the RT-PCR assays. In fact, a recent study demonstrated that false-negative rate for SARS-CoV-2 RT-PCR testing can be as high as 67% in individuals tested up to 5 days after exposure and 21% in cases tested 8 days post exposure [10] .

Acceptable nasopharyngeal (NP) and oropharyngeal (OP) swabs are made with materials such as dacron and rayon, since they do not inhibit the PCR reaction. While specimens collected with NP and OP swabs differ in tip size and flexibility, both have been used to successfully collect specimens for identification of SARS CoV-2 [11] . Other specimens validated by different laboratories include nasal swabs, nasopharyngeal or nasal washes/aspirates, sputum, saliva and bronchoalveolar lavage [12] . Since each of the specimen types examines different anatomic areas with variable levels of viral inoculum, the possibility of false negative results should be ruled out for optimal patient management. The NP swab remains the "gold standard" specimen source.

Transport medium for swabs are reagents that retain virus viability in the specimen and minimize bacterial overgrowth for the time necessary to transport it to the clinical laboratory. Evaluation of different types of transport media including but not limited to viral transport media and universal transport media, showed that specimens consistently yielded amplifiable RNA with mean Ct differences of <3 over the various conditions assayed, thus supporting the use and transport of alternative collection media [13] . For SARS CoV-2, the FDA has strongly recommended that viral culture not be performed. Thus, alternatives to classical viral transport media have been validated in light of media shortages. These include normal saline, Amies transport media and Hanks balanced salt solution.

A caveat to interpreting molecular results is that it can be difficult to ascertain whether a patient has an active infection or was previously infected. Molecular assays can detect viral RNA both when patients are actively shedding the virus (current infection) or have residual viral RNA present. Therefore, these assays are most useful in acute settings to detect patients with SARS-CoV-2, where the results can optimize potential therapy and isolation protocols to ensure that appropriate personal protective equipment protocols are utilized for containment of the virus.

In addition to RT-PCR, reverse transcription loop-mediated isothermal amplification (RT-LAMP) technologies with increased levels of sensitivity, have shown utility, in resource-limited settings [14] . Notably, the first test using CRISPR (clustered regularly interspaced short palindromic repeats)-Cas12 based technology for SARS-CoV-2 detection was recently granted EUA (Sherlock Biosciences). The test has a limit-of-detection of 100 viral copies and involves a two-step process, where SARS-CoV-2 RNA undergoes RT-LAMP followed by transcription of the amplified DNA which activates CRISPR cleavage of reporter genes resulting in a fluorescent readout. The entire process can be completed in an hour [15] . Tests that use high-throughput sequencing of the SARS-CoV-2 genome are also being utilized in a research setting. These tests give additional information on viral mutations and can trace the global evolution of the pandemic.

Point-of-care (POC) testing is beginning to be available for SARS-CoV-2. POC testing refers to a broad category of diagnostic tests that can be performed where patient care occurs. However, due to evidence that samples collected in transport media may fall below the assay's limit of detection [16] [17] [18] [19] , the EUA for this test was modified for testing only from direct swabs.

Preliminary data though indicates that despite this modification, the Abbott ID NOW TM COVID-19 had a significant false negative rate when using dry nasal swabs [20] . Some additional rapid assays that are commonly used are the Xpert® Xpress SARS CoV2 (Cepheid) and the BIOFIRE® COVID-19 test.

The other major type of diagnostic assay is serological. These assays determine a patient's exposure history. At this time, it is unknown whether antibody detection equate to immunity. These assays detect the presence of antibodies against SARS-CoV-2 antigens in a patient's serum. There is a delay between the initial viral infection and the production of antibodies by the immune system. During this time, termed the "window period," a patient who is infected with SARS-CoV-2, but has not yet produced antibodies, would test negative on such an assay. As the immune system mounts a response against the virus, IgM antibodies are initially produced, which are short lived, followed by a more durable IgG antibody response [ Figure 3 ]. Therefore, serological tests may be unique to one class of immunoglobulins or detect multiple and can typically be completed in 1-2 hours.

Currently, there are at least 12 EUA serology assays, some of which are automated [6] . Most commercial serological SARS-CoV-2 assays use a lateral flow assay technique and format, and for many of these there are unsubstantiated, or even false, claims about test performance [21] .

The estimated median seroconversion time is 7-12 days with virtually all COVID-19 patients producing detectable antibodies approximately 15 days after onset of symptoms [22] [23] [24] .

Therefore, these assays will be most helpful in determining an individual's exposure status and perhaps in assessing their immune response to SARS-CoV-2. Going forward, these assays can be particularly helpful in identifying SARS-CoV-2 cases in individuals who may have had symptoms consistent with SARS-CoV-2 but were never tested with an RT-PCR assay, as well as individuals who may have had asymptomatic infection. Given that ~80% of SARS-CoV-2 cases are mild to moderate in severity [24, 25] , and that molecular testing has predominantly been restricted to the most severely ill patients, the true number of SARS-CoV-2 cases is likely to be vastly greater than that available from molecularly-confirmed case counts. Thus, serological testing will help identify the number of past infections, which can help epidemiologists better understand the true burden of disease to model viral dynamics.

