Testing for COVID-19

Since the first cases of COVID-19 were identified in December 2019, in Wuhan City, China, the disease has spread rapidly around the world, claiming the lives of thousands of people. Efforts to contain the disease and reduce the number of those infected, have included travel restrictions and social distancing measures such as school closures and quarantine.

However, Dr Tedros Adhanom Ghebreyesus, WHO Director-General, remarked in a media briefing on March 16, 2020, that countries needed to do more by increasing the rates of testing, isolation and contact tracing, which he referred to as “the backbone of the response.”

In his speech, he added: “You cannot fight a fire blindfolded. And we cannot stop this pandemic if we don’t know who is infected. We have a simple message for all countries: test, test, test.”

What can testing tell us?

“Traditionally there are two types of diagnostic test for infectious organisms – tests for the presence of the virus itself (current infection) and tests for antibodies to the virus (prior infection),” Eleanor Riley, Professor of Immunology and Infectious Disease, University of Edinburgh.

Testing to identify whether someone is infected with the SARS-CoV-2 virus at that point in time (testing for active infection) is important to:

• Ensure patients receive appropriate treatment.
• Enable rapid isolation of infected patients and undertake contact tracing to prevent further spread of the virus.
• Identify how many people are infected on a given day.
  
  

Testing to reveal if a person has been infected with the SARS-CoV-2 virus in the past could be used to:

• Allow people who are likely to be immune to safely return to work.
• Identify who should be prioritized for vaccinations, when they become available.
• Identify potential donors for convalescent plasma therapy.
•  Track the spread of the disease.
  
  

What types of testing methods are there?

Testing for the presence of the virus

Polymerase Chain Reaction (PCR)

PCR is currently the “gold standard” for viral diagnostic testing.

“PCR testing is important early in infection because it can help us isolate infected people and contain the outbreak. PCR testing is also important later in the outbreak because it shows us how the virus is transmitting through the population and we can then calculate the percentage of people that develop serious complications, which reveals how dangerous this virus is for different people,” Dr Zania Stamataki, Viral Immunologist, University of Birmingham.

Most of the currently available COVID-19 diagnostics are based on this technology, including:

• The first diagnostic assay to be published by the WHO as a guideline for COVID-19 diagnostic detection. The test was developed at the German Center for Infection Research in January 2020.
  
  
• The CDC’s testing protocol (CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel), which received Emergency Use Authorization (EUA) for the diagnosis and detection of COVID-19 on February 4, 2020.

PCR tests such as these detect specific SARS-CoV-2 viral genetic material (RNA) in a patient sample.

First, the sample is obtained from a swab taken from the patient’s nose or throat. If present, the SARS-CoV-2 genetic material is then isolated using an extraction kit and a complimentary DNA (cDNA) copy made from the viral RNA template. Primers amplify these virally derived DNA sequences. A range of primers can be used to target nucleic acid sequences specific to the SARS-CoV-2 genome. 

Sequence specific probes are designed to bind downstream of one of the primers and to give a fluorescent signal during the reaction.  One end of the probe is labelled with a fluorescent reporter molecule, whilst the other has a quencher molecule. When they are physically close, fluorescence is low, but if the target sequence is present in the sample, the probe binds and is cleaved by the polymerase enzyme during the reaction. This separates the fluorescent reporter and quencher so the quencher no longer dampens the fluorescent signal. Emitted fluorescence is then detected at every PCR cycle, the more target cDNA is present, the more probe is cleaved and the stronger the signal becomes

If no SARS-CoV-2 cDNA is present, the probes do not fluoresce, indicating that the patient does not have COVID-19.

PCR-based methods can detect very low levels of viral RNA, so are highly sensitive and can enable early diagnosis of COVID-19 a few days after infection, even before clinical signs arise. However, they require skilled personnel and sophisticated, often expensive equipment, with turn-around times often in excess of 24 hours (including shipment of samples) meaning they are not suitable for rapid, point-of-care situations. Issues with poor sampling technique can also cause a relatively high rate of false negatives. Reagent shortages are causing bottlenecks in many countries, limiting the amount of tests that can be carried out.

Alternative molecular methods in development

Feng Zhang, Omar Abudayyeh, and Jonathan Gootenberg have recently developed open-access SHERLOCK research protocols and design resources which could be used to develop a CRISPR-based COVID-19 test using paper strips.

Several other research groups are also currently working on developing tests in this area, and a number of preprints have recently been submitted. Preprints are articles which have not yet been peer-reviewed. Results are therefore regarded as preliminary and should be interpreted as such.

• The lab of Dr Changchun Liu at the University of Connecticut Health Center submitted a preprint detailing the All-In-One Dual CRISPR-Cas12a assay method for rapid, ultrasensitive, specific and visual detection of viral nucleic acid.
  
  
• Another preprint describes the potential for Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) to be used as a faster and cheaper point-of-care test.

Methods such as these could offer advantages including being faster and more suited to point-of-care use, without the requirement for expensive equipment. However, possible limitations include the need for different reaction systems and multiple manual operations.

