If you haven’t seen all the chatter that states PCRs using more than thirty-something (someone even suggested 25 to me!) cycles will suffer from false positives and so is useless, then read no further. For those who have seen this and wondered, I have two bits of data to show and discuss with you on why the false-positive PCR problem is not a problem at all for SARS-CoV-2 diagnostics. And then there’s a rant.
False positives are really uncommon in practice
Below I’ve included an RT-PCR ‘run’ performed some years ago. It was screening nucleic acids extracted from sick human respiratory swabs and aspirates, looking for human parainfluenza virus type 4 (HPIV-4).
The RT-PCR was run for 55 cycles. Not 30. Not 40. Not 45. 55.
That’s excessive, but I actually liked to do that in my research days to a) make sure the test wasn’t doing anything weird and b) that there weren’t really weak positives I was missing (if a sample came up late (never saw one above 45) I would use a suite of other PCR tests to investigate further.
The point is that after 55 cycles of amplifying nucleic extracts from human respiratory tracts samples, the only thing that crossed the threshold (red line in the figure below) was the positive control (a known extract of HPIV-4 RNA). I have the same sort of data for other viruses.
To say that any PCR run for more than thirty-something cycle will be a false positive, or increase the likelihood of a false positive, is misleading and wrong. The false-positive PCR problem is not a problem.
Thirty-something and biological variability
Look, I fully agree that the later (higher the numerical value) the threshold cycle (CT “cycle number”) of the RT-PCR result, the less target RNA is present at the start of the reaction. That RNA is a surrogate marker for the amount of virus but not an actual measure because PCR methods can’t tell you a virus is infectious.
People are very chuffed to have learned this fact in 2020 by the way. You can tell because they say so. A lot. But despite their newfound out-of-context knowledge they actually know very little about what they’re talking about.
NOTE: while successful detection of viral RNA or DNA doesn’t guarantee the presence of culturable virus, please don’t be so silly as to suggest it must then mean there isn’t any infectious virus present, as someone just did on Twitter. Let’s make a bold call – a positive PCR detection means infectious virus is usually present. And if not at the time of sampling, it was only hours or days ago. You are infected by that virus and that virus is a thing not normally in your body. This is a new and important result at a time when the world is trying to reduce virus transmission – which it can’t do unless it knows where it is.
Most of the time, if you pick two different labs to test a panel of the same samples, you’ll get two sets of subtly (sometimes not so subtly) different results. Molecular biology people each have their favoured way of doing things and they get quite dogged in their ways.🙄
A single CT isn’t comparable
I’ve lifted a figure from an article. It highlights this variability, which definitely also extends into the much more volatile realm of cell culture and virus isolation (growing virus from a patient’s sample using immortal cell preparations grown in flasks).
The figure shows that if you were to blindly and ignorantly rely on single CT values to proclaim lack of infectious risk – let’s say 35 or greater – you would miss people who were still shedding infectious virus.
If you surveyed more labs with expertise in cell culture and virus isolation and labs that could get hold of fresh samples and samples from the actual site of virus replication, I’m sure you could find even later CTs. You can also find labs who can’t isolate virus from samples with CTs this late. This biological variability means we need to step back from these stupid single number cut-offs.
Ummmmm…what false positives?
When presented with a conspiracy theory – I like to ask for the evidence. I generally reject YouTube video sources and weird websites. They never have facts just belief and opinion.
So the real question is: where, oh theorists of the conspiracy type, are all these false positives that lots of PCRs all over the world are generating?
(h/t to Klaus Hentrich for this idea)
Let’s look at Australia.
We have no reported local transmission of SARS-CoV-2 or cases of COVID-19 disease to speak of as I write this. I reckon that’s pretty low prevalence in a country of 25 million.
Australia’s COVID-19 cases right now are almost exclusively in quarantine hotels and among Aussies returning, or special-purpose travellers visiting, from a world on fire. We’ve had a few clusters
This has only been manage in a handful of locations. Specifically, Wuhan (and China in general which peaked at around 4,000 daily cases) and Singapore which contained a dormitory outbreak that peaked at 1,400 cases per day as well as more widespread community cases (outside that crowded foreign worker dorm environment) which never reached higher than 50 cases per day. Both of these locations have now had multiple doughnut days (or months).
Australia hasn’t seen its combined national daily tally of almost exclusively PCR-based tests, fall below 20,000 since June. These aren’t just test results from hotel quarantine by the way – this is mostly from among the community in each of Australia’s eight jurisdictions.
