Julia Jenkins, PhD
The ability to test for SARS-CoV-2 is fundamental to any public health strategy aimed to curb the coronavirus pandemic. Testing identifies those who have been infected, allowing treatment, isolation and contact tracing, and enumerates the true scope of the outbreak and the velocity of transmission. The World Health Organization’s director-general has been abundantly clear about the need for comprehensive testing, “The most effective way to prevent infections and save lives is breaking the chains of transmission. And to do that, you must test and isolate. You cannot fight a fire blindfolded.”
The US capability to diagnose SARS-CoV-2 infections has been expanding; however, there remains an urgent need to perform more testing. This article examines the most important considerations for any lab preparing to develop and/or run SARS-CoV-2 assays.
In response to this crisis, the US food and drug administration (FDA) issued an emergency use authorization (EUA) for COVID-19 tests on February 29. This has accelerated the development and use of assays in addition to those provided by the US Centers for Disease Control and Prevention (CDC) and is described in the following guidance.
Laboratories with clinical laboratory improvement amendment (CLIA) designation may submit an EUA to the FDA. However, another approval route exists in which, the test is developed under the authority of the state in which the lab resides (if applicable). For example, certain laboratories in New York State are authorized to test under the auspices of the Wadsworth Center of the New York State Department of Health. Several states have adopted this route; however, testing laboratories should confirm their state-wide arrangements for approval as this situation may change.
In vitro diagnostics made under an EUA will not undergo the normal FDA review process. Companies have two weeks to send internal validation data to the FDA, but in the interim, they can use the assay to test and report on patient samples (the specifics of the approval process for companies requesting EUA are detailed in sections C and D of the FDA guidance).
The US institutions that tests may be marketed to have different capabilities; county and State public health laboratories may have different equipment and expertise than research institutions. PCR testing, for example, requires thermocyclers that may not be routinely present in hospital laboratories. Some approved diagnostic kits are platform specific, such as the cobas® SARS-CoV-2 test, and others can be adapted to standard thermocyclers, such as the WHO assay.
Laboratories must determine whether developing assays based on in-house designed test kit components (e.g., probes and primers), or purchasing reagents from third-party manufacturers is the most time- and cost-efficient strategy. Commercial RT-PCR testing kits are retailing at approximately $15 USD per test. A lab developed test using a standard intercalating dye such as SYBR green would be cheaper, costing approximately $5 USD per test.
Some of the commercially available testing kits run on specific platforms. Any potential capital investment should be weighed against the reimbursement rates for testing. The Centers for Medicare and Medicaid Services announced Medicare reimbursement for the CDC test and non-CDC testing at approximately $36 and $51, respectively.
The coronavirus has caused disruption of the supply chain for vital reagents and kits that may impede the development or distribution of testing systems. The swabs used to obtain samples are in extremely short supply. A well-publicized bottleneck in SARS-CoV-2 PCR testing is the availability of RNA extraction kits. A limited number of RNA extraction kits are listed in the CDC protocol, and there is an unprecedented level of demand for these kits, limiting the supply of these and other vital reagents.
Labs must ensure they are equipped to adequately preserve samples prior to testing; for example, nasopharyngeal swabs must be taken into stabilizing media and stored between 2-8 ?C. Currently, viral stabilizing media is in critically short supply. Assessment of proteins, such as antibodies, will require reliable low-temperature storage to prevent degradation
Access to the positive and negative control patient specimens that would ideally be used to validate diagnostic assays can be problematic. Initially, these specimens were difficult to source as samples had to be obtained from China. Currently, access may still be limited by complicated shipping regulations and patient privacy laws.
There are numerous permutations of qPCR, but all require a method to extract RNA, materials to run the PCR reaction (including excess nucleotides, thermostable dsDNA polymerase, optimized buffers, and specific primers), a way of quantifying the specifically amplified DNA (e.g., quantification of fluorescent probes), and access to a thermocycler.
