Laboratory Testing for SARS-CoV-2: Current Landscape and Lessons Learned
by S. Anderson, B. Finger, M. Grodman, S. Hersey, B. Terbrueggen, Jorge Villacian and D. Zimmerman COVID-19 Testing Consortium
The COVID-19 pandemic has accounted for more than 65 million infections and over 1.5 million deaths globally since first reported in December of 2019.1 The pandemic has dramatically disrupted individual lives and businesses leading to severe global health challenges and economic hardships. A vaccine(s) is on the near-term horizon for late 2020 or early 2021 for healthcare workers and those at high risk, but today virus spread is primarily being managed through public health measures such as social distancing, use of masks, and broad-scale testing programs.
In this publication we will review testing methodologies, key analytical and clinical performance attributes, regulatory considerations, potential use cases, access to testing, and lessons learned from the urgent attempts to develop and implement appropriate testing for SARS-CoV-2. These topics are important not only for our understanding of the current state of COVID-19 testing, but also will aid in preparing for the next wave(s) of the pandemic as we move into the 2020/2021 respiratory virus/flu season.
The COVID-19 pandemic has created a unique and challenging environment for the diagnostic testing industry. Diagnostic tests had to be developed in record time, while simultaneously ramping up testing capacity to unprecedented levels, and entertaining new point-of-care and at-home sample collection modalities. In general, COVID-19 testing falls into two major categories: 1) molecular detection of the virus (SARS-CoV-2) nucleic acids or viral antigens and 2) immunologic testing for an individual’s antibody response to a COVID-19 infection. As illustrated in Figure 1, there is a distinct time course of viral infection and response, which helps inform testing solutions.
A nucleic acid test, like a polymerase chain reaction (PCR) assay, with high sensitivity is most appropriate for detection of viral infection in the first days after infection. However, it should be noted that even the most sensitive PCR tests cannot detect the virus within the first few days following exposure. Therefore, those asymptomatic individuals who were in contact with a confirmed case should wait approximately 5 days post exposure prior to testing.2,3
The main nucleic acid based testing method, PCR (Polymerase Chain Reaction), relies on the use of an enzyme (Taq Polymerase) to amplify SARS-CoV-2 viral sequences for the sensitive detection of an infection. This technology and related methods such as Transcription Mediated Amplification (TMA) and newer CRISPR (Cluster Regulated Interspaced Palindromic Repeat sequences) methodologies usually require sending a sample to a central laboratory because of the complex nature of the testing.4 Some point of care testing methods and instruments are now also available for on-site testing with an assay turn-around-time of hours not days as in referral lab testing. A recent survey of over 100 molecular amplification tests used for SARS-CoV-2 has shown that 90% utilize Reverse Transcriptase-PCR, 6% isothermal amplification methods (i.e. TMA), 2% hybridization technologies and 2% CRISPR based technologies.5
Antigen tests rely upon direct detection of viral protein, such as the nucleocapsid or spike proteins, with colorimetric label antibodies.6 Rapid antigen tests have the advantage of providing results in a very short time period and therefore have multiple potential uses, i.e. screening in healthcare or school and college settings. One of the disadvantages is that the sensitivity is in general often less using an antigen test versus a nucleic acid based test. As a result, the timing of the antigen test becomes a critical consideration. In order to minimize the risk of false negatives, the FDA Emergency Use Authorization (EUA) for antigen tests restrict their use to “within the first 5-7 days of the onset of symptoms”, when an individual’s viral load is highest. In the same approach, other testing methods with an EUA designation also require disclaimers regarding the interpretation of results by a physician, such that they consider the test results along with other potential clinical findings.