All that testing provides a good number of opportunities for those so-called false-positive PCR results to show up in our daily reported numbers. Surely more testing should mean a steady flow of false positives if these conspiracies are correct? And others love to add that it should be even more of a problem in a low prevalence setting. Australia certainly fits that bill. Plus, we’re testing mostly sick people but also asymptomatic and presymptomatic people.
But lo and behold there is no steady stream of false positives. We don’t have an issue because once again, this is an over-oxygenated amateur conspiracy theory and not an actual issue.
If we have a look at the next figure, we can see no constant stream of positives among the local testing. Yes, cases are occurring (as far as I know, mostly the same RT-PCR-based testing) among those coming from outside Australia but these are – as far as I know – linked to ill travellers diagnosed as having COVID-19. Actual clinical cases. The false-positive PCR problem is not a problem.
That’s not to say there are never false positives. There are. But they are very, very rare events that are almost always caught by the process involved in reporting test results. This process considers the lab results alongside clinical and epidemiological context and checks itself before reporting.
It may be that some parts of the world are too overwhelmed to use that sort that process. And in that instance, other testing might prove helpful. For example the less sensitive overall, but contagiousness-detecting rapid antigen tests may be helpful. Or they might not; there isn’t a lot of real-world evidence for or against their helpfulness. But in the US – what is there to lose?
Nonetheless, an overwhelmed testing system doesn’t make PCR results useless it just makes them less obtainable.
The false-positive PCR problem is not a problem
There clearly isn’t a CT crisis or a false positive plague or a “casedemic”. The false-positive PCR problem is not a problem. There is, however, a crisis in the lack of understanding about a heretofore obscure branch of science that toils in relative obscurity and a tool it relies on; pathology testing and PCR.
But hey, is anyone actually saying there is a problem? Sadly, yes. These are is a couple of the many I’ve read. One is a comment currently in holding, submitted to this blog. The other a tweet at me last night. And yet despite sounding so sure of itself, it isn’t supported by facts and is simply absolute rubbish.
Don’t blame PCR for lots of positive people – blame your government
Just about every aspect of the COVID-19 pandemic has at one time or another during this pandemic, been hauled up as a reason to deflect from the reality of 2020. The reality is that ineffective, unprepared, slow-to-act and cloth-eared leadership – supported by the cult of wish-it-away – have slowed the response to this pandemic, making things worse. Not just in 2020 but across decades of profit-over-people decision making.
Leadership needs to lead
Governments that won’t provide universal healthcare can’t look after the mental and physical wellbeing of their populations so they haven’t been able to safely take the harsh measures which most effectively break widespread transmission while also maintaining suitable care for the multitude of health needs of those under their care.
Governments who won’t get serious about financial protections for the lives and livelihoods of frontline workers have failed to protect their communities. Instead, they’ve doubled down against the most effective restrictions to prevent bad economic numbers.
Governments who had in place convoluted lab test development regulations and who hadn’t built up testing capacity have instead had to settle for less ideal ways to test people because the gold standard is unachievable. Or it’s just too late.
Governments who failed to invest and listen to science and public health have not been able to use the power of contact tracing to help contain more transmission and have generally made poor decision putting their communities at greater risk.
Governments who have failed to learn from successful countries around subjects like safer return to school, how to reduce restrictions carefully, the importance of multi-lingual and multilayered communication and eductaion on all facets of the pandemic and changing the dogma of “droplet” verse “airborne” in response to the screaming pleas of expert aerosol scientists.
There are also the loud angry supporters of those governments who consciously hold fast to and amplify these failings for their own benefit and fame, or because they don’t know any better, or they simply follow other malicious agents of chaos down a dark path of often angry and usually selfish fantasy.
Individual rights over societal good is a doctrine that yields disastrous results it seems, for the health of an interconnected world. Who could have guessed this?
But I’ve digressed.
The PCR test works to find infections. It can find them before you become sick. That helps in containing spread if you have a process for isolation.
PCR testing can find infection if you were sick recently but missed a timely test. Whether you feel sick or not, that means contact tracing can be used to contain transmission chains that you may have started.
PCR can actually be used to slow SARS-CoV-2 spread and contribute to bringing an epidemic under control. Of course only if you actually do something to respond to those results.
If you miss transmission chains and cases or find them but don’t do anything to interrupt them, then you simply have to rely on hope and faith that things will work out in the end. So far, hope and faith haven’t helped much.
- Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020
- Charting the COVID-19 spread in Australia
- Statement From The Premier, Victoria, 22NOV2020
- Antigen-based testing but not real-time PCR correlates with SARS-CoV-2 virus culture
- Singapore’s COVID-19 Interactive Situation Report
- China COVID-19 cases via the World Hleath Organization dashboard
- 28DEC2020: corrected some typos