How to avoid false positive COVID test

Viral TikTok videos showing teenagers hacking rapid COVID-19 tests by inserting sodas and other acidic solutions into the tests’ inlet are causing quite a stir, especially in the UK, where these easy-to-use, at-home tests are currently used for regular screening of students and teachers in secondary schools and colleges. 

While PCR tests remain the gold standard for SARS-CoV-2 detection, so-called rapid COVID-19 tests employ lateral flow technology instead, and can return a result in 30 minutes or less. 

In addition to their speed, these rapid tests can also be performed anywhere, increasing the accessibility of testing. However, preliminary research posted to the preprint server medRxiv on July 5 confirms that certain beverages can cause false positives. And because anyone in the UK who obtains a positive rapid test result must immediately self-isolate for up to 10 days, report the result, and follow up with a PCR test—or face a fine—education leaders in the UK are concerned such false positives are being used to get out of school. Geoff Barton, the general secretary of the Association of School and College Leaders in the UK, tells the news outlet i that such hacking is limited to “a very small minority of pupils.”

The sudden explosion of rapid test hacking videos has added to existing concerns about their accuracy. “People are saying ‘well, these tests are hopeless,’” explains Alexander Edwards, who researches and develops clinical and microbiological tests at the University of Reading School of Pharmacy in the UK. 

See “COVID-19 Diagnostics: How Do Saliva Tests Compare to Swabs?” 

The Scientist spoke with Edwards to understand how sodas can cause false positives and the broader implications of this kind of test hacking—as well as why these tests aren't hopeless.

Courtesy of Alexander Edwards

The Scientist: What is a lateral flow test, and how does it work?

Alexander Edwards: A lateral flow test is a rapid way of measuring one particular target, which is useful for clinical diagnosis, in a complex mixture. And that mix could be something coming off a swab, or it could be blood, or something like that. The way it works is it binds to that target using antibodies. And if the target is present, the antibodies present in the system allow you to get a visual line, which is your positive line, because the antibodies and the antigen trap the target on that stripe on that little line.

TS: How accurate are they?

AE: Lateral flow tests can be extremely accurate, and they’re very powerful for detecting particular targets in complex mixtures. The only catch is that they’re only as accurate as how they’re being used.

The great benefit of lateral flow tests is they’re portable, they’re cheap, they’re disposable, and they’re easy to use near the patient, but that does come at a slight cost in terms of their analytical performance. . . . The good news is that lateral flow tests can be extremely specific, and what that means is that if you get a positive result, it’s very likely to be a real result. What we mean by that is, we don’t tend to get a huge number of false positives for COVID-19 lateral flow tests. 

The manufacturers would say that this is quite simply because you have these gold particles, which forms the colored element, and if that’s treated with an acidic solution, you may get it clumping together or aggregating.

There is a final challenge about the accuracy of these tests, which is that . . . sometimes the tests are less accurate when they’re used in a different way. And one really good example of that is that if you take a test and use it in a trained supervised environment, you tend to get very reproducible, reliable results—they work the same way every time. But if you then roll it out in a community setting, when perhaps the people using them aren’t as experienced, what you start to see is more false negatives and more false positives, sometimes simply because the people operating the test might make a mistake, the instructions may not be very clear, or even something really silly, like the test has gone out of date, or it’s just broken in some way.

TS: Why might something like sodas return a false positive result?

AE: The biochemistry of it’s quite interesting, because it seems like there’s something odd there at first sight. You might say, ‘well, you should only be able to get a positive result if there is the virus particle there, because that’s the whole point of the test; it should only be able to detect the virus particle.’ But there are some ways that these tests can go wrong, and one really obvious one is if you put something into the test which isn’t supposed to go in the test, particularly something like a strong acid or an acidic solution. What you can do is you can mangle up, if you like, or mess up the antibodies in the test. And when you do that, you can get unexpected results. 

What I’ve been able to see—and there’s even some papers that have looked at this experimentally—is that under some circumstances, you can get the appearance of a positive line. The manufacturers would say that this is quite simply because you have these gold particles, which forms the colored element, and if that’s treated with an acidic solution, you may get it clumping together or aggregating. That makes it sticky, and then it sticks where it shouldn’t stick. 

Anytime you get a false positive, especially if it’s a false positive that’s arising from improper use of the test, you’re actually damaging the . . . public health system that’s there to protect us.

To be fair to the manufacturer, they never intended those things to go into the test. So it’s certainly not a weakness or a criticism of the test, because if you follow the instructions that come with a test, and you don’t put soda or a slice of kiwi fruit or whatever it is that people have tried onto the test, and you actually follow the rules and do the test thirty minutes after eating and drinking, if that’s what the instructions say, then you won’t get these unexpected results.

TS: Why might false positives, whether intentional or not, be a concern?

AE: If you have a testing program where you test people a lot of the time . . . especially if you’re screening people without symptoms, every time you get a false positive, the person involved and also all those people who’ve been in contact with them have to be treated as if they have this very dangerous, highly contagious infectious disease. In many countries, it is a legal requirement to report that result, because the pathogen is considered to be so dangerous. . . . So anytime you get a false positive, especially if it’s a false positive that’s arising from improper use of the test, you’re actually damaging the system, and the system is the public health system that’s there to protect us. This is obviously a bad thing.