Figure 2. Reported LODs of 79 EUA authorized tests kits with reported LODs from 180 copies/mL to 600,000 copies/mL
Antibody or serological testing, evaluates the antibody profile in response to a SARS-CoV-2 infection. Antibodies against SARS-CoV-2 can appears within 5 days, but can often take a minimum of 2 weeks post-infection for antibodies to appear. Antibodies can often be detected for several months post-infection (Figure 1). Some antibody tests evaluate for IgM and IgG antibody subtypes, while others do not distinguish between the subtypes. The potential relevance of assessing the immunoglobulin subtypes is that IgM antibodies show up earlier in the course of infection than IgG antibodies, and the presence of only one subtype (IgM only or IgG only) can be useful for determining the approximate time post-infection and degree of infectiousness. During the period where a person is IgM positive only and PCR positive, they would most likely still be infectious. Antibody assessment helps in determining not only the nature of the immune response but the potential protective nature of the presence of antibodies to reinfection.7,8 The nature of that relationship is not currently known but still is of importance not only for patient evaluation but also for vaccine development. In addition to the timing and titer of the immune response to vaccination, the ability to assess neutralizing antibodies to the SARS-CoV2 virus is also important for the determination of the potential efficacy of new vaccines.9
Separate but related to the method of testing is the method of sample collection. PCR testing is usually performed using a nasopharyngeal swab (NP), a nasal swab, and recently some tests can now use saliva collection. Saliva testing is only used with PCR testing, and not antigen testing, owing to the sample processing that is required. Several home collection kits are now available that either use a self-collected nasal swab or a self-collected saliva sample that are then shipped back to a central laboratory for processing. Antibody tests require blood and testing is performed using either a finger-prick blood sample point of care testing or by a venous blood sample sent to a central lab. Quick antigen tests all use a nasal swab, with the most more common utilizing swabbing of the lower nose/nostrils.
Regulatory and Analytical Performance Considerations
The commercialization of a new diagnostic test kit is generally a complex process requiring regulatory review and approval. It normally takes the test manufacturer a significant time period (i.e. up to several years) to complete the required clinical studies and validation of manufacturing processes to demonstrate quality, reproducibility of manufacture, and overall test performance including both analytical and clinical validation.
In order to assist in the rapid scale-up of SARS-CoV-2 diagnostic tests, the US Food and Drug Administration (FDA) implemented a streamlined Emergency Use Authorization (EUA) process requiring a minimum of analytical studies to speed the process and ensure availability of tests. Manufacturers of PCR test kits were initially allowed to use in-silico analysis to demonstrate tests specificity, and laboratory studies with synthetic templates to demonstrate the limit of detection (LOD: a measure of how many copies of virus must be present in a sample to elicit a positive result) along with comparative testing of 30 banked positive and 30 negative patient samples relative to a previously authorized SARS-CoV-2 test. Almost all tests that received EUA designation showed sensitivities and specificities of greater than 95% for the banked samples. The approved tests reported similar accuracy despite reporting LODs that varied by more than 1,000-fold. Scientifically, tests with a 1000-fold difference in LOD would be expected to have very different clinical performance. It is also not clear LOD can truly be compared one against another since manufacturers used different synthetic specimens for their studies. For those reasons the regulatory agency has taken additional measures to try and ensure adequate analytical performance and comparability of molecular testing across sites and methods.
For example, the FDA SARS-CoV-2 Reference Panel was developed consisting of one heat-inactivated SARS-CoV-2 strain, and one heat-inactivated MERS-CoV strain. Manufacturers were sent 5 tubes, a standard at a known concentration along with four blinded unknowns for testing, and assay developers were provided a very specific protocol for establishing LOD, Figure 2 shows a chart of the reported LODs of 79 EUA authorized tests kits with reported LODs from 180 copies/mL to 600,000 copies/mL, a range of more than 1,000-fold.9
The FDA has also performed evaluation studies to verify the sensitivity and specificity of serology/antibody tests kits, and this resulted in the removal10 of several antibody tests that lacked adequate specificity and sensitivity. In addition, only 3 molecular detection (2 PCR kits and 1 antigen kit) have been removed from the market, in large part due to the inherent sensitivity and specificity of PCR. As of the end of September 2020, 242 SARS-CoV-2 tests currently have Emergency Use Authorization including 187 molecular tests, 4 antigen tests, and 50 serological/antibody tests.10
Laboratory Testing Considerations
One current debate in the scientific community is the importance of limit of detection (LOD) of SARS-CoV-2 tests and how LOD effects real world clinical performance. Conventional thinking is that having a low LOD (i.e. less than 1000 copies/mL of sample) is very important11,12 and a recent publication estimated that every 10-fold increase in LOD leads to a 13% increase in false negative rates. However, there is an argument to be made that it is better to test more frequently (e.g., twice weekly) with a less sensitive rapid antigen test than infrequently (e.g., every other week) with a high sensitivity PCR test. In a recent New England Journal of Medicine paper12 the authors suggest that the “false negatives” missed by the rapid antigen test are actually most likely to be individuals in late stage of their infection and non-infectious, so missing them may not contribute to the spread of the virus. One point that scientists do agree on is that frequent testing for viral infection is key to controlling the pandemic.
