Ct Values: What Do They Mean? Can They Be Used?

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Ct values:What do they mean?Can they be used?Mark W. Pandori PhD HCLD(ABB)Director, Nevada State Public Health LaboratoryAssociate Clinical Professor of Pathology and Laboratory MedicineUniversity of Nevada, Reno School of Medicine

PCR is a method ofamplifying a targetDNA molecule-For SARS-CoV-2, the target is thevirus’ genome; ---it is made of RNA;but it is an easy process to convertRNA into DNA-PCR takes place in cycles; eachcycle, temperature is changed fromcold to hot to warm and then back tocold.With each cycle, the amount of target (theoretically) doubles.This is amplification, and gives PCR its extreme sensitivity

PCR--With each cycle, if target is present, theamount of target is (essentially) doubledPositive specimen--amplified targets are measured byfluorescent light that they give off--”positive” and “negative” specimens aredifferentiated by whether the amount offluorescent light given off passes athreshold--The cycle where that amount offluorescence is reached is a “Ct” or “Cyclethreshold”.Negative specimen

PCR--lab tests therefore use Ct value as a measure ofwhether to call a specimen Positive or Negative--low Ct values are achieved when there is a largeamount of target present; high Ct values areachieved when there is a low amount of targetpresent--think of Ct as a measure of “effort” that the testhas to make to detect a positive specimen:if there is very little target (virus) in the sample,then you have to do a lot of cycles ofamplification to find it; and vice versaPositive specimenNegative specimen

PCR testing, the components:RNA (viral genome) extractionConvert all RNA toDNAAmplify byPCRPositive or NegativeResultbased on Ct value

Ct values correlate with a specimen’s ability toinfected cells in laboratory culture La Scola B, Le Bideau M, Andreani J, Hoang VT, Grimaldier C, Colson P, Gautret P, Raoult D. Viral RNA load as determined bycell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards. Eur J Clin MicrobiolInfect Dis. 2020 Jun;39(6):1059-1061. doi: 10.1007/s10096-020-03913-9. Epub 2020 Apr 27. PMID: 32342252; PMCID:PMC7185831. Jefferson T, Spencer EA, Brassey J, Heneghan C. Viral cultures for COVID-19 infectious potential assessment - a systematicreview. Clin Infect Dis. 2020 Dec 3:ciaa1764. doi: 10.1093/cid/ciaa1764. Epub ahead of print. PMID: 33270107. Bullard J, Dust K, Funk D, Strong JE, Alexander D, Garnett L, Boodman C, Bello A, Hedley A, Schiffman Z, Doan K, Bastien N,Li Y, Van Caeseele PG, Poliquin G. Predicting infectious SARS-CoV-2 from diagnostic samples. Clin Infect Dis. 2020 May22:ciaa638. doi: 10.1093/cid/ciaa638. Epub ahead of print. PMID: 32442256; PMCID: PMC7314198. Laferl H, Kelani H, Seitz T, Holzer B, Zimpernik I, Steinrigl A, Schmoll F, Wenisch C, Allerberger F. An approach to lifting selfisolation for health care workers with prolonged shedding of SARS-CoV-2 RNA. Infection. 2020 Oct 6:1–7. doi:10.1007/s15010-020-01530-4. Epub ahead of print. PMID: 33025521; PMCID: PMC7538033. CDC, unpublished data I’ve personally done this/seen this myself, here at the NV State Public Health Lab

What is “culture”?Another kind of lab test--infectious virus can be detected usingwhat are called “cell culture”techniques--Cell culture involves using cells derivedfrom humans or animal tissue that is /was cancerous--cancer cells live forever in culture--viruses can be detected / propagatedby adding them to cell culturesCells grow adherently to bottom of dish--”Vero” cells are commonly used forSARS-CoV-2 infection/propagationVero cells: African Green Monkey Kidney cancer: have been growing since 1962;

“high” Ct specimendon’t grow in culture--low amount of virus?Uninfected cells--”broken”, junk particles?-CDC showed no ability to infect cells in Veroculture after Ct value 33.00 (on their PCR assay)So --do such specimens present a public health threat?Infected--do PCR assays go too far?No commercial or CDC assay used inNevada with EUA uses cutoffs higherthan 40.

