Interpreting a lateral flow SARS-CoV-2 antigen test

What is a lateral flow device?

Lateral flow devices can detect the presence of a target substance in a liquid, typically in a single use disposable device. Their use is well established for home pregnancy testing. For covid-19, these devices are detecting a SARS-CoV-2 antigen, consequently sometimes they are termed “rapid antigen tests.” A large number of SARS-CoV-2 antigen LFD tests are available internationally.9 In the UK the Innova test is approved by the Medicines and Healthcare products Regulatory Agency to identify covid-19 in people who do not have symptoms and is the only test widely used.10 The US drug and device regulator, the Food and Drug Administration, has not approved the Innova test and in June 2021 issued a safety communication warning the public not to use the test based on concerns that its performance had not been adequately established.11

Other LFDs have met minimum standards and are being field tested in the UK.12 This article focuses on interpreting the Innova LFD; the underlying principles will be similar for the interpretation of other tests.

What is the UK policy?

Currently the government is making LFDs freely available to all adults and secondary school children (age 11+) in England, with advice to test twice each week to detect cases in people without symptoms.1 In addition, visitors to care homes in England are also expected to undertake an LFD before their visit. Northern Ireland, Wales, and Scotland are also making these tests widely available, although the recommendations between countries vary.234 Independent of government, some employers are establishing their own testing schemes.

Anyone who has a positive LFD test result is advised to act as if they have symptoms of covid-19—ie, they and their household should isolate and arrange a confirmatory polymerase chain reaction (PCR) test within two days. If the confirmatory test is negative, they are advised that they do not need to isolate.13 If the confirmatory test is positive, then they are advised to continue to isolate for 10 days.

If people have symptoms of covid-19, they are asked to book a PCR test to rule out covid-19, rather than use an LFD. Typically, people who have had a positive result from a recent PCR test are advised not to participate in regular LFD testing for 90 days.1415 People who have been vaccinated are still encouraged to test. The performance of these tests in people who have been vaccinated has not been directly evaluated. Concerns have been raised that people who have been vaccinated may have a lower viral load, therefore will be less likely to test positive, although people who have been vaccinated are also less likely to transmit the virus.16 Given the LFD is an antigen test, vaccination will not trigger a positive test result.

Recommendations for use of LFDs are changing rapidly. Trials include “test to enable” (eg, testing before attending a large cultural or sporting event) and “test to release” (eg, daily testing of contacts of cases, with a negative test enabling a partial relaxation of the 10 day isolation requirements).517

How do LFD tests perform?

Some of the concern around LFDs relates to their ability to correctly pick up cases of SARS-CoV-2 infection, particularly when deployed in home and community testing. Test accuracy is defined in terms of sensitivity and specificity.18 Real world performance depends on test characteristics but also the likelihood of disease in the individual and the quality of the testing.

Data on the sensitivity and specificity of the Innova LFD test are limited. A Cochrane review synthesised the current evidence for a wide variety of LFDs.19 However, all the studies on the Innova LFD included in the review had not been peer reviewed, and some more recent, relevant evaluations were not included as they were published after the end date of the searches (30 September 2020).2021 The Cochrane review reports a range of estimates for sensitivity and specificity in different contexts of use.18 The sensitivity of LFDs (the proportion of people with disease who have a positive test, or the true positive rate), according to the review, ranged from 28% (when used in an outbreak investigation) to 79% (when used by laboratory scientists), and the specificity (proportion of patients without the disease who have a negative test, or the true negative rate) from 99.5% to 99.9%.19 Recent analysis by Public Health England, not included in the Cochrane review, suggests the specificity may exceed 99.9%.21

These estimates of sensitivity and specificity are based on evaluating the LFD tests against a gold standard of PCR.6 However, the PCR test has limitations as the gold standard test for diagnosing SARS-CoV-2 infection. Firstly, it is not 100% sensitive, meaning that some people will be missed when relying on PCR testing for diagnosis.18 Secondly, a PCR test can detect very low levels of virus present in a sample, meaning a positive PCR test does not necessarily equate with people being infectious. The median time for which an individual will test positive with a PCR test is the range 22-33 days,22 longer than the typical infectious period. In a clinical setting this high sensitivity to a low concentration of the virus in the sample may be helpful, by facilitating a diagnosis even if a poor sample is taken or if viral levels are low in the person being tested. But if PCR tests are used to test large numbers of people without symptoms to identify those who are infectious and to prevent further spread, the PCR test will register positive for people who are highly infectious, but it will also register positive for people who recently had the infection but are no longer infectious.

