Information

Are specific primers or detectors, or both, used in COVID-19 tests?


I am trying to learn about the rRT-PCR testing procedure used to test for COVID-19, but I am slightly confused on one point. Are highly specific primers used with a non-specific detector, or are highly specific detectors used, or are both specific primers and detectors used? Some of the sources say that multiplex PCR is used, which indicates a specific detector, but as I am still a beginner on the subject, I am not sure. I would also really appreciate it if somebody could also point to a source that details the some of the testing procedures.

Thank you


When you run a PCR reaction, you are making copies of a DNA sequence of interest (cDNA in case of retrotranscription, since retrovirus have RNA as the genetic material). The sequence of interest is presumed to be in a sample, that you take from the patient, or some organism (I say 'presumed' because the PCR is precisely to confirm this). These copies are made by a group of 'generalist' enzymes, called polymerases, and by generalists I mean that they do not discriminate on which DNA sequence they are copying. But, they cannot copy DNA de novo (from scratch). The copying process needs to be 'primed' by a short sequence of nucleotides that is complementary to the DNA of interest. These are called primers, and they effectively serve as probes to target the copying of the specific sequence you need to amplify. And this is where scientists put a lot of care on: primer design. Bad primers will not be very specific, or will not be very efficient during the amplification process. As for the 'machine' that actually detects the copies, there are many variants. The most typically used in research and diagnostic laboratories, involves staining the amplified DNA with some molecule that binds nucleic acids (which there are many), and visualize it via DNA electrophoresis. What you visualize is a population of DNA copies (via the staining you make), so you need to know beforehand what to expect. For instance, do you expect a small fragment (a couple hundred basepairs) or a big one (a few thousands?). You have a 'ladder' (also called marker) that gives you a reference to compare, like in this image:

People know what to expect because they already know the DNA (or RNA) sequence they are amplifying (otherwise they could not have designed the target primers). They know the sequence because they've done some form of nucleic acid sequencing (labs, health institutes, and so, do this all the time and deposit the sequences in some platform, for instance GeneBank).

A 'positive' amplification will give you a band in the expected size range, and no other bands (because your primers are targeted for a particular DNA sequence). But as you mentioned, there exists also a multiplex PCR reaction. In it, you design multiple primers to target different sequences (using the same DNA sample -e.g. from a patient or organism-), but the verification process is basically the same. Here people are extra careful to avoid cross-interactions among the primers added to the reaction.

PCR is a family of techniques, not a single one. There are variations in the actual process, and also in the 'detection' (for instance, qPCR uses real time detection of copy numbers). But the general principle is the same: primers are specific, while the 'detection device' is not really.


COVID-19 ONE-STEP RT-PCR KIT

Reverse transcription polymerase chain reaction (RT-PCR) is a standard PCR variant that consists of amplified forms of particular mRNA, attained from minute samples. It eradicates the requirements for the burdensome mRNA purification procedure needed in the traditional cloning practices. In RT-PCR, in addition to the standard PCR reagents, reverse transcriptase and an RNA sample are used. The reaction mixture is heated to a temperature of 37 ˚C, which permits cDNA production from the RNA sample through the mechanism of reverse transcription. This cDNA hardens to one of the primers leading to first-strand synthesis. Standard PCR continues, producing dsDNA.

The primers used in the procedure must counterpart the project. Oligo (dT) primers are sufficient in case amplification of the entire mRNA of a cell is required because they get attached to the poly(A) tails. On the contrary, if a particular mRNA needs to be amplified, a primer specific to the coding region can be used.

RT-PCR is a general virology diagnostic procedure, often combined with quantitative real-time PCR (qPCR), which is extensively used for quantification of RNA transcript levels in cells and tissues. The combination of real-time PCR (qPCR) and reverse transcription PCR is referred as quantitative RT-PCR or qRT-PCR

What is Multiplex PCR?

Multiplex polymerase chain reaction refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously. This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler.

Meril COVID-19 one Step RT-PCR Kit

Due to the novel coronavirus (SARS-CoV-2) outbreak, Meril Diagnostics has worked extensively to develop a new one step multiplex real-time RT-PCR kit to allow laboratories to diagnose COVID-19 caused by SARS-CoV-2 infection with fast and easy to use assays. Meril Covid-19 one step Rt PCR kit is a fast, highly sensitive multiplex diagnostic kit that contains both the assays and controls needed for the real-time PCR detection of RNA from the SARS-CoV-2 virus.

It is a One tube Multiplex PCR reaction for identification and detection of 2019-ncov. Its Primer and Probe mix based on dual-target gene design the two confirmatory gene ORF 1ab gene and nucleoprotein N gene for the detection of Sars COV 2 in human sample, with an analytical Analytical Accuracy of < 5 RNA copies/ reaction it makes Meril COVID-19 one Step RT_PCR Kit a highly sensitive and specific kit for the detection of SARS COV 2 RNA.

The kit is highly compatible with Open ended RT PCR instruments with FAM, HEX/VIC, RED/ROX channels. The easy protocol, minimum assay time and high specificity and sensitive makes it an ideal choice for your laboratory for the detection of COVID 19 suspected patients.

