What you need to know about COVID-19 testing

1. Detection of the virus itself or of viral materials

a. Detection of viral RNA by RT-PCR.

The SARS-CoV-2 genetic sequence was revealed by researchers from China around 10-12 January 2020 (1, 2) and deposited in Genbank (accession MN908947). This has allowed the very rapid design of primers and other reagents for Reverse Transcription Polymerase Chain Reaction (RT-PCR) methodologies to detect SARS-CoV-2 viral RNA. Many of the tests can simultaneously distinguish SARS-CoV-2 from other similar viruses such as SARS and MERS as well as the more common corona viruses endemic in humans 229E, NL63, OC43, and HKU1 and sometimes, various influenza viruses. RT-PCR is a well-established, and very commonly used technology for the diagnosis (and monitoring e.g. of HIV) of viral infections and is widely available in clinical diagnostic laboratories.

As of 7 June 2020, there were at least 146 commercially available in-vitro diagnostic (IVD) PCR kits (combinations of consumables and reagents for PCR machines in clinical laboratories) – an increase of 38 over the previous month (3). These kits are generally based around the detection of one or two conserved genes: RdRP – the RNA dependent RNA polymerase gene found within the orf1ab region; the E (Envelope protein) gene; and the N (nucleocapsid protein) gene.

As would be anticipated for such a well-established technology, all but eight of these manufacturers claim some sort of regulatory approval in Europe (CE-mark) or the USA (EUA) for their tests; however, the majority of these “approvals” are actually Emergency Use Authorisations (EUA) issued by the FDA or occasionally the US CDC. Other tests (around 50) claim CE-mark, but it should be remembered that this will generally be by self-certification. The implications of this are that any laboratory using these tests will need to establish for themselves the functionality of the kits on their devices and maintain rigorous quality control procedures including the processing of standard positive and negative samples on a very regular basis. It also means that switching between kits may not be possible (because the kits will have been analytically validated for specific PCR machines), or at a minimum a switch will require extensive revalidation of the laboratory processes and work-flows.

Key questions for any diagnostic test are what sample type (tissue or fluid) should be collected, and when and how to collect those samples. In cohorts of symptomatic patients, viral nucleic acid is reported to be detected in respiratory samples (nasopharyngeal and/or oropharyngeal swabs, aspirates, sputum) at symptom onset and for up to two to four weeks (occasionally longer) (4, 5). Initial viral loads in posterior nasopharyngeal specimens are reported to be high, but somewhat lower in saliva (and not always detectable); load is reported to decline slowly in recovering patients and to be somewhat higher at presentation in more severe patients and older patients. Viral nucleic acid was detected in stool or rectal swabs in 59% of patients and in serum in 41%.

For obvious reasons, most of the literature describing studies of PCR based viral nucleic acid detection have concentrated upon symptomatic patients (in many countries, the presence of symptoms has been, at least for some periods, a pre-requisite for testing). However, there are reports of asymptomatic carriers testing positive (6, 7, 8, 9). These individuals were typically contacts of known infected patients. In these reports many (but not all) of the subjects became symptomatic; in the pre-symptomatic individuals viral nucleic acid was detected on average 3 days before the emergence of symptoms.

A key matter is the turnaround time between obtaining a sample and the delivery of results to the patient and their physician. Although RT-PCR is a relatively quick technology (running the test manually from sample to result can take 3-4 hours, and automated systems can reduce this time) delays can occur due to logistical issues with sample transport, results review, and return of the results. Other delays occur due to machine down-times, and reagent supply issues. Queues of samples can build up at centralised testing laboratories. It ought to be possible when testing is performed in a local laboratory for results to be returned within less than 24 hours, but in territories where testing has been centralised, there have been reports of considerable delays.

Returning to the list of clinical utility questions above, the following conclusions regarding RT-PCR seem sensible:

1. RT-PCR has the characteristics of a very good diagnostic test; it has excellent sensitivity and specificity (at least with respiratory samples) and meets the needs of all parties when the results are returned quickly.
2. The situation regarding infectivity is less clear. A positive test probably means someone is potentially infectious although viral nucleic acid is detectable long after the symptomatic period for many patients. More research will be needed to confirm whether current guideline in the UK, for example, of 7 days self-isolation is sufficient. A negative test means only that the individual is unlikely to be infectious at the timepoint that the sample was taken. Given the reports that asymptomatic individuals exist and pre-symptomatic individuals harbour virus for three or more days before showing symptoms, rather frequent testing will be required (perhaps twice weekly) to guarantee that an individual is not infectious. Again, this guarantee of non-infectivity requires rapid turn-around of the results.
3. RT-PCR is probably excellent for its use to evaluate the current prevalence of disease and to locate infection hot-spots.
4. It is not useful as a method to determine how many people have been previously infected nor who might be immune.

b. Detection of viral nucleic acid by methods other than RT-PCR

The need for a more rapid turnaround time of results and perhaps for greater sensitivity in samples such as saliva has driven the invention and development of alternatives to classical RT-PCR.

Four such EUA approved tests with substantially reduced turnaround times use isothermal amplification technology. One example is the Abbott ID-Now near patient device, which detects the RdRp gene in less than 15 minutes directly from throat and nasopharyngeal swabs. This technology seems potentially extremely useful as a means to demonstrate non-infectivity, and possibly useful in situations where rapid confirmation of a clinical diagnosis is required. The Abbott kit and device seem slightly less sensitive in situations of reduced viral quantities through dilution in, for example, viral transport media. Other comparable tests are in development and are expected to become available in the future. Another very interesting technology for the rapid detection of viral nucleic acid which has become commercially available in the last month uses a CRISPR technique to enzymatically detect and signal the presence of the nucleic acid. This technique is reported to provide results within one hour from nasopharyngeal and throat swabs, and other respiratory samples (10).

Some rapid PCR systems run panels of tests simultaneously, e.g. for lower respiratory infections, and can identify which causative agent is present, rather than giving only a yes/no answer for a single test, and in approximately 1 hour. These systems do not yet include COVID-19 in FDA, EUA or CE-marked panels but it is only a matter of time.

Droplet digital PCR is a technology to improve the sensitivity in samples with relatively fewer copies of viral nuclei acid and reduce the rates of false negatives. One such test is available (BioRad, FDA EUA) and others are in development or awaiting approval. The clinical utility of this kind of test is not yet completely clear.

c. Detection of viral material which is not nucleic acid.

On May 9th, the FDA granted emergency use authorisation for a very exciting alternative to nucleic acid testing for the SARS-CoV-2 virus – a device and reagents that qualitatively detect the presence of the nucleocapsid protein. The Sofia 2 SARS Antigen FIA is lateral flow immunofluorescent sandwich assay to be used with the Sofia 2 Fluorescent Immunoassay Analyzer from Quidel (11). This is a near patient or point of care test which needs to be carried out in suitable facilities with trained staff, but which can provide results in a matter of minutes from throat and nasopharyngeal swab samples. The precise performance characteristics of this IVD are not yet clear, but the manufacturer warns that similar antigen tests are known to be less sensitive than nucleic acid tests. In the last few days, a similar test has gained a CE-mark in Europe (Maripoc from ArcDia).

Antigen tests in lateral flow devices are also becoming available and may well have a place in circumstances such as cruise ships and offshore establishments, e.g. oil rigs, where a rapid test for current infection is highly valued. Such tests for Influenza and Norovirus have been used for some years, and, although less sensitive than PCR techniques, can provide a fast confirmation to help limit outbreaks in these confined and remote locations.