TS: What can be done to minimize the impact of false positives?

AE: The advice is really very simple. . . . Try and follow the instructions that come with the kit as closely as possible. And the reason I say that is because when we have data to trust the test, that data is always generated when using the test according to the manufacturer’s instructions. So it may well be possible that you could change the way you do the test, and it would still work fine, but we don’t know that.

That sounds kind of boring, to just follow the instructions. . . . But because it’s a medical test, it’s actually really, really important that you do follow the instructions—otherwise the system tends to break down.

Editor's note: This interview was edited for brevity. 

Two COVID-19 cases previously linked to Melbourne’s current outbreak have now been reclassified as false positives.

They’re no longer included in Victoria’s official case counts, while a number of exposure sites linked to these cases have been removed.

The main and “gold standard” test for detecting SARS-CoV-2, the virus that causes COVID-19, is the reverse transcriptase polymerase chain reaction (RT-PCR) test.

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The RT-PCR test is highly specific. That is, if someone truly doesn’t have the infection, there is a high probability the test will come out negative. The test is also highly sensitive. So, if someone truly is infected with the virus, there is a high probability the test will come back positive.

But even though the test is highly specific, that still leaves a small chance someone who does not have the infection returns a positive test result. This is what’s meant by a “false positive”.

First off, how does the RT-PCR test work?

Although in the age of COVID most people have heard of the PCR test, how it works is understandably a bit of a mystery.

In short, after a swab has been taken from the nose and throat, chemicals are used to extract the RNA (ribunocleic acid, a type of genetic material) from the sample. This comprises a person’s usual RNA and RNA from the SARS-CoV-2 virus, if present.

This RNA is then converted to deoxyribonucleic acid (DNA) — this is what the “reverse transcriptase” bit means. To detect the virus, the tiny segments of the DNA are amplified. With the help of some special fluorescent dye, a sample is identified to be positive or negative based on the brightness of the fluorescence after 35 or more cycles of amplification.

What causes false positive results?

The main reasons for false positive results are laboratory error and off-target reaction (that is, the test cross-reacting with something that’s not SARS-CoV-2).

Laboratory errors include clerical error, testing the wrong sample, cross-contamination from someone else’s positive sample, or problems with the reagents used (such as chemicals, enzymes and dyes). Someone who has had COVID-19 and recovered might also show a false positive result.

How common are false positive results?

To understand how often false positives occur, we look at the false positive rate: the proportion of people tested who do not have the infection but return a positive test.

The authors of a recent preprint (a paper which hasn’t yet been peer-reviewed, or independently verified by other researchers) undertook a review of the evidence on false positive rates for the RT-PCR test used to detect SARS-CoV-2.

They combined the results of multiple studies (some looked at PCR testing for SARS-CoV-2 specifically, and some looked at PCR testing for other RNA viruses). They found false positive rates of 0-16.7%, with 50% of the studies at 0.8-4.0%.

The false positive rates in the systematic review were mainly based on quality assurance testing in laboratories. It’s likely that in real world situations, accuracy is poorer than in the laboratory studies.

A systematic review looking at false negative rates in RT-PCR testing for SARS-CoV-2 found false negative rates were 1.8-58%. However, they point out that many of the studies were poor quality, and these finding are based on low quality evidence.

No test is perfect

Let’s say for example, the real-world false positive rate is 4% for SARS-CoV-2 RT-PCR testing.

For every 100,000 people who test negative and truly don’t have the infection, we would expect to have 4,000 false positives. The problem is that for most of these we never know about them. The person who tested positive is asked to quarantine, and everyone assumes they had asymptomatic disease.

This is also confounded by the fact the false positive rate is dependent upon the underlying prevalence of the disease. With very low prevalence as we see in Australia, the number of false positives can end up being much higher than the actual true number of positives, something known as the false positive paradox.

Because of the nature of Victoria’s current outbreak, authorities are likely being extra vigilant with test results, potentially making it more likely for false positives to be picked up. The Victorian government said:

Following analysis by an expert review panel, and retesting through the Victorian Infectious Diseases Reference Laboratory, two cases linked to this outbreak have been declared false positives.

This doesn’t make clear whether the two people were retested, or just the samples were retested.

Either way, it is unlucky to have two false positives. But given the large numbers of people being tested every day in Victoria at present, and the fact we know false positives will occur, it is not unexpected.

The broader implications

For an individual who received a false positive test result, they would be forced to go into quarantine when there was no need. Being told you have a potentially lethal disease is very stressful, especially for elderly people or those at risk because of other health conditions. They would also likely be worried about infecting other members of their family, and could lose work while in quarantine.

Particularly given authorities initially pointed to these two cases as examples of transmission of the virus through “fleeting” contact, no doubt many people have wondered whether without these cases, Victoria might not be in lockdown. This is just conjecture and we can’t really know one way or the other.

False negative results are clearly very concerning, as we don’t want infectious people wandering around the community. But false positives can also be problematic.

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