The Live-Dead qRT-PCR problem, the testing industrial complex and its impact on society.

I never thought the work I did for the human genome project would be weaponized to lock down society. We are now ruled by qPCR right and the transparency on the process is shameful. Image
I hope to convey the need to push for public transparency on the CQ values being utilized to count “case” #'s. I also hope to convey the need for home testing and how this regains our medical privacy. It is clearly required for any personalized medicine dream to materialize.
Why listen to me?
I’ve done a lot of PCR (25 yrs). Below is factory floor our R&D team designed, programed, and maintained at The Whitehead Institute/MIT Center for Genome Research. It processed 40M Sanger sequencing reactions/year (a more complicated version of linear PCR) Image
What is the live dead PCR problem? PCR doesn’t count infectious virus. It counts RNA molecules which can be from live or dead virus. Initial infections contain lots of live virus but as you clear the virus the Live-dead ratio shifts more towards the dead.
Jaafar et al addressed this by correlating CQ levels in qPCR with Plaque Forming units (PFUs) on Vero cells. Plaques form only when virus is infectious. Dead Virus forms no plaques.
But culturing virus on Vero cells takes too much time to track pandemics and is hazardous to lab employees as you are replicating live virus. Image
Jaafar et al. showed that Cqs after 33 were mostly dead. Most testing facilities report a positive result if under CQ 40 (but this should be verified in your jurisdiction).…
Due to many diff qPCR tests in the market , one cannot simply lift Jaafar et al equation onto your test but its an important data point.
Cqs are an inverse Log2 scale. Each Cq digit is a factor of 2 so a Cq of 10 is twice as much RNA as a CQ of 11. There are ~3.3 Cqs for every factor of 10. One would use 2^7 or 128 fold to estimate how much difference there is for 7 Cq offset from 33 to 40. Image
Limit of Detection.
Take RNA, perform a serial dilution in qPCR and measure how low you can go.
Most tests crap out 50 copies of SARs RNA at around 37 cycles. Calling positives past your Limit of detection is shady. I think there is a lot of this going on.
This can create False positives that can transform a pandemic into a “case-ademic”. There are a few papers circulating that demonstrate this effect on other outbreaks that have occurred in the past. Please post them to this thread if you have them at your fingertips.
Sampling limitations.
LODs are measured putting RNA straight into the qPCR. Reality requires qPCR of a nasal swab usually soaked into 1ml of fluid and no-one PCRs the whole milliliter. They take 1/25th - 1/50th of this into the qPCR.
If you have 1 copy on that swab and you only take 1/50th of the volume into qPCR.. you have a poisson sampling problem are unlikely to find the single molecule unless 50 tests are performed. This can create false negatives. SNPs under primers can create false negatives as well.
SNPs under primers are something we need to watch as the virus mutates. I think this is a smaller problem right now but needs to be considered when looking at re-infection data.
To understand these have a look at an EUA for the Roche qPCR assay. FDA RUO for Roche as an example
FDA EUA links and a few studies comparing different kits performance in the market place.………
These FP rates may be low but when the disease prevalence is low, the FPs become a higher % of the total number of positive tests. 1% population disease prevalence with a 1% FP rate would deliver 10 FPs and 10TP out of 1000 tests but you wouldn’t know which of the 20 are T or F.
For some tests the FP rate must be below 1:1000 as we see positivity rates like this at some universities.
See Northeastern university rates (150/~300,000)…
If you assume those NE tests are FPs, this is an upper bounds on the FP rate. Reality is they are probably a mixture but public transparency on the FPs and FN of the test being run is sorely lacking. This is clinically irresponsible.
Why Live-Dead matters.
The majority of the time a patient is qPCR positive (<40Cq) is the tail end of the disease where the virus is shedding and more dead than alive RNA. I call this the lower infectivity long tail. People detected in this window are usually non-infectious
These poor folks are quarantined, track & traced, medical privacy violated, constitution nullified and shamed from society. But this creates a lot more testing volume! I doubt there is motivation to tighten the dial, despite there being cheap old-school tools which address this. Image
Everyone caught in the long tail create a chain reaction of contact tracing testing and when these people are young and have less risk than the flu... Great Barrington Declaration... @aier…
A good paper on this.…
How does one address this?
Does every test need a Jaafar correlation curve? Maybe. There is another Maniatis/Sambrook trick one could use. This is getting old school, open source and cheap. Image
Viruses evolved from viroids. They differ in that viruses evolved protein coats to protect the RNA from nucleases. Life has evolved many tools to target and destroy naked viroids and one of them are RNases. These digest RNA into smaller nonfunctional pieces. Image
RNases are cheap and as ubiquitous as chalk in a classroom in the genomics field. They are easily used and deactivated with RNase blockers. Image
A person skilled in the art would be tempted to RNase treat the nasal sample prior to phage lysis to resolve the non-infectious RNA from the infectious RNA. qPCR could be performed before and after this treatment to measure total RNA and RNA that is nuclease resistant.
Figure 13 in our manuscript makes an attempt at this. It needs more validation with a Jaafar like calibration but derisks much of this approach.… Image
With the proper internal controls this assay would provide a ratio of Live-Dead viral RNA and provide more clarity on who to retest 24 hours later. Testing 2 days apart can inform you if the total load is going up or going down and rule out FPs and FNs on the first test.
2X the testing but significantly reduce # of people trapped in the long tail and vacuuming in all of their contacts map into a testing frenzy. Net:Net, this would probably mean less testing and I suspect that is why this simple and obvious Live-Dead trick is being ignored.
Here is a paper from Quest Diagnostics looking at testing multiple time points.…
Now that I have your attention regarding Costs... This video from @ThomasEWoods is a must watch.