Meril COVID-19 RT-PCR Kit compatible with popular Applied Biosystems (ABI) 7500, BioRad CFX 96, Shanghai Hongshi SLAN-96P, QIAGEN Rotor Gene Q and other PCR instruments with FAM, HEX/VIC, RED/ROX channels.

The primer and probe mix for this kit accepts the dual-target gene design, which aims the particularly conserved sequence encoding the ORF 1ab gene and the nucleoprotein N gene. The template&rsquos amplification can be quantitatively monitored by the increasing fluorescence signal detected by a real time PCR instrument, as the PCR reaction mix is readily provided.

The PCR detection system comprises of an endogenous internal control primer and probe mix. The result of internal control provides precise sampling and extraction process, to avoid false negative results.

Intended Use:

This kit is designed to detect COVID-19 disease using real time PCR. The results can be used to assist diagnosis of patients with COVID-19 infection, and provide molecular diagnostic basis for infected patients.

This RT-PCR kit can be utilized by clinical and public health laboratories to rapidly assess up to 94 patient specimens with a single kit, within a span of 3 hours. The kit is approved for use with RNA taken out from nasopharyngeal swabs, nasopharyngeal aspirate (nasal aspirate), and bronchoalveolar lavage (BAL) from patients at risk of getting exposed to the SARS-CoV-2 virus or having signs and symptoms of COVID-19.

Results are for recognizing SARS-CoV-2 RNA. During the acute phase of infection, the SARS-CoV-2 RNA is usually evident in any of the above specimens. Positive results are suggestive of the SARS-CoV-2 RNA presence albeit, clinical link with patient history and other diagnostic information is essential to decide the stage of patient infection. Positive results do not exclude bacterial infection or co-infection with other viruses. The identified agent may not be the certain cause of disease.
Also, negative results do not disqualify SARS-CoV-2 infection and hence, should not be used as the only basis for deciding patient management. Negative results have to be pooled with clinical observations, patient history, and epidemiological information.

Evaluation with the Meril COVID-19 One Step RT PCR Kit is meant for use by qualified and trained clinical laboratory personnel, specially instructed and trained in the real-time PCR procedures and in vitro diagnostic procedures.

The test results of this kit are meant for clinical reference only and should not be used as the sole reference for clinical diagnosis. Conducting an inclusive analysis is suggested by combining the test results with patients&rsquo symptoms and other laboratory tests.


How do antigen and molecular tests work?

Viruses require a host to replicate. The virus hijacks the host’s cells to produce more viral copies of itself. The genomic material for the SARS-CoV-2 virus is ribonucleic acid (RNA), which remains in the body while the virus is still replicating and reproducing. Diagnostic tests look for evidence of this replication process—that more viruses are being made—to diagnose an active infection of COVID-19.

Antigen diagnostic tests detect structural features of the outside of the virus called antigens—small proteins that make up the virus—that may be present in a patient’s sample.

Antigen diagnostic tests work by:

  • Finding larger pieces of evidence that the SARS-CoV-2 virus is actively infecting a person
  • Detecting specific, 3-dimensional antigens on the outside of the SARS-CoV-2 virus

Molecular tests amplify bits of viral RNA so that viral infection can be detected using a specialized test. These tests are also referred to as nucleic acid amplification tests (NAAT). The procedure begins by taking a sample from a potentially infected person’s nose or mouth (saliva), where virus might be found. If SARS-CoV-2 is present in the sample, then even low levels of virus genomic material can be amplified into millions of copies detected during a molecular diagnostic assay. If a person is infected, the viral RNA will be detected and produce a positive test result if a person is not infected, no viral RNA will be copied or detected, which will produce a negative test result. Amplifying the signal allows for even small amounts of virus to be detected. This category of diagnostic test includes polymerase chain reaction (PCR) tests, loop-mediated isothermal amplification (LAMP), and clustered, regularly interspaced short palindromic repeat (CRISPR)-based assays. There are a wide variety of molecular diagnostics, and some provide faster results than traditional PCR-based methods. These rapid molecular tests include LAMP, which can provide results in minutes rather than hours. Rapid molecular tests that use techniques like LAMP are very specific, but also very sensitive because they amplify the genomic material in the patient sample. Importantly, not all rapid diagnostic tests are antigen tests—some are rapid molecular tests that are highly sensitive but provide results in minutes.

Molecular diagnostic tests work by:

  • Detecting that the SARS-CoV-2 virus is actively infecting a person
  • Creating millions of copies of small segments of the SARS-CoV-2 virus, if it is present in the patient’s sample, amplifying the signal
  • Detecting those millions of copies on specialized machines

Testing for SARS-CoV-2 Infection

Many categories of tests are used to detect SARS-CoV-2, 1 and their performance characteristics vary.

