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Response to Tomconroy (Reply #65)

Wed Aug 25, 2021, 06:31 PM

67. A "Ct" is an abbreviation for "Cycle threshold"

And again, as I noted earlier, the Wisconsin report was not designated "a study". It was their reported data that had background info associated with it, and analysis of what they found with their outbreak.

The "Ct" reference value is used in PCR (polymerase chain reaction) tests that amplify the amount of collected sample through a number of "cycles" to get enough of it to be detected by a fluorometric detector. A bunch of years ago I ran these as part of a couple training courses that I was one of the instructors for. I am sure the instruments today are much more sophisticated than those back then but the concept is the same. I believe the sample gets a marker substance that binds to the molecules that would cause the fluorescence that would eventually get detected.

Basically the more cycles needed to get enough of it to detect, the less there is of the targeted material in that sample. Alternately, the lower the number of cycles needed to detect the marker, the more there is in that sample. Variations will inevitably occur with samples based on the collection techniques and how much was actually collected using the swab, as well as the transfer techniques to preserve that sample for eventual marking, and analysis. I.e., sometimes not enough is collected or something happened between collection and transfer or sample prep that results in getting an "inconclusive" result.

University of Wisconsin describes it here (PDF file) - https://www.wvdl.wisc.edu/wp-content/uploads/2013/01/WVDL.Info_.PCR_Ct_Values1.pdf

In general, across the hundreds of different manufacturers of test kits/schemes, the number of cycles for amplification is generally cut off at ~35.

I posted about the guidance regarding these tests here - https://www.democraticunderground.com/?com=view_post&forum=1014&pid=2778994 and will re-post below -


FDA publishes a reference standard for the current EUA-approved tests - https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/sars-cov-2-reference-panel-comparative-data

which indicates the minimum detected viral load (particles per ml) for each of the approved test systems. A good summary of that is here -

Op-Ed: Why PCR Cycle Threshold Is Useful in Coronavirus Testing
It can help better triage patients, physician argues

by Robert Hagen, MD January 4, 2021


The FDA has given lab manufacturers a wide latitude in determining the cycle threshold cut-off number of their qualitative tests to determine positive versus negative. These tests were approved under Emergency Use Authorization and have not been subjected to typical FDA scrutiny. With this in mind, the state of Florida has required all laboratories doing COVID testing to report the cycle threshold numbers used in qualitative and quantitative tests.

So how does a qualitative RT-PCR test work? Basically, the manufacturer sets the test to turn off the cycling or amplification process when a certain number is hit. For a qualitative test set at 40, after 40 amplification cycles, if any viral material is detected, it turns off and is reported as positive. If none is detected, it would be reported as negative. If the number of amplification cycles was really 15 or 25, it would still run until it gets to 40 and be reported as positive. With these type of tests, it's critical to use an agreed-upon cycle threshold value such as 33 (CDC) or 35 (Dr. Fauci) rather than setting it at a potentially misleading 40 or 45.

Many of the current tests in use are preset by the manufacturer to these higher numbers.The World Health Organization issued a notice last week telling the labs "the cut-off should be manually adjusted to ensure that specimens with high Ct values are not incorrectly assigned SARS-CoV-2 detected due to background noise." Could this be a reason why many people test positive but remain asymptomatic? In that same memo, WHO said all labs should report the cycle threshold value to treating physicians.

A quantitative test is designed to come up with the actual cycle threshold value as the cycling process turns off when detecting any virus. There is not a preset value, so a quantitative measure is obtained. A test that registers a positive result after 12 rounds of amplification for a Ct value of 12 starts out with 10 million times as much viral genetic material as a sample with a Ct value of 35. Above that level, Fauci has said the test is just finding destroyed nucleotides, not virus capable of replicating.


(emphasis mine)

The number of cycles (Ct) to reach a detectable amount with forced replication (amplification), tends to vary (due to sampling type and consistency, and obviously due to sample prep required for the various test systems and their actual detector thresholds). But from a bunch of research things that I looked at, some kind of "positive" result will be detectable within a range from ~13 - 35 cycles.

Supposedly many of the test systems are set to run for up to 40 cycles to catch the most minimal of particles, but generally going that far didn't seem necessary. For example, a simple description of that was this (from October 2020 regarding viral shed) -

The correlation of SAR-CoV-2 viral loads and PCR cycle thresholds (Ct) values with isolation of viable virus is a topic of interest. The Ct value upper bound cutoff that determined a positive PCR was inconsistent among studies reporting this threshold, though most reported positive values at ≤35 or ≤40. (49-52,54,72,77) Bullard et al (5) compared PCR Ct value with culture positivity and found that the ability to isolate virus in culture was reduced when Ct value was ≥24. They reported that the odds ratio for infectivity decreased by 32% for every 1 point increase in the Ct value. (5) La Scola et al (8) report significant correlation between Ct value and culture positivity rates. Positive cultures occurred in all samples with Ct values 1317 but culture positivity decreased to 12% at a Ct value of 33. (8) Isolating virus in culture with positive PCR samples containing viral loads