Some estimates on the cost of track and trace to prevent a single death are staggering.…
This brings up an important economic point. When we print excessive money we end up with Cantillon effects.…
You see inflation in the price of goods through the artery in which the money is being injected. Don’t believe me.. qPCR test are usually under $10. CLIA regs might inflate that cost to $50. Money Printer go BRRRR ...The rapid COVID tests in Mass are $140. Image
I hope this thread encourages you to ask the testing labs for their FP/FN rates. Push for public transparency of the CQ values to be reported with the Case data. Without this, the FP rates will ensure the pandemic never ends. At home private testing would change this as well.
A final paper from the Broad Institute on CQ values over time and how they creep out towards the LOD.… Image
A recap based on some tweets I'm seeing fly over this.
I am a strong believer in qPCR for tracking pandemics but its limitations need to be known and better communicated to the public. At the moment it feels like a parlor trick to seize people rights.
Some have used my arguments to claim we should go back to 50 year old plating techniques. There are even more problems with cell culture. The biggest issue is that it lacks specificity. When you see a plaque you have to assume its from SARs.. but what about other viruses?
Not all viruses or microbes can be cultured. Many publications claim 95% of microbes cant be cultured. This leaves PCR as your only option.
Many pathogens are endophytes. They live inside the organism and the only way to get them out of the organism is to lyse open all of the cells and now nothing is viable and plating is again out the window. This is main problem in Cannabis testing. Aspergillus is an endophyte.
Culture can also take 5-7 days. By the time you get the result, the patient could be dead. In cannabis space, getting an answer a week later begs the question of what has grown on the cannabis during the wait period? The time domain is an exponent in pandemics and you cant wait.
In summary, qPCR is invaluable but...
1)Needs inclusion and exclusion testing to demonstrate your test doesn't cross react off target.
2)Public LODs and no calling after your LOD
3)Public FP and FNs
4)Attention to Live-Dead and endophytes
5)Public awareness of the sampling bias.
Notice when the CDC attempts to culture the virus they perform this on Vero cells which are monkey cells that are logs scales prone to higher PFU counts than many human cell lines tested by the CDC.

This implies the Jaafar curves are conservative.…
Another source for links related to culturing the virus and correlation with CQ.…
Raoults lab automates the Vero cell culture process. More data points on Cycle thresholds and viability.