The WHO lists several optimized RT-PCR protocols, including that of the CDC and its virus-specific primers and probes. The CDC primers target two unique regions of N1 and N2 that encode a viral capsid protein; another primer targets a ubiquitous gene common to the SARS-like viruses, and the final primer targets a human housekeeping gene, which acts as a positive control.
If novel primers are to be designed, then the FDA mandates an in silico analysis indicating a 100 percent identity match of primers against publicly available SARS-CoV-2 sequences. The FDA recommends that laboratories characterize the limit of detection (LoD) of their SARS-CoV-2 assay by spiking positive RNA control samples into a biological matrix, e.g., bronchoalveolar lavage or an artificial matrix. A dilution series with three replicates at each concentration should be prepared and confirmation should be made of the lowest concentration with 20 replicates. The LoD is defined as the lowest concentration at which 19 of the 20 replicates are positive. To determine the cross-reactivity of the test with other similar pathogens, the FDA requires in silico comparison between the primers and probes with other common respiratory flora and viral pathogens. Cross reactively is defined as > 80 percent homology.
PCR assays are used to detect acute COVID-19 infections; however, they cannot provide any information on patient antibody responses. Serological assays provide crucial information on the rate of infection and enumerate the total number of sufferers, including asymptomatic patients. This information can be used to determine whether COVID-19 non-pharmaceutical interventions (NPIs) are effectively limiting disease spread. These assays will help to identify donors for serum therapy and identify those who are potentially immune, allowing their strategic deployment (i.e., health care workers) into high-risk situations. Researchers have developed a serological ELISA, for example, where antigen from the SARS-CoV-2 spike protein is directly bound to immunoassay plates. Any anti-SARS-CoV-2 human antibody present in the sample will bind and is detected by anti-human IgG HRP conjugated antibodies and a colorimetric substrate.
The regulatory submission for serological assays is less burdensome than RT-PCR testing, but the FDA will require cross-reactivity, class specificity, and clinical agreement data based on known positive clinical samples, as detailed in the FDA guidance.
Rapid systems that require limited technical expertise to run are being developed for SARS-CoV-2 testing. For example, Cepheid has developed a small PCR cartridge for SARS-CoV-2 testing which it recently obtained FDA EUA approval for. The module simply slots into a dedicated GenXpert system, and provides diagnostic readout within 45 minutes. The advantages of this assay are considerable, given the short run duration and its “plug and play” system that can be used without extensive training. However, the system requires financial investment, and the system cannot simultaneously run batches of > 80 samples. Similarly, Abbott has gained approval for their ID now SARS-CoV-2 RT-PCR tests that can provide results in 10 minutes.
Novel testing methods
A rapid 30-minute CRISPR (clustered regularly interspaced short palindromic repeats)-Cas12 assay for SARS-CoV-2 has recently been described. This assay performs simultaneous, reverse transcription, and isothermal amplification using loop-mediated amplification (RT-LAMP). The presence of viral RNA is confirmed by Cas12 detection of specific SARS-CoV-2 sequences and the cleavage of a reporter molecule liberating a color
Direct sequencing of genetic material and comparison to the SARS-CoV-2 sequence may be used as a diagnostic. This high-speed gene sequencing, such as Oxford Nanopore’s, approach can be used as a diagnostic in remote areas without access to complex laboratory equipment. These handheld devices have been deployed in other disease outbreaks, such as Ebola. Sequencing the SARS-CoV-2 genome will also give valuable information on viral mutation.
Point-of-care diagnostic tests produce rapid and accurate clinical results without the need for complex lab equipment and immediately provide results to health care providers. Currently, PCR testing requires shipment of the clinical sample to a specialist facility, and turnaround is usually at least one day. Rapid (~15-minute) immunochromatographic antibody (IgM and IgG) tests for COVID-19 are in development and awaiting EUA approval. The test card contains labeled recombinant SARS-CoV-2 antigen and immobilized anti-human monoclonal IgG and IgM antibodies. The card works like a pregnancy testing strip; the blood sample is spotted onto the strip and moves via capillary action across the card. Colored lines will form on the strip if the sample contains human anti-SARS-CoV-2 antibodies.