In addition to robust performance, turn-around-time for testing is critical with the need to return results within 48-72 hours of testing, as results returned after a much longer time period are of minimal value from a public health perspective. With adequate turn-around-time, twice weekly testing of individuals is sufficient when combined with social distancing, masking, hand-washing and other behavioral elements in the attempt to manage the pandemic.
Another area of significant study is how to distinguish between a COVID-19 positive patient that is infectious versus non-infectious, which is important to address the asymptomatic spread of the virus. Scientific evidence collected to date through epidemiological investigations suggests that transmission is possible just prior to symptom onset and that pre-symptomatic spread could account for approximately 50% of transmission across populations.13 Other publications indicate that infectiousness may peak on or before actual symptom onset and supports pre‑symptomatic spread as a major contributing factor to transmission.13,14 For symptomatic patients, individuals are considered infectious immediately prior to symptom onset and for the first 10 days post‑symptom onset, until their symptoms resolve.
For asymptomatic patients, the infectious status of an individual is less clear. A single positive time-point, without a prior near-term negative test result, leaves the physician with little guidance of the disease state of the patient. Are they pre-symptomatic infectious or actually late-stage non-infectious. In this context, a potential consideration for the physician is to utilize the viral load of the patient as indicated by PCR testing. A recent article looked at the relationship between viral load (as determined by PCR Cycle threshold (CT) value) and the ability to grow SARS-CoV-2 virus in culture. Samples taken from individuals with a high viral load (CT <25) could be grown in culture 70%, but only 3% of the time in low viral load samples (CT>35).13
Another potential solution for determining infectiousness is using antibody testing along with nucleic acid testing when appropriate. For example, individuals that are PCR positive, but antibody negative are likely still infectious. Additionally, during the period where a person is IgM positive only (not IgG) and PCR positive, most patients are likely still infectious.14
In Figures 3 and 4, there are two commonly deployed testing pathways depicted for use of the different methodologies that are frequently used for patient testing.
◆ Option 1: molecular (detection of the RNA from SARS-CoV-2)
◆ Option 2: detection of the antigen (antigen test)).
As can be seen in the Figure 3 pathway, one should consider that a false negative could occur as part of this testing algorithm and as the FDA has clarified that in many of the Emergency Use Authorizations, “Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions,” The CDC has issued similar guidance and recommendations as well. As a result, if individuals have a negative PCR result and their physician is still concerned about their SARS-CoV-2 infection status, options are shown in Figure 3 regarding repeat testing and/or using an antibody test at a subsequent date to confirm the original testing result. Antibody tests may be used in some cases to supplement the clinical assessment of individuals who may present later in the course of the infection.
For asymptomatic/pre-symptomatic testing of patients, the same two types of tests have been deployed (Figure 4); however, there is limited data to date on the performance of many of these tests in the asymptomatic/ pre‑symptomatic patient population to guide the use of these tests. The advantage to actively monitoring populations for asymptomatic/pre-symptomatic disease is that potential infections could be detected prior to symptom onset, guiding decisions on quarantine strategies to prevent/minimize the potential for transmission. It should be acknowledged that with asymptomatic/pre-symptomatic testing it is essentially a “snapshot” of the viral status within the individual on that given day. It is quite possible an individual could be negative one day and positive on a subsequent day if their viral load was not sufficient for detection, based on the sensitivity of the test used. As such, it is important that test results for molecular/antigen are obtained within 48-72 hours. Furthermore, with asymptomatic testing, there has been some debate as to what actions should be taken for individuals who test positive with low viral loads (e.g., high CT values on molecular tests) and remain asymptomatic, as there is limited to no clinical data regarding what level of viral load confers transmission. Until additional clinical data and quantitative assays are more readily available, these subjects will likely be treated similarly as all other individuals who test positive.
Across the world, governments and individuals all have had challenges to face during this pandemic, with one important area being the access to testing. During the first 9 months of the pandemic in the U.S. many factors contributed to the initial lack of adequate access to testing including availability of critical supplies and reagents, access to sample collection sites, laboratory testing capacity, adequate turn-around-time for testing, and effective data reporting and aggregation tools. While many of the early supply issues have been resolved testing capacity and expansion of that capacity to address additional surges and waves of infection continues to be a concern. In addition to more testing labs, the validation of multiple sample types for specific tests, the use of drive-through testing centers, and approved home collection kits have improved access. Testing capacity is also challenged by the various use cases for testing which range from diagnostic applications in symptomatic patients, to policy decision making for school or work considerations, and lastly contact tracing activities to slow the community spread of infections.