  • Some tests provide results rapidly (within minutes) others require time for processing.
  • Some must be performed in a laboratory by trained personnel, some can be performed at the point-of-care, and others can be performed at home.
  • Some tests are very sensitive (i.e., few false-negative results or few missed detections of SARS-CoV-2) others are very specific (i.e., few false-positive results or few tests incorrectly identifying SARS-CoV-2 when virus is not present) and some are both sensitive and specific.
  • Some tests can be performed frequently because they are less expensive, easier to use, and supplies are readily available.

The selection and interpretation of SARS-CoV-2 tests should be based on the context in which they are being used, including the prevalence of SARS-CoV-2 in the population being tested (See Table 1) and the status (signs, symptoms, contacts) of the person being tested.

Test Types

Viral tests, including nucleic acid amplification tests (NAATs) and antigen tests are used as diagnostic tests to detect infection with SARS-CoV-2 and to inform an individual&rsquos medical care. Viral tests can also be used as screening tests to reduce the transmission of SARS-CoV-2 by identifying infected persons who need to isolate from others. See FDA&rsquos list of In Vitro Diagnostics Emergency Use Authorizations external icon for more information about the performance of specific authorized tests.

    NAATs, such as real-time reverse transcription-polymerase chain reaction (RT-PCR), are high-sensitivity, high-specificity tests for diagnosing SARS-CoV-2 infection. NAATs detect one or more viral ribonucleic acid (RNA) genes and indicate a current infection or a recent infection but, due to prolonged viral RNA detection, are not always direct evidence for the presence of virus capable of replicating or of being transmitted to others. Most NAATs need to be processed in a laboratory and time to results can vary (

Correct interpretation of results from both antigen test and confirmatory NAATs, when indicated, is important.

Positive test results allow for identification and isolation of infected persons, as well as a case interview to identify and notify the case&rsquos close contact(s) of exposure and the need to quarantine.

Negative test results in persons with known SARS-CoV-2 exposure suggest no current evidence of infection. These results represent a snapshot of the time around specimen collection and could change if the same test was performed again in one or more days. Unvaccinated individuals with a negative result should continue to quarantine for 14 days or for the period established by local public health authorities. Fully vaccinated people with no COVID-like symptoms do not need to quarantine or be tested following an exposure to someone with suspected or confirmed COVID-19, as their risk of infection is low. For guidance on quarantine and testing of fully vaccinated people, visit Interim Public Health Recommendations for Fully Vaccinated People for more information.

Negative test results in persons without symptoms and no known exposure suggest no infection. All persons being tested, regardless of results, should receive counseling on the continuation of risk reduction behaviors that help prevent the transmission of SARS-CoV-2 (e.g., wearing masks, physical distancing, avoiding crowds and poorly ventilated spaces).

Antibody (or serology) tests are used to detect previous infection with SARS-CoV-2 and can aid in the diagnosis of Multisystem Inflammatory Syndrome in Children (MIS-C) and in adults (MIS-A) 2 . CDC does not recommend using antibody testing to diagnose current infection. Depending on the time when someone was infected and the timing of the test, the test might not detect antibodies in someone with a current infection. In addition, it is not currently known whether a positive antibody test result indicates immunity against SARS-CoV-2 therefore, at this time, antibody tests should not be used to determine if an individual is immune against reinfection. Antibody testing is being used for public health surveillance and epidemiologic purposes. Because antibody tests can have different targets on the virus, specific tests might be needed to assess for antibodies originating from past infection versus those from vaccination. For more information about COVID-19 vaccines and antibody test results, refer to Interim Clinical Considerations for Use of mRNA COVID-19 Vaccines Currently Authorized in the United States.

Overview of Testing Scenarios

Diagnostic testing is intended to identify current infection in individuals and is performed when a person has signs or symptoms consistent with COVID-19, or when a person is asymptomatic but has recent known or suspected exposure to SARS-CoV-2.

Examples of diagnostic testing include:

  • Testing people who have symptoms consistent with COVID-19 and who present to their healthcare provider
  • Testing people as a result of contact tracing efforts
  • Testing people who indicate that they were exposed to someone with a confirmed or suspected case of COVID-19
  • Testing people who attended an event where another attendee was later confirmed to have COVID-19

Screening tests are intended to identify infected people who are asymptomatic and do not have known, suspected, or reported exposure to SARS-CoV-2. Screening helps to identify unknown cases so that measures can be taken to prevent further transmission.

Examples of screening include:

  • Testing employees in a workplace setting
  • Testing students, faculty, and staff in a school or university setting
  • Testing a person before or after travel
  • Testing at home for someone who does not have symptoms associated with COVID-19 and no known exposures to someone with COVID-19

Public health surveillance is intended to monitor population-level burden of disease, or to characterize the incidence and prevalence of disease. Surveillance testing is primarily used to gain information at a population level, rather than an individual level. Surveillance testing results are not reported back to the individual. As such, surveillance testing cannot be used for an individual&rsquos health care decision making or individual public health actions such as isolation or quarantine.

An example of surveillance testing is wastewater surveillance.