(emphasis mine)

So looking at the 5 pages of "approved" tests, the most sensitive vs the least sensitive -

NDU/ml (NDU/mL = NAAT Detectable Units/mL)

180 | PerkinElmer, Inc. | PerkinElmer New Coronavirus Nucleic Acid Detection Kit
600000 | Boston Medical Center | BMC-CReM COVID-19 Test

So the above represents the "lowest detectable" by those systems (as the most sensitive vs the least sensitive for the approved list) but either would be considered a "low" viral load. And within the ranges of each of these tests, there would be a "high" load value that is detected with the minimum amount of amplification cycles, and based on a number of papers, those with "high" loads happen fairly soon in the number of cycle runs, but more often than not, after about 12/13 cycles. And apparently once they hit a "positive", they cease any further runs.

=======================END RE-POST===========================

And with respect to the UK study that you cite, this summarizes what they found - https://www.nature.com/articles/d41586-021-02187-1

One massive analysis of Delta transmission comes from the UK REACT-1 programme, led by a team at Imperial College London, which tests more than 100,000 UK volunteers every few weeks. The team ran Ct analyses for samples received in May, June and July, when Delta was rapidly replacing other variants to become the dominant driver of COVID-19 in the country. The results suggested that among people testing positive, those who had been vaccinated had a lower viral load on average than did unvaccinated people. Paul Elliott, an epidemiologist at Imperial, says that these results differ from other Ct studies because this study sampled the population at random and included people who tested positive without showing symptoms.

But that is not unexpected and it indicates that the vaccine "is working".

Here is a link to that UK study's summary and what it said in the abstract - https://spiral.imperial.ac.uk/handle/10044/1/90800

Background The prevalence of SARS-CoV-2 infection continues to drive rates of illness and hospitalisations despite high levels of vaccination, with the proportion of cases caused by the Delta lineage increasing in many populations. As vaccination programs roll out globally and social distancing is relaxed, future SARS-CoV-2 trends are uncertain. Methods We analysed prevalence trends and their drivers using reverse transcription-polymerase chain reaction (RT-PCR) swab-positivity data from round 12 (between 20 May and 7 June 2021) and round 13 (between 24 June and 12 July 2021) of the REal-time Assessment of Community Transmission-1 (REACT-1) study, with swabs sent to non-overlapping random samples of the population ages 5 years and over in England. Results We observed sustained exponential growth with an average doubling time in round 13 of 25 days (lower Credible Interval of 15 days) and an increase in average prevalence from 0.15% (0.12%, 0.18%) in round 12 to 0.63% (0.57%, 0.18%) in round 13. The rapid growth across and within rounds appears to have been driven by complete replacement of Alpha variant by Delta, and by the high prevalence in younger less-vaccinated age groups, with a nine-fold increase between rounds 12 and 13 among those aged 13 to 17 years. Prevalence among those who reported being unvaccinated was three-fold higher than those who reported being fully vaccinated. However, in round 13, 44% of infections occurred in fully vaccinated individuals, reflecting imperfect vaccine effectiveness against infection despite high overall levels of vaccination. Using self-reported vaccination status, we estimated adjusted vaccine effectiveness against infection in round 13 of 49% (22%, 67%) among participants aged 18 to 64 years, which rose to 58% (33%, 73%) when considering only strong positives (Cycle threshold [Ct] values < 27); also, we estimated adjusted vaccine effectiveness against symptomatic infection of 59% (23%, 78%), with any one of three common COVID-19 symptoms reported in the month prior to swabbing. Sex (round 13 only), ethnicity, household size and local levels of deprivation jointly contributed to the risk of higher prevalence of swab-positivity.

Discussion From end May to beginning July 2021 in England, where there has been a highly successful vaccination campaign with high vaccine uptake, infections were increasing exponentially driven by the Delta variant and high infection prevalence among younger, unvaccinated individuals despite double vaccination continuing to effectively reduce transmission. Although slower growth or declining prevalence may be observed during the summer in the northern hemisphere, increased mixing during the autumn in the presence of the Delta variant may lead to renewed growth, even at high levels of vaccination.

(the link went to something that was jammed into one big paragraph so I separated out based on where section headers were)

The above regarding the reduced effectiveness over time is also why you see the U.S. and other countries going with the boosters and again, it confirms what was found from the data from Wisconsin and Provincetown - i.e., "fully vaccinated people" were found to be able to contract the virus over time. And in the case of the UK, they were actually testing everyone - both symptomatic and asymptomatic, whereas in the U.S. they are not really tracking asymptomatic infections (unless someone believes they were exposed and gets tested and that result gets reported). So the number of "positives" in the U.S. is probably much higher, but people who have little or no symptoms are generally not getting tested (and have been dissuaded from getting tested).

And with that, some Thomas Dolby (came out my senior year in college and I still have the 45 in a crate somewhere ) -

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