SARS-CoV-2 testing is also important for identifying potential convalescent plasma donors for clinical trials. Studies are currently underway where patients who have recovered from SARS-CoV-2 and have detectable antibodies against SARS-CoV-2 can donate plasma, which can then be transfused to patients who are currently critically ill with COVID-19. Theoretically, the neutralizing antibodies against SARS-CoV-2 present in the plasma will help patients currently infected overcome the illness. Serologic testing to identify anti-SARS-CoV-2 antibodies are now part of the donor work-up to determine eligibility for clinical trials. At some institutions in New York City, potential donors also require RT-PCR testing to determine if they are still actively shedding virus and, therefore, contagious.

There is now one antigen detection assay available from Quidel which uses a lateral flow clinical laboratory improvement amendment (CLIA) of SARS-CoV and SARS-CoV-2. Information provided by the manufacturer in the package insert indicates an 80% concordance when compared to PCR. Historically, antigen detection kits for viruses have not performed well so the utility for SARS CoV-2 remains to be determined. Such tests are used routinely for other viruses -HIV p24 antigen as part of 4th and 5th generation HIV tests, and also for hepatitis B surface antigen [26] .

Laboratories regulated by Clinical Laboratory Improvement Amendment (CLIA) were able to get EUA for Laboratory Developed Tests (LDT's) either directly from the FDA or, the Wadsworth Center of the New York State Department of Health, as in the case of several laboratories in New York. The EUA route permitted the laboratories to implement the LDT's for routine clinical diagnostics. Unfortunately, in spite of these sanctions, the inability to provide broad diagnostic testing, was widely seen as a failing effort to contain the virus. This setback of optimal testing in a crisis, was largely due to lack of a national laboratory testing strategic plan that brings together the major players in diagnostic testing, including public health, clinical / hospital-based, and commercial laboratories. Clinical hospital-based laboratories play a major role in identification and containment of infectious threats and a coordinated laboratory network would likely be more effective at damage control earlier in pandemics such as the current one [27] .

In summary, testing has been critical to understanding and managing the SARS-CoV-2 pandemic. While both molecular and serological tests provide meaningful data for treating patients with SARS-CoV-2, each methodology has a different clinical utility. As we move forward, clinical laboratories will continue to be on the forefront of combating this pandemic by developing new assays and implementing increased testing capabilities to meet the high-volume demands necessitated by this pandemic. A concerted rather than isolated effort may be the best approach to accomplish mass-scale testing.

World Health Organization Coronavirus disease (COVID-19) Pandemic

Impact of viral multiplex real-time PCR on management of respiratory tract infection: a retrospective cohort study. Pneumonia (Nathan)

Coronavirus and the race to distribute reliable diagnostics

New York State Department of Health COVID-19 Tracker

Food and Drug Administration Emergency Use Authorizations

Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Persons for Coronavirus Disease

Comparison between Saliva and Nasopharyngeal Swab Specimens for Detection of Respiratory Viruses by Multiplex Reverse Transcription-PCR

Patient-collected tongue, nasal, and midturbinate swabs for SARS-CoV-2 yield equivalent sensitivity to health care worker collected nasopharyngeal swabs

Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction-Based SARS-CoV-2 Tests by Time Since Exposure

Virological assessment of hospitalized patients with COVID-2019

Laboratory diagnosis of emerging human coronavirus infections -the state of the art

Evaluation of Transport Media and Specimen Transport Conditions for the Detection of SARS-CoV-2 Using Real Time Reverse Transcription PCR

Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus 2

Point-of-care testing for COVID-19 using SHERLOCK diagnostics

Comparison of Cepheid Xpert Xpress and Abbott ID Now to Roche cobas for the Rapid Detection of SARS-CoV-2

Comparison of Abbott ID Now and Abbott m2000 methods for the detection of SARS-CoV-2 from nasopharyngeal and nasal swabs from symptomatic patients

Evaluation of the COVID19 ID NOW EUA assay

Clinical Evaluation of Three Sample-To-Answer Platforms for the Detection of SARS-CoV-2

Performance of the rapid Nucleic Acid Amplification by Abbott ID NOW COVID-19 in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a New York City academic institution

Food and Drug Administration Beware of Fraudulent Coronavirus Tests, Vaccines and Treatments

Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019

Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study

Immune responses and pathogenesis of SARS-CoV-2 during an outbreak in Iran: Comparison with SARS and MERS

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. The Lancet

p24 revisited: a landscape review of antigen detection for early HIV diagnosis

Laboratories and pandemic preparedness: A framework for collaboration and oversight

Figure 2: Number of individuals tested and number of positive cases from

Figure 3: Schematic of the antibody response to SARS CoV-2

Why do we need antibody tests for COVID-19? Available at