Choosing a Test

When choosing which test to use, it is important to understand the purpose of the testing (e.g., diagnostic, screening), analytic performance of the test within the context of the level of community transmission, need for rapid results, and other considerations (See Table 1). For example, even a highly specific antigen test may have a poor positive predictive value (i.e., high number of false positives) when used in a community where prevalence of infection is low. As an additional example, use of a laboratory-based NAAT in a community with high transmission and increased test demand may result in diagnostic delays due to processing time and time to return results. Positive and negative predictive values of NAAT and antigen tests vary depending upon the pretest probability. Pretest probability considers both the prevalence of the level of community transmission as well as the clinical context of the individual being tested. Additional information on sensitivity, specificity, positive and negative predictive values for antigen tests and antibody tests and for the relationship between pretest probability and the likelihood of positive and negative predictive values is available. Also see FDA&rsquos letters to clinical laboratory staff and healthcare providers on the potential for false-positive results with antigen tests external icon and the potential for false-negative results with molecular tests if a genetic variant of SARS-CoV-2 external icon occurs in the part of the viral genome assessed by the test.

Table 1 summarizes some characteristics of NAATs and antigen tests to consider for a testing program. Most antigen tests that have received EUA from FDA external icon external icon are authorized for testing symptomatic persons within the first 5, 7, 12, or 14 days of symptom onset. Given the risk of transmission of SARS-CoV-2 from asymptomatic and presymptomatic persons with SARS-CoV-2 infection, use of antigen tests in asymptomatic and presymptomatic persons can be considered. FDA has provided a list of FAQ for healthcare providers who are using diagnostic tests in screening asymptomatic individuals external icon external icon , and the Centers for Medicare & Medicaid Services will temporarily exercise enforcement discretion external icon to enable the use of antigen tests in asymptomatic individuals for the duration of the COVID-19 public health emergency under the Clinical Laboratory Improvement Amendments of 1988 (CLIA). Laboratories that perform screening or diagnostic testing for SARS-CoV-2 must have a CLIA certificate and meet regulatory requirements. Tests that have received an EUA from FDA for point of care (POC) use can be performed with a CLIA certificate of waiver.


Antigen tests

Antigen tests can turn around results in minutes&mdashbut speed comes with tradeoffs.

Like PCR tests, antigen tests usually require a nose or throat swab. But unlike PCR tests, which look for genetic material from the SARS-CoV-2 virus, antigen tests look for proteins that live on the virus’ surface. This process is a little less labor-intensive than PCR testing, since there isn’t as much chemistry involved, but it’s also less sensitive. Mehta says that opens the door for possible false positives (if the test picks up on proteins that look similar to those from SARS-CoV-2) or negatives (if it misses proteins entirely). False positives are rare with antigen tests, but as many as half of negative results are reportedly inaccurate. If you test negative but are showing symptoms or have had a risky exposure, your doctor may order a PCR test to confirm the result.

While antigen testing is becoming more common in the U.S., only a few such tests have been approved by the FDA so far. Much like with rapid genetic tests, some experts argue that fast-moving antigen tests could help ease testing bottlenecks enough to compensate for their reduced accuracy.


RT-PCR as a Frontline Diagnostic Method for COVID-19 Diagnosis

Molecular diagnostic approaches are appropriate as compared to other syndromic testing approaches because molecular diagnosis targets the genome or proteome of the pathogen thus making it a specific and reliable method of diagnosis (Zhou etਊl., 2020b). For a novel pathogen sequencing and diagnosis becomes imperative to recognize the nature of the pathogen and its genomic composition. Random amplification and deep sequencing strategies played a critical role in early identification of the SARS-CoV-2, which was further confirmed to be a member of the coronavirus family via different bioinformatics approaches (Briese etਊl., 2014). Using metagenomic sequencing, the first genomic sequencing was conducted for SARS-CoV-2 (Miller etਊl., 2019 Sheridan 2020a). On 10 th January 2020, the findings were made public and the sequences submitted to the sequence repository of GenBank (Wu etਊl., 2020). Release of whole genome sequence of SARS-CoV-2 to public databases made it easy for scientists to design primers and probes for conducting laboratory diagnosis of COVID-19 (Corman etਊl., 2020). After the identification of this virus, WHO recommended real time reverse transcription polymerase chain reaction (real time RT-PCR), which is a nucleic acid-based technique, as the frontline diagnostic approach to detect SARS-CoV-2 infection in suspected patients. RT-PCR is highly sensitive and can detect infection at minute levels of pathogen present in the patient sample. It is a nucleic acid-based technique used to amplify a target gene/nucleotide present in a sample, which helps in detecting a specific pathogen and discriminating it from other related pathogens. There are usually two possible ways of performing RT-PCR including one-step assay or two-step assay. One step assay consolidates reverse transcription and PCR amplification in a single tube thus making the process of detection rapid and reproducible however, this assay provides a lower target amplicon generation. In case of two-step assay the reactions are carried out sequentially in two separate tubes making it time-consuming, but a sensitive assay compared to the one-step assay format (Wong and Medrano, 2005).

