COVID testing explainer 2: What is a false negative?

virus

I’ve seen a lot of comments on various Isle of Man facebook groups over the last few weeks where people are claiming their negative SARS-CoV-2 test result must be a “false negative”. So let’s delve into the testing and what “false negative” really means.

The first thing to know is that a SARS-CoV-2 PCR test result is reported as either:

  1. SARS-CoV-2 DETECTED
  2. SARS-CoV-2 NOT DETECTED

As you can see the lab doesn’t use the words “positive” or “negative”. When a PCR test is carried out, it either detects the virus or it doesn’t and that’s how the results are reported to your doctor.

This paper shows that infectiousness starts around 2.5 days before COVID-19 symptoms start and the peak viral load (the amount of virus) occurs about half a day before symptoms start. By the time you start to show symptoms (and therefore become eligible for a test through the IoM 111 line) there should be an awful lot of virus present at the back of your nose and throat for detection. Detectable (but possibly non-infectious) viral RNA also hangs around in there for at least two weeks and even up to a month. PCR testing helps to differentiate people with COVID-19 from people with colds and flu so that they can be contact traced and the spread of SARS-CoV-2 contained.

All PCR tests have something called a limit of detection which is the lowest number of copies of the virus that the test can detect. The test we use on the Isle of Man has a limit of detection of 10 copies of the virus, which is very sensitive.

np_swab

A possible weak point in SARS-CoV-2 testing is the swabbing. The nasopharyngeal swab that needs to be taken to collect the virus has to go really far back into the nose. It’s not very pleasant (but thankfully only takes a few seconds) and there’s the potential for a false negative result if the swab doesn’t collect enough cells. However, the testing we do on the Isle of Man actually takes this into consideration and tests the amount of human RNA present on the swab as well as the SARS-CoV-2 RNA. If the level of human RNA is below the threshold we established during our validation testing the result is reported as INSUFFICIENT SAMPLE and the patient is asked to have a new swab taken.

If we didn’t do the human RNA test then a small proportion of the low quality samples could be from patients with COVID-19 but could be reported as negative because the lack of material in the swab reduced the amount of virus below the limit of detection of the test. These would be false negatives and given the potential for a false negative person to spread the disease thinking they’ve had a negative test, we introduced the human RNA check on the Isle of Man to minimise the risk of false negatives as much as we possibly can.

So what does “false negative” really mean?

Well, there’s bit more to it than just that term and what most people assume it is. There are really four different terms to understand: true positive, true negative, false positive and false negative. 

positives-negatives

Let’s look at hypothetical Bob who was thought up by me and doesn’t exist in real life.

Bob has had a cough for a few months, sees the symptoms of COVID-19 on the news and decides to ring 111 for a test. He says the cough is new. The test result is “SARS-CoV-2 NOT DETECTED”. A few days later he gets infected with SARS-CoV-2 after a sneaky visit to his friend’s house during lockdown (bad Bob). At day 5 he becomes infectious and at day 7 he starts to show symptoms of COVID-19. He calls 111 for a second test and his result this time is “SARS-CoV-2 DETECTED”.

Bob assumes that his first test is a false negative and tells his mates on facebook that the test is rubbish.

But bad-boy Bob is wrong. His first test was a true negative and his second test was a true positive.

So no false negative for Bob.

As you can see, a real false negative is a patient who has COVID-19 disease but where the PCR test does not detect any SARS-CoV-2 virus (usually due to insufficient swabbing) rather than a true negative followed by a true positive. Despite the facebook rumours, real false negatives are actually pretty rare because of the human RNA test we carry out, the limit of detection and the known sensitivity of the test in patients with COVID-19 disease (99.4%).

So in conclusion, the likelihood is that if your test came back negative you had something else like a cold or the flu, not COVID-19.

COVID19 testing explainer 1: What is a virus?

virus

This post is intended as an explainer for the general public

To understand how the testing works we first need to understand what a virus is and how each species of virus differs.

Viruses can infect all types of life, not just humans. There are viruses that infect animals, plants, and even ones that infect bacteria. Viruses infect life forms, but they’re not considered to be either alive or dead themselves. They’re really quite clever little hijackers. They gain entry to a host cell and then use the cell’s own molecular machinery to make more copies of the virus using a genetic blueprint of instructions contained inside the virus. The cell then becomes a microscopic virus factory which ultimately bursts and releases all the new viruses it just made. The new viruses then go on to infect other cells as the infection progresses. The ultimate aim of a virus is to make as many copies of themselves as possible, and often cause chaos along the way which we call disease.

We classify organisms by splitting them into groups – a taxonomy – based on their characteristics. The groups get split into smaller and more specific sub-groups until all the similar organisms are grouped together. Virus taxonomy splits viruses by how they look (morphology), how they copy themselves, what kind of genetic material they have, which organisms they infect and even the type of disease they cause.

The first big split in virus taxonomy is whether the virus genetic material is DNA or RNA. You’ve probably heard of DNA because it’s the same language our own genetics are written in. It’s made of four letters: A, C, T and G. RNA is a similar molecule but the T is replaced with a U. DNA and RNA can be both double stranded or single stranded.

dna_vs_rna

SARS-CoV-2 (the proper name for the virus causing COVID-19 disease) is a single stranded RNA (ssRNA) virus. This type of virus tends to mutate quite quickly and rather than “strains” they tend to form something we call a quasispecies. The mutations accumulate over time as the virus infects more people and this means that we can track the infections using genomic epidemiology techniques by comparing the full genome sequence of the virus (the A, C, U and G letters) of all the different isolates. If you’d like to see more info on this have a look at the Next Strain website, it’s where the scientists are putting the genome data during the outbreak. I’ll put a post about genomic epidemiology on my to-do list.

The letters in the SARS-CoV-2 genome code for the instructions to make the proteins of the virus. This is comparable to having the blueprints to build a house. The virus hijacks the host cell and uses it’s molecular machinery to make more copies of itself. As an aside, this is why computer viruses got their name as they’re malicious code which likes to replicate itself. The RNA inside SARS-CoV-2 codes for a number of proteins which sit on the membrane of the virus as well as for enzymes which do the copying of the instructions themselves (the RNA-dependent RNA polymerase, or RdRp).

SARS-CoV-2

Once we know the sequence of letters in the viral genome we can start to design tests to detect it using those letters to our advantage.

It’s alive!

The Rachomics Blog has started!

I’ve been wanting to set up a blog for about, oh, 12 years or so. I was previously a scientist within the UK scientific civil service so blogs weren’t exactly something that were welcomed.

These days, I run a molecular diagnostics company (Taxa Genomics) and my collaborative work with the Isle of Man Department for Health and Social Care during #COVID19 has prompted me to look again at finding the time to do some science outreach. Plus, 93% of you said you’d read it. I’m going to hold you to that.

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Let’s face it, I love science and think that everyone else should too, given how much it impacts our daily lives (even if we don’t realise it some times).

I promise I won’t swear (too much).

//Rachel