The COVID-19 pandemic presented a variety of global healthcare challenges that center on two main considerations that are important for managing through the pandemic-namely, global and local preparedness and timeliness of response. What was clear in the first waves of the pandemic is that relying on science and facts to drive policy decisions, developing and implementing adequate communication channels and sharing of best practices can and should contribute to managing through the current and future waves of the pandemic. These areas which were not always followed or employed, magnified gaps in both early preparedness and timeliness of response. In addition to the impact on global healthcare, the pandemic also provided substantial challenges for global economics and policy decision making. Policy implementation such as social distancing measures and the balance between lockdown and reopening, continue to impact a variety of business and personal life choices. Regarding the implementation of testing and the impact for global healthcare, the areas of continued importance are applications of testing for diagnostic purposes along with, surveillance measures and drug development (therapeutics and vaccines) applications. Diagnostic testing continues to be a critical element of managing individual and community health concerns. Surveillance and tracking of the waves of the pandemic is also crucial to policy decision making such as when to open businesses and schools and when they should be closed or restricted. Also, the use of nucleic acid testing and antibody testing has been vital to vaccine development. The use of antibody titers and the presence of neutralizing antibodies have been used to show early efficacy of the current vaccine candidates.
Some of the key lessons learned regarding the pandemic include are the value of public and private partnerships in addressing preparedness and enabling transfer of information and technology to address the need for diagnostic testing capacity. Furthermore we note the importance of partnerships and collaborations between global health organizations (i.e., WHO, CDC), public health labs and private testing labs in the US, scientific personnel and public policy decision makers, in vitro diagnostic manufacturer’s and laboratories. Gaps that were revealed early in the pandemic limited both the ability to respond appropriately and in a timely manner. In some areas relationships and collaborations have improved over the last several months, but in other areas they continue to be a challenge. Lessons learned on how better to leverage such partnerships and collaborations will be important as we prepare for the next wave(s) of the pandemic, as we move into respiratory virus season, and as we await therapeutic and vaccine development and deployment.
The development of effective and frequent testing strategies are key attributes to keeping countries and local municipalities open for business and children back in school. Commercial labs and diagnostic companies have developed a range of testing options for identifying individuals infected with SARS-CoV-2, and new consensus is developing around best practices for diagnostic testing, quarantine strategies, as well as workplace and school testing programs. Initially in the COVID-19 pandemic here were limited options for testing and personal protective equipment. Today the US and the world is better positioned to combat the virus but it still requires us to enhance our collective levels of cooperation and collaboration among all partners to address the ongoing challenges of the pandemic.
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9. https://www.fda.gov/medical-devices/coronavirus-covid-19- and-medical-devices/sars-cov-2-reference-panel-comparativedata
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12.Mina MJ et al, Rethinking Covid-19 Test Sensitivity – A strategy for Containment, NEJM 383:22
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Steven M. Anderson, PhD
Senior Vice President and Chief Scientific Officer, Covance Drug Development
Bob Terbrueggen, PhD
Founder and CEO, DxTerity Diagnostics
Marc D. Grodman, MD
Co-founder and CEO, Genosity Inc.
Mr. Bill Finger
Chief Operating Officer, Interpace Pharma Solutions
Jorge Villacian, MD
Sr. Scientific Director, Infectious Diseases Th erapeutic Area, Janssen R&D, a Johnson & Johnson Company
Sarah Hersey, PhD
Vice President, Precision Medicine, Bristol Myers Squibb
Dirk Zimmermann, PhD, MBA, MSc
Global Business Area Head for Oncology and Infectious Diseases, Biocartis Group NV
The COVID-19 Testing Industry Consortium
The consortium brings together a diverse group of companies with synergistic areas of expertise, including precision medicine, diagnostics, occupational health, pharmaceuticals and clinical testing laboratories, to help provide clarity and potential solutions to COVID-19 testing challenges. By creating the COVID-19 Testing Industry Consortium, 22 organizations from the healthcare industry have joined forces with a goal to help inform, improve, innovate, and accelerate various aspects of testing, ranging from research to clinical diagnostic applications.
To find out more, please visit: https://news.bms.com/news/details/2020/19-Organizations-from-the-Healthcare-Community-Unite-to-Form-COVID-19-Testing-Industry-Consortium/default.aspx