Although eleven nucleic acid-based protocols and eight antibody detection kits have been approved by the National Medical Product Administration (NMPA) in China, PCR was considered as a preferred diagnostic technique. The US Centers for Disease Control and Prevention (CDC) uses a one-step PCR format to diagnose COVID-19 (https://www.fda.gov/media/134922/download). The assay is carried out by isolating RNA from the sample and adding to the master mix containing forward and reverse primers, nuclease-free water, reaction mixture (reverse transcriptase, polymerase, nucleotides, magnesium and other additives). A PCR thermocycler is loaded with extracted RNA and mastermix, and the temperature is set to run the PCR reaction (https://www.fda.gov/media/134922/download). Cleavage of a fluorophore quencher probe during this reaction generates a fluorescence signal that is detected by the thermocycler, and the progress of amplification is recorded. Positive and negative controls must be included whenever running any RT-PCR reaction, which makes the interpretation of results easy and stringent (Chan etਊl., 2020). RT-PCR and some biosensor based diagnostic kits can detect SARS-CoV-2 nucleotides in fecal samples or sewage water that can be a warning of an infectious disease outbreak in the particular area. SARS-CoV-2 can survive from hours to days in the untreated sewage water (Orive etਊl., 2020).

RT-PCR is a sensitive and rapid detection tool in molecular diagnostics. It can detect and amplify even a few copies of specific genomic sequence in a variety of samples, but it depends upon certain aspects to deliver reliable results like proper collection, transport, storage, and processing of samples (Afzal, 2020). It has been used for detection of diverse viruses like Adenovirus, Rotavirus, Astroviruses and many enteric viruses isolated from fecal samples (Kowada etਊl., 2018). A Major drawback of this technique is the need for a well-equipped laboratory and technical personnel for handling the experiment, which cannot mitigate the increased demand of rapid testing during pandemic situations like COVID-19 (Bustin and Nolan, 2004). The RT-PCR based kits are highly expensive and take much time to deliver results thus making it essential to look for other rapid and reliable diagnostic methods (Hofman etਊl., 2020 Sheridan 2020b).


By Rachel West, Gigi Kwik Gronvall, and Amanda Kobokovich | February 2, 2021

A few months ago, concerns about COVID-19 diagnostic testing were mostly a matter of test availability and whether the results would be returned in soon enough to make a public health difference.

Now, emerging SARS-CoV-2 variants and the COVID-19 vaccination campaigns have increased complexity for COVID-19 testing.  Many wonder if the tests will accurately diagnose infection with a variant strain of SARS-CoV-2, and whether vaccination will lead to inaccurate results with a diagnostic or serological test. The good news is that most diagnostic tests now in use will remain accurate with the variant strains, and vaccination should not interfere with diagnostic or antibody tests. However, maintaining diagnostic test accuracy is yet another reason why surveillance for new variants of SARS-CoV-2 is critically important, and why we should move forward with vaccination programs as expeditiously as possible. With concerns growing over testing results and how they relate to vaccinations, it’s important to understand the accuracy and relevance of testing.

SARS-CoV-2 Variants and molecular diagnostics: are they still accurate?

SARS-CoV-2 variants are circulating widely in at least 37 countries, including the United States. Particular variants of concern, which appear to be more transmissible, include B.1.1.7, originally sequenced in the UK, and 501Y.V2, originally sequenced in South Africa. The emergence of these variants raises concerns about molecular diagnostic tests that may be used to identify SARS-CoV-2 infection if the variant has a different genetic sequence in the area probed by the test, it will not diagnose SARS-CoV-2, potentially leading to a false negative result. In fact, the initial identification of B.1.1.7 was partially due to a diagnostic test issue, called S-gene target failure. This technical failure raises the question of the ability of currently used molecular diagnostics to reliably identify future SARS-CoV-2 infections. Both variants of concern have mutations in the spike protein, including position N501Y in both and Δ69/70 in B.1.1.7. The FDA has already issued warnings of potential target failures in the following tests: the TaqPath COVID-19 Combo Kit by Thermo Fisher Scientific, the Accula SARS-CoV-2 test by Mesa Biotech, and the Linea COVID-19 Assay kit by Accula.

Because of the diversity and breadth of tests currently available, most diagnostic tests can still be reliably used to diagnose the variant strains. Based on publicly available data and tracking of the 246 molecular diagnostics which have FDA Emergency Use Authorization (EUA), the majority (85.4%) of diagnostic tests have targets other than the spike gene, so they should still be effective for these variants, and would not produce a “failed” test if the infection is caused by a variant with mutations in the spike gene. Of the remaining 14.6% of tests, 7.3% have multiple targets within the SARS-CoV-2 genome in addition to the spike gene, such as ORF1ab and N genes, so they should continue to yield accurate results. Furthermore, the majority (90.1%) of rapid antigen tests with EUA detect nucleocapsid protein, rather than spike protein, so they should be unaffected. It should be noted that 5.7% of tests have no clearly stated genomic target, but may have that stored as proprietary information. Of the 4 tests which target S gene alone, it is not yet clear if these tests would identify an individual infected with a variant primer sequences should be compared to the variant sequences by the manufacturer to screen analytical sensitivity.  Moving forward, it will be vital to watch for notices from the FDA regarding the efficacy of the EUA diagnostic tests, and to follow up on test failures to investigate potential variants’ impacts. Organizers of COVID-19 testing centers must remain informed of any potential changes to test efficacy that might affect their operations or purchasing agreements.