Recall Vero cells are log scales more infective than human cells according to the CDC.… Image

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More from @Kevin_McKernan

21 Jan
Lets discuss the WHO release...
This is the anniversary of the WHO populating their web site with the Drosten test that caused many of these problems.

Document penned on Jan 13th but released during Biden's inauguration on the Jan 20th.
@Bobby_Network Image
This is a deflection to the labs to understand their LODs.
This is good advice but not very specific advice.
Keep in mind labs running 40 cycles are not necessarily calling at 40 cycles and this is a reminder for labs to scrutinize their IFU/EUAs for the correct LOD. Image
Protocol that ask to collect out to 45 cycles should be scrutinized as its a sign that something is off in their assay if it requires more cycles to capture Cts out at 40.

Here they speak about Viral Load. Lets dissect this term as its being mis-used. Image
Read 19 tweets
14 Jan
1\But but... you ran it to 40-45 cycles?
This is an argument that needs dissection.

The protocols you see in EUAs are speaking to the Camera time not the Call time. To call at 37Ct... you need to see the slope of the curve out to 40. It doesn't mean they are calling at 40.
2/Summertime positivity rates cannot be used as a proxy for wintertime false positive rates.

The virome in the winter is rich in HCoVs not present in the summer. I would assume the background microbiome is as well.
But we tested the Primers against HCoV in our exclusion analysis!

Sure.... but Just 4.

We just witness the S-gene amplicon drop out in the second season of C19 and we should expect HCoVs to have similar diversity.
Read 15 tweets
13 Jan is getting a good face-lift.
Samples now have variant tables with annotation on their impact to the gene.
There is also an IGV integration. You need IGV installed locally but the Start|Jump links will open up you BAM file in IGV.
You must have IGV open and then click the start link.
The sample's BAM will show up for viewing in IGV.
Once open, you can use the jump link to move swiftly around to each variant. This is a variant in THCAS.
You can jump to other variants in the same gene. In this case we have one non-synonymous variant in phase with a synonymous variant in THCAS.
Read 9 tweets
11 Jan
We have written another 60 page dissection of the Drosten PCR protocol.

This should end all of the criticism of the initial retraction request not having enough “wet-lab” proof.

20 peer reviewed papers showing catastrophic problems.

@Eurosurveillanc Image
Ironically, The origin of this quote is even debated but rightly describes the problem in society of Fake News.

It starts with Fake Science.

We will never end fake science if don’t address the lack of transparency in peer review. It must be decentralized and subject to audit. Image
The largest fatality in C19 was the public trust in science.

They have every right to be pissed.

For too long peer-review has remained ancient, ossified and secret.

It is not fit for our current world of rapid communication and transparency.

🦕 thrashing in tar pits Image
Read 10 tweets
11 Jan
I have finally read this in full.
Have not hit 255 references.

Anyone who tries to debunk this in a day... sorry. This would take many weeks to fact check

The PCR section is on point.
However, there is not enough attention paid to the significant false negatives also at play
While a lot has been written about false positives, the nasal swabs can vary 1,000-10,000 fold in their collection efficiency (Dahdouh et al) and many jurisdictions are using human gDNA internal controls to normalize for this large variance.
This variance implies many false negatives mixed in with many false positives.

More sensitive PCR tests won’t fix false negatives that are a result of poor swabs.

They aggravate the problem as track and trace becomes a completely futile boondoggle.
Read 4 tweets
11 Jan
We spend a lot of time calibrating our qPCR assays to replication competent organisms.

When we do this, we don't simply use a single culturing system as the results can vary significantly depending on the media used.

Here is DRBC(left) vs PDA (right) with Aspergillus niger. Image
You can see many more colonies on PDA.
This is likely due to DRBC having Chloramphenicol in it.

Chloramphenicol is an antibiotic with know inhibitory effects on Yeast and Molds.……
This begs the ? of which plating system to calibrate qPCR to. Also begs the ? of what a "gold standard" is.

95% of microbes cant be cultured, leaving DNA based methods as the only universal gold standard for absolute detection of wild samples.
See... Image
Read 4 tweets

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