How sensitive are LFDs at identifying people who are infectious?

In the UK, LFDs are being used primarily to prevent spread of SARS-CoV-2 by finding cases among people who do not have symptoms of covid-19. Key to this is the sensitivity of LFDs in identifying people who are currently infectious. In this context, LFDs’ poor ability to detect people who are not infectious (but who recently had the infection) is not a concern. The PCR test, in contrast, identifies those who are currently infectious, and those who were previously infected but are no longer infectious. We need to be mindful of this when assessing data evaluating the accuracy of LFD tests against a PCR reference standard.

Assessing the performance of LFDs in identifying people who are infectious depends on having a good measure of infectiousness. One proxy measure of infectiousness is the Ct value from a PCR test. The Ct value is the number of PCR cycles required to detect the virus, with a low Ct value indicating a large concentration of virus present in the tested sample. The measured Ct value is only likely to be a proxy for the viral load in the patient, as it will depend on the sample quality.23 Evidence shows a strong correlation between Ct values and in vitro infectiousness24 and some evidence for increased risk of transmission from patients with lower Ct values.25 However, no agreed Ct threshold exists for infectiousness. Transmission is also likely to be influenced by other factors (eg, host immunity, social distancing, and mask wearing) as well as how infectious the case is.

Some of the variation in reported sensitivity is explained by variation in the Ct value.62627 For example, a large evaluation of community testing in Liverpool (not included in the Cochrane Review) among people without symptoms found relatively high sensitivity when testing people with a lower Ct (>80% for samples requiring fewer than 20 PCR cycles to detect the virus—ie, Ct <20), but very low sensitivity at high Ct values (6% for a Ct value of 30-35).27 Taking a conservative threshold for an infectious sample (a Ct value ≤25) from the Liverpool study would suggest that LFDs have sensitivity of 67% for identifying a person who is infectious (95% confidence interval 41% to 87%).27 The Liverpool study (n=5869) was relatively large and reflected real world use, with trained lay testers, and although it was primarily an evaluation of supervised testing rather than home testing, it is more relevant than data from the Cochrane review,19 which comes exclusively from the early Public Health England (PHE) evaluation.26

Other factors affecting test sensitivity

The Liverpool and PHE evaluations suggested that some of the reported variation in sensitivity could be explained by the quality of the testing undertaken.2627 That includes taking the sample, processing it, and reading the test. For example, the PHE evaluation reported higher sensitivity (79%) when the testing was undertaken by laboratory scientists compared with non-scientists (58%).26 This was based on limited data, but nonetheless raises the possibility that home testing (which increasingly predominates) may be less sensitive than testing performed in supervised test centres.

Interpreting a negative test

To determine the reliability of a negative LFD test, pre-test probability needs to be taken into account. For most people being tested who do not have symptoms this is likely to be low or very low. A negative LFD test result will reduce the post-test probability of having disease (table 1), but it does not eliminate fully the possibility of infectiousness. If we assume a sensitivity of 50%, a negative test result will approximately halve the post-test probability of disease.

Identify any factors that might indicate a higher pre-test probability. For example, people with symptoms or who have been a contact of case will have an elevated risk of covid-19, potentially greater than the background prevalence in the community, and a negative LFD should therefore be treated with caution. If covid-19 is suspected clinically, arrange a PCR test. More generally, when a patient states they have had a negative covid-19 test, the clinician should check whether this was an LFD test or a PCR test and reinforce the need to isolate if symptomatic until the patient has received a PCR test result.