The current diversity of molecular diagnostics targets bodes well for the ability to identify SARS-CoV-2 infections of future variants, but also underscores the importance of widespread, regular sequencing of clinical samples. CDC is increasing surveillance of SARS-CoV-2 samples to understand the spread of the current variants as well as future variants. Test target failure or negative results with clinical symptoms should be accompanied by sequencing to understand if failures are due to sequence divergence. Cataloguing the genomic targets of SARS-CoV-2 diagnostics will be important to understand testing limitations now, as well as in the future should new variants arise.  

After vaccination, will the accuracy of diagnostic and serology tests change?

Vaccination against SARS-CoV-2 will not result in a positive diagnostic test. The Moderna and Pfizer/BioNTech vaccines consist of non-replicating mRNA, so the maximum level of SARS-CoV-2 specific mRNA a vaccine recipient will have is the small amount present in the vaccine. In a SARS-CoV-2 infection, the virus replicates so that mRNA levels increase to much higher levels and persist for weeks. In addition, both EUA vaccines contain mRNA specific to the spike gene of the SARS-CoV-2 virus. The majority of diagnostics target multiple genes other than spike, if they target spike protein at all. Therefore, vaccination with an mRNA vaccine will not result in a positive diagnostic test.

People who have been vaccinated may be interested in getting a serology test, to see if the vaccine “worked,” but a vaccinated person is very likely to get a negative result from a serology test, even if the vaccine was successful and protective. Serology tests are typically used to determine whether a person has been exposed to SARS-CoV-2 in the past and developed antibodies against the virus. Different serology tests detect antibodies to different parts of the virus, but after vaccination with Pfizer and Moderna vaccines, the antibodies formed will only be to one part of the virus: the spike protein. Some serology tests do not detect antibodies specific to spike protein at all, while others are specific for antibodies that target regions within the spike protein (like the receptor binding domain, or RBD). For example, the Roche Elecsys Anti-SARS-CoV-2 S assay detects antibodies to spike RBD, while the the Platelia SARS-CoV-2 Total Ab assay from Bio-Rad detects antibodies to the nucleocapsid protein. Commercially available serology tests should not be used to seek a positive antibody result after vaccination given the differences in vaccine targets, and current EUAs do not authorize individual serology testing for measuring vaccine efficacy. Therefore, a negative serology test after vaccination does not necessarily mean the vaccine failed and reinforces that serology tests should not be used for this purpose. To understand if vaccination stimulated an antibody response, a test specifically designed for the antibodies of interest would need to be used.

While the diagnostic testing news is good for now-- infections by current SARS-CoV-2 variants are likely to be detected by  tests on the market-- it is important to increase surveillance and monitor the emergence of future variants, which may be more problematic. Thankfully, the Biden administration’s plan includes expansion of testing and increased genomic surveillance--two goals that can work hand in hand to monitor SARS-CoV-2 spread and inform public health interventions. It will be vital to implement surveillance and test performance monitoring quickly, so that emerging variants that may impact therapeutics and vaccines are detected and addressed in real time.

Rachel West, PhD, is a post-doctoral associate with the Center for Health Security and the Johns Hopkins Bloomberg School of Public Health Department of Molecular Microbiology and Immunology.

Gigi Kwik Gronvall, PhD is a senior scholar and associate professor with the Center for Health Security and the Johns Hopkins Bloomberg School of Public Health Department of Environmental Health and Engineering.

Amanda Kobokovich, MPH is a senior analyst and research associate with the  Center for Health Security and the Johns Hopkins Bloomberg School of Public Health Department of Environmental Health and Engineering.


The Science of SARS-CoV-2 Testing: What Tests Are Available and What This May Mean for You

COVID-19 testing equips individuals with the information they need to protect themselves and others, and arms public health professionals with data that can inform response efforts.

Recently, leadership across NIH articulated why widespread testing is necessary, important, and achievable. Equally important is understanding the different types of testing available. As a leader and pioneer in the development of clinical data standards, NLM supports the electronic exchange of clinical health information data, including those related to COVID-19 testing, for approved purposes and with appropriate privacy protections.

Three types of testing are available to identify COVID-19 (the disease caused by the SARS-CoV-2 virus).

1) Nucleic acid amplification tests (NAAT), also called molecular tests, detect the virus’s genetic material

2) Antigen tests detect parts of specific proteins produced by the virus and

3) Antibody tests detect COVID-19 antibodies in the blood (serum) that infected people develop to fight off the virus.

NAAT tests are dependent upon a method used to multiply the relatively few copies of viral nucleic acid that might be present in a specimen into a very large number of copies — making it much easier detect the virus. At present, most NAAT tests use an amplification method called polymerase chain reaction (PCR).