Ct values are not ready to beused diagnostically, or routinelySeven considerations in this regard, follow:

1. Different assays,different Ct--NSPHL Ct data between two assays--efficiencies of PCR vary--where would you draw the line?--e.g. what would you say about Ctvalues of, say, 32 or 34, if your cutoffwas 33?TaqPath ThermofisherTaqPath CDCSampleN 0% of specimens show 4-fold difference in load (i.e.greater than 2 Ct differences)

2. Extraction methodsaffect Ct values-swabs pulled out of noses and throats haveviral loads on them-to measure it, the viruses must be destroyedand their RNA molecules removed-the RNA is removed, and ‘washed’ for PCR tofollow-this is called “extraction”-there are many kits on the market for thisgenetargetQiagen Viral Mini KitMagMAX Viral NA Iso KitMag-Bind Viral NA Extraction 1832.1335.7Arrows indicate where different extraction methods led todifferent Ct values from the SAME specimens in the final PCRby at least a factor of 2 (est: a 4-fold change in measured viral load)

3. Is lab cell culture aproper surrogate for thereal infectious process?--cancer cells in a dish vs. primary humansystems: are they equal ?--evolution of SARS-CoV-2 occurs(ed) inreal tissue, not in cell cultures--very hard to do actual infectivityexperiments without volunteer humansubjectsWe don’t know yet. For other viruses (e.g. HIV), there are vast differences in infectivity

4. Collection andstorage variability cancause Ct variabilityKeep in mind:What is tested by PCR may not reflect whatwas in the nose at the time of collection:PCR / Ct generationAfter collection, specimens are put intomedia, stored for 1-3 days, at roomtemperature or cold packs, sometimes theyare transported long distancesRNA ExtractionSo: what was an infectious virus at time ofcollection , may not be after PCR testing hasoccurredLots of handling steps.1-3 days

5. Most positivespecimens detected arein an “infectious” Ctrange--pandemic was not caused by high Ct values--sampling 1,264 specimens from our CDC assay data*:mean Ct: 27.55SD: 6.11So: 84% of specimens tested have had a Ct value lessthan 33.66.According to CDC data, this means thatThe strong majority of specimens we haveascertained at NSPHL likely were infectious in cell culture*using N-gene detection

6. Note: viral loaddoesn’t tell you whetherinfection is new or old--high Ct, low load specimens can be“coming” or “going”-misclassification of a new infectionas an old infection could becatastrophic

6. Note: viral loaddoesn’t tell you whetherinfection is new or old--high Ct, low load specimens can be“coming” or “going”Low-load,high Ct-misclassification of a new infectionas an old infection could becatastrophicNo longerinfectiousBecominginfectious}Same Ct

7. Published Work showing Ct values 36 canharbor infectious virus Romero-Gómez MP, Gómez-Sebastian S, Cendejas-Bueno E, Montero-Vega MD, Mingorance J, GarcíaRodríguez J; SARS-CoV-2 Working Group. Ct value is not enough to discriminate patients harbouringinfective virus. J Infect. 2020 Nov 26:S0163-4453(20)30720-9. doi: 10.1016/j.jinf.2020.11.025. Epub ahead ofprint. PMID: 33248218; PMCID: PMC7688433. -they show that perhaps timing of specimen collection after symptoms can affect infectivity of specimen

What is going to happen ? Truth: there is a correlation with infectivity!Potential ways forward: Standardization of viral loads Antigen tests as “clearance” tests? Per-assay cutoffs? Whatever it is, the FDA will have a major say in how and when!

in an “infectious” Ct range--pandemic was not caused by high Ct values--sampling 1,264 specimens from our CDC assay data*: mean Ct: 27.55 SD: 6.11 So: 84% of specimens tested have had a Ct value less than 33.66. According to CDC data, this means that The strong majority of specimens we have ascertained at NSPHL likely were infectious in .

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