Antigen tests

Rather than detecting the presence of viral RNA, antigen tests detect viral proteins, such as those found on the virus’ surface. These tests will reveal if a person is currently infected with the virus - once the infection has gone, the antigen also disappears.

They are likely to be devised for use with swab samples and could be used at the point-of-care in place of molecular-based tests such as PCR.

However, according to Dr Michael Skinner, Reader in Virology at Imperial College London: “The complexity of identifying and producing the required antibodies for the kit means that development of the tests lags well behind that for antibodies.”

A group of European photonics scientists are currently developing an ultrasensitive laser sensor that detects SARS-CoV-2 antigens from a saliva or nasal swab in minutes.

Testing for prior infection

Antibody tests

Antibody-based tests reveal if a person has been exposed to a virus by detecting antibodies in a person’s blood or serum. They are rapid and simple tests, that are well suited to point-of-care use.

The challenge with an antibody test is knowing when to do the test. IgM antibodies are produced first, and IgG antibodies follow a few days later. A test which can distinguish between IgM and IgG could give information about the phase of infection.

It is still not clear if a person’s own antibodies will protect them against re-infection from SARS-CoV-2, and if so, how long this protection will last.

A number of antibody tests are currently being pursued:

• A preprint from researchers at the Icahn School of Medicine details the development of an ELISA assay capable of detecting SARS-CoV-2 antibodies as early as 3 days post onset of clinical signs.
  
  
• EUROIMMUN’s antibody-based COVID-19 diagnostic test recently received CE Mark.
  
  
• Beckman Coulter is developing tests to detect coronavirus IgM and IgG antibodies.
  
  

Accelerating the availability of COVID-19 diagnostic tests 

Efforts are underway around the globe to increase testing, both by scaling up and overcoming bottlenecks in current methods, as well as fueling research into the development of new, superior tests. Measures such as EUAs can help to expedite this process.

An EUA enables unapproved medical products or unapproved uses of medical products to be used in the U.S. in an emergency when there are no adequate, approved, and available alternatives. The Secretary of the US Department of Health and Human Services determined on February 4, 2020, that COVID-19 was a public health emergency, and subsequently declared the circumstances justified the authorization of emergency use of in vitro diagnostics for detection and/or diagnosis of the virus that causes COVID-19. On February 29, 2020, the FDA issued guidance enabling laboratories certified to perform high-complexity testing under CLIA to use their validated COVID-19 diagnostic tests before receiving FDA authorization. The guidance was further updated on March 16, 2020 to include a policy enabling States to authorize laboratories within their State or territory to develop their own COVID-19 tests and perform specimen testing, without submitting an EUA request.

Current EUA COVID-19 diagnostics include:

• CDC 2019-Novel Coronavirus (2019-nCoV) Real-time Reverse Transcriptase (RT)-PCR Diagnostic Panel (CDC)
• New York SARS-CoV-2 Real-time RT-PCR Diagnostic Panel (Wadsworth Center, NYSDOH)
• cobas SARS-CoV-2 (Roche Molecular Systems, Inc.)
• TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific, Inc.)
• Panther Fusion SARS-CoV-2 (Hologic, Inc.)
• COVID-19 RT-PCR Test (Laboratory Corporation of America)
• Lyra SARS-CoV-2 Assay (Quidel Corp.)
• Quest SARS-CoV-2 rRT-PCR (Quest Diagnostics Infectious Disease, Inc.)
• Abbott RealTime SARS-CoV-2 assay (Abbott Molecular)
• Simplexa COVID-19 Direct (DiaSorin Molecular LLC)
• ePlex SARS-CoV-2 Test (GenMark Diagnostics, Inc.)
• Primerdesign Ltd COVID-19 genesig Real-Time PCR assay (Primerdesign Ltd)
• Xpert Xpress SARS-CoV-2 test (Cepheid)
• BioFire COVID-19 Test (BioFire Defense, LLC)
• Accula SARS-Cov-2 Test (Mesa Biotech Inc.)
• PerkinElmer New Coronavirus Nucleic Acid Detection Kit (PerkinElmer, Inc.)
• AvellinoCoV2 test (Avellino Lab USA, Inc.)
• Real-Time Fluorescent RT-PCR Kit for Detecting SARS-2019-nCoV (BGI Genomics Co. Ltd)
• ID NOW COVID-19 (Abbott Diagnostics Scarborough, Inc.)
• NxTAG CoV Extended Panel Assay (Luminex Molecular Diagnostics, Inc. Luminex Molecular Diagnostics, Inc.)
• NeuMoDx SARS-CoV-2 Assay (NeuMoDx Molecular, Inc.)
• QIAstat-Dx Respiratory SARS-CoV-2 Panel (QIAGEN GmbH)
• Cellex Inc. qSARS-CoV-2 IgG/IgM Rapid Test (Cellex Inc.)
• ·         COV-19 IDx Assay (Ipsum Diagnostics, LLC)