PCR uses small segments of DNA, called primers, to pick out the DNA that it needs to multiply. The PCR instruments process the sample in repeated cycles of heating and cooling. During each cycle, the number of copies of the targeted nucleic acid doubles. From a few original copies, it can generate up to a billion new copies to make the virus easier to see in the final detection step.

The FDA recently authorized a different NAAT test method called loop-mediated isothermal amplification (LAMP). This test method warms the sample to a constant temperature and uses six different primers to drive the replication of different segments of the novel coronavirus’s genome. It does not require multiple cycles of heating and cooling. By many accounts, this method is faster and easier to use than real-time PCR. Other methods of COVID-19 detection are under development.

Different SARS-CoV-2 NAAT testing products target different parts of the virus, use different primers to start the PCR reaction, apply to different specimens, and differ in the ability to detect the virus.

The primary methods for collecting a sample are through nasal, throat, and saliva (spit). Nasopharyngeal (NP) samples are believed to be the most sensitive for detecting the virus, but pushing the swab through the nostril into the nasopharynx at the base of the skull can be uncomfortable. The collection of other samples from nasal swabs and saliva can be easier on the person being tested and are becoming increasingly accessible.

The spread of SARS-CoV-2 is particularly challenging to manage because people can be contagious and spread the infection to others, even before they begin to show symptoms. NAAT tests can sometimes detect the virus in early stages before symptoms appear, but not always, and do not necessarily turn positive immediately with the onset of symptoms.

One strategy with NAAT tests involves the use of pooled samples. Pooled sampling involves mixing several samples together in a batch, or pooled sample, then analyzing the pooled sample with a diagnostic test. If the test on the pooled specimen is negative, then all the individuals who contributed to the pool are considered negative for COVID-19. If the pooled sample is positive, the lab must run separate tests on each of the samples to determine who is positive and who is negative. When the prevalence of COVID-19 in a population is low (in the 1-2% range), the total number of tests needed is reduced, and an organization’s testing capacity increases.

Antigen tests for COVID-19 detect the presence of a protein that is part of the SARS-CoV-2 virus. Today, the NP and mid-nasal samples are the primary sampling methods used for antigen testing, but the development of antigen tests for saliva are underway.

Antigen tests are relatively inexpensive and provide results almost immediately. These tests perform best in the early days after an infection begins. While they are not as sensitive as NAAT tests, some have suggested that repeated testing with a fast, although less sensitive test, may do more to help end the epidemic more quickly than perfect tests done infrequently.

Antibody SARS-CoV-2 tests detect the antibodies, or the “virus fighting proteins”, that a person’s immune system produces to fight infection. Antibody testing is generally done on the serum component of a blood sample. Antibodies may appear just a week or so after symptoms of SARS-CoV-2 infection appear. Antibody tests are not used to diagnose an active COVID-19 infection however, they are useful for detecting whether someone has had a past infection.

Two different kinds of antibodies can be measured: IgM (immunoglobulin M) and IgG (immunoglobulin G). IgM antibodies appear early after infection (usually after the first week or so). Somewhat later, IgG antibodies, a more durable antibody, is produced. Today, there is no clear advantage of IGM or IgG antibody testing and not everyone will develop antibodies after a known COVID-19 infection. Importantly, scientists do not know how well or for how long antibody levels might protect someone against a future infection.

All three types of tests can be evaluated locally with a point-of-care (POC) machine or sent to laboratory for processing (in-lab testing). POC tests are carried out in close proximity to a patient and typically take 5-15 minutes, but only one or a handful of samples can be processed at a time. Not all POC machines have the capability to communicate electronically to public health and other reporting systems. In-lab testing machines can process hundreds of samples at time and, with the right safeguards, can deliver results electronically to patients, providers and public health reporting systems. However, in-lab testing has built-in delays due to its batch testing nature and the time it can take to deliver samples to laboratories.

There are many opportunities for innovation in testing methods to improve upon the efficiency, specificity, and scalability of currently available tests. Having a good set of well performing tests for SARS-CoV-2 is very important, but we also need to be able to deliver the results of such tests accurately and quickly (electronically) to the responsible care providers and to public health authorities.

To facilitate electronic delivery of such content, NLM has long supported the development of formal health care terminologies including LOINC (Logical Observation Identifiers Names and Codes), RxNorm, along with SNOMED CT, and more recently, communication structures such as HL7 FHIR (R) . These capabilities are especially important during this time of COVID-19. In the last six months, the FDA has authorized more than 80 SARS-CoV-2 test products for emergency use, the CDC has defined a COVID-19 Case Report Form, and the Centers for Medicare & Medicaid Services has specified content that should accompany every SARS-CoV-2 test. NLM-supported LOINC codes have been defined for all of this content, as well as SNOMED CT codes for coded test values. The FDA, CDC, and industry have produced a compendium of the all SARS-CoV-2 tests and their standard codes. The use of standardized test codes for test results is essential to smooth delivery of test results into electronic health records and for the aggregation of test results for research and public health purposes.

Testing for COVID-19 is important, safe, and easy. Getting tested early and often and following best practices, such as wearing a mask, washing hands often, and limiting social contact will help get us back to normal.

Did you learn something new about testing methods? How else can NLM help support testing activities?

Clem McDonald, MD, is the Chief Health Data Standards Officer at NLM. In this role, he coordinates standards efforts across NLM and NIH, including the FHIR interoperability standard and vocabularies specific to clinical care (LOINC, SNOMED CT, and RxNorm). Dr. McDonald developed one of the nation’s first electronic medical record systems and the first community-wide clinical data repository, the Indiana Network for Patient Care. Dr. McDonald previously served 12 years as Director of the Lister Hill National Center for Biomedical Communications and as scientific director of its intramural research program.


Monitoring of emergent strains

The mutations that accumulate in the SARS-CoV-2 genome can alter the viral phenotype and confer a selective advantage that gives rise to new strains. Genomic epidemiology showed that one strain, distinguished by a non-synonymous D614G mutation in the spike protein, first emerged in Europe before expanding to become the predominant strain worldwide 79 owing to a selective fitness advantage conferred by the mutation that increased viral transmissibility 80 .

The integration of genome sequencing within population-scale testing can enable monitoring of the viral strains circulating within a population. Numerous countries have mandated that a proportion of positive samples is subjected to whole-genome sequencing, thereby providing ongoing surveillance of emerging and circulating variants. This sequencing information can identify emergent SARS-CoV-2 variants with differing transmission or pathogenicity, with resistance to antiviral treatment or that are at risk of vaccine escape 81 . In late December 2020, a new SARS-CoV-2 strain known as B.1.1.7 rapidly increased in prevalence throughout the UK, apparently outcompeting existing variants and prompting the rapid imposition of restrictions on travel to other countries 82 . Additional variants that might increase transmissibility and pathogenicity or reduce the efficacy of vaccines have similarly arisen in South Africa (B.1.351) 83 and Brazil (P.1) 84 . As the effect of these variants on the viral phenotype has become apparent, authorities have recognized that global testing will be increasingly needed to monitor the emergence and circulation of new variant SARS-CoV-2 strains.

Variant diversification identified by genomic surveillance is also important to assess the influence of new mutations on the ongoing performance of molecular diagnostic tests 85,86 . The emergent B.1.1.7 strain harbours a large number of mutations that might prevent the binding of some primers to the spike gene and thereby reduce the sensitivity of RT–qPCR tests 87 (Box 1). In response, numerous variant-specific primers have been developed, illustrating that strain diversification will require ongoing updates and validation of testing reagents.


TESTING COSTS AND PRIORITIZATION

A new law mandates that Medicare, Medicaid, other government health care and insurance plans, and most private plans cover COVID-19 testing in the United States without copays or deductibles. On 5 March 2020, the Centers for Medicare and Medicaid Services (CMS) announced new Healthcare Common Procedure Coding System (HCPCS) codes for health care providers and laboratories to test patients for SARS-CoV-2. Starting in April, laboratories performing the test could bill Medicare and other health insurers for services, using a newly created HCPCS code (U0001). This code applies to all tests that were developed by the CDC. Laboratories performing non-CDC laboratory tests for SARS-CoV-2 can bill for them using a different HCPCS code (U0002). Current test prices are $35.91 for U0001 and $51.31 for U0002. The overall costs should take into context how a diagnostic test is used in practice. For example, if a test is restricted to very sick patients, the cost is small compared to the overall medical care. Conversely, if a test is used for broad screening, the cost per positive result could be high depending on prevalence.

There are different indications for diagnostic testing for individuals with a proven or suspected case of COVID-19. Given the limited testing capacity in the United States, priority lists have been established. Priority 1 includes hospitalized patients and symptomatic health care workers. Priority 2 includes symptomatic patients in health care facilities, >65-year-old patients with underlying conditions, and first responders. Priority 3 includes symptomatic patients, including critical infrastructure workers. Individuals without symptoms are currently not prioritized for any testing (28). Specific use cases for different tests have also been laid out (29), but they are likely to change as more testing capabilities become available and societal needs change, such as identification of infectious individuals versus seropositive individuals returning to work.

When carried out broadly and repeatedly, NAT results have consequences for individuals, communities, and the entire population. These tests not only permit the identification, isolation, and treatment of infected individuals but also diagnose presymptomatic and asymptomatic carriers and thus more accurately define the infection rates across populations. Serological testing should be used in parallel with NATs to determine which individuals have acquired immunity and how long it lasts. Serosurveys may also help efforts to develop vaccines. By extension, serological testing that is performed frequently and on a wide scale should help determine what fraction of the population may be immune to COVID-19 and which individuals may rejoin the workforce. The lack of longitudinal testing is problematic because it inhibits our ability to understand the evolution of the disease.

Containing COVID-19 will likely require combinations and concomitant use of the different types of diagnostic tests discussed above. Excitingly, more sensitive and specific kits have become available from major vendors. To be successful, these assays will need to be deployed in such a way that broad and repeated testing becomes routine. Last, there is a need to develop test kits that simplify lengthy purification steps and yield results in much shorter time frames than is currently available. A variety of new approaches are currently being tested experimentally to achieve such results.