I tested positive for SARS-CoV-2

hi everybody i'm vincent raquiniello and recently i tested positive for sars cov2 i wanted to explain what that means how the test is done and why i'm not worried as many of you know i'm a professor at columbia university and with the back to work that started a few months ago the university instituted a testing plan i'm at the medical center an area was set up here where people could be sampled and you can go in and have a test done or you can be randomly selected by the university to get an idea of who might be infected on october 22nd i was selected for random testing i went in that day and had the test done and what it involves is going into this area you have an appointment they scan a barcode on your phone they print out a label they put it on a tube and you go and you sit down at some carol elf desks they're all separate about a dozen desks separated from each other and you take a q-tip out of a sterile packet you swab each nostril three times right at the front take the q-tip you put it back in the tube you give it to the lady at front she puts it in a box and then every day they're shipped up to the broad institute in boston and they do the polymerase chain reaction or pcr so this was on a thursday morning friday night i got a call saying that i was positive and that my ct value was 34.7 so let's take a look at what all of this means so let's take a look at where this virus reproduces in us so here's a diagram of the upper and lower respiratory tract and the virus typically enters your nose it enters these cells that line your respiratory tract the respiratory epithelial cells which are covered with mucus the virus gets into these cells it reproduces in them and then the cells produce tons and tons of virus which spread throughout the tract in some individuals the virus spreads down all the way down to the alveoli in the lungs and of course there it can cause serious disease covid19 so i sampled the very anterior part of my nostrils here and so the virus is clearly reproducing in cells there it also shows you why you can transmit this virus simply by touching your nose contaminating your fingers with the mucus that you always have on these mucosal epithelial cells and if it has virus in it you could transf transfer it to others the early tests for saurus cov2 infection involved nasopharyngeal swabs so you put the swab in the nose and you go all the way up into the nasopharynx the top portion and you rub it there and that's very invasive of course so this little anterior nary sampling is is is welcome because it doesn't hurt at all so when we sample the nose for sores cov2 with the q-tip as i just told you what you're sampling is a mixture of virus particles and dead and dying cells chronovirus particle of course contains an rna genome so you could potentially have some virus particles on the swab and the rna genome is a very long rna as shown at the top here but you also have most likely cells dead and dying cells is that are present in the swab sample and the dead and dying cells can also contain viral rna as well as viral messenger rnas so when you do a polymerase chain reaction you're going to be detecting both viral rna and viral messenger rnas now the pcr primers that are used in the particular test being used here at columbia and which is being done by the broad institute are detecting the n gene right here so it's all the way at the right end of the viral genome and the nmrna down here as well it's only a single primer set some assays use two primer sets for one gene typically the engine maybe the spike gene or some other gene as well but the key here is that we're extracting rna from my q-tip swab and the next step is to do polymerase chain reaction which will only work on dna so the viral rna on the swab has to be extracted and then converted to dna so here is the schematic of the polymerase chain reaction we start with cyrus cov2rna which is on my q-tip it's extracted the rna is extracted then it's converted to dna by an enzyme called reverse transcriptase and then the polymerase chain reaction or pcr can proceed and this is a diagram of what's happening in the pcr so we can understand what ct is cycle threshold so here is the dna that was made from sars cov2rna we put it in a tube with the four nucleotides a t c and g and we have the two dna primers which i told you in this case are specific for the end gene of the virus and the first step is to separate the two strands of dna by heating the sample now we have the two single strands we then bring down the temperature so the oligonucleotides the dna primers will anneal to the dna and then there's a dna polymerase present in the mixture which will then make a dna copy of the dna that we've put in using those primers to start so now we have two double-stranded dna copies where we started with one so we go from one to two and that is one cycle one cycle of the polymerase chain reaction and then it's repeated the whole thing is repeated you have these two dnas that you've made they're denatured the primers anneal and then you make more dna so after the first cycle the second cycle here you have four copies of the original target and for each cycle you double the number of dnas that you have so you can see eventually if you go 40 cycles you can detect a very small amount of dna to start with so the cycle threshold or ct tells you how many cycles you need to get a signal now a signal is typically read on a machine there are a number of ways you can do that one is to use a dye called cyber green which will insert into only double-stranded dna and then it fluoresces so the cyber green itself is not fluorescent but when it inserts into double-stranded dna which is a product of the pcr of course it will fluoresce green and that can be measured by a machine so at every cycle at every one of these cycles you could measure the amount of fluorescence and so you could be incubating a sample cycle one two three et cetera and then cycle 12 bingo you get a reading of the fluorescence that's your ct your ct would be 12. so my ct was 34.7 what could it mean to have a ct of 34.7 well first of all it's not very much viral rna we know already that a number of studies have shown that when you get into the high 30s there's not very much rna and as i'll show you in a moment there's very little infectivity on the other hand a very low or a lower ct means there's more rna there and you're more likely to be infectious so i had a ct at the higher end of the scale and typically these reactions are done for 40 cycles i could have been at the very beginning of an infection i might have recently been infected and it was just starting i could have been at the very end of a asymptomatic infection so the amount of rna is low in the beginning it goes up and then it comes down so here's a slide that daniel showed on twiv 677 showing the different phases of saris cov2 infection te is the time of exposure so that's when you acquire the virus and the purple line is synthesis the level of virus or viral rna and there's an incubation period and then there is a period where the viral rna starts to go up and then there's ts is the time at which you have symptoms so you can see the viral rna peaks at or around the time of symptom onset and then the viral titus decrease in in the next week or so in terms of ct values you would have very high ct values here in the beginning then the ct values would drop and then the ct values would go up again so my ct of 34.5 could have been either at the very beginning of an infection as i said at the end of a asymptomatic infection so that's two of the possibilities to explain it and the third possibility is that it was a false positive of course and that something went wrong and i really did not contain any sars cov2rna what how what would cause a false positive well an error of some kind if samples were mixed or if samples were contaminated or if i happen to contain some nucleic acids that to which the primer is hybridized and they amplified uh some of my mrnas not viral rna all of those things could explain if a false positive now here is a experiment that was published recently here's the link for it at the bottom here which is a nice way to correlate ct with infectivity so what was done here is over 3000 individuals were studied and they were given pcr tests similar to the one i've just described and then the the samples from their noses were placed on cells in culture in the laboratory to see if they contain any infectious virus and that's what's shown on the graph it's a little complicated but let me walk you through it so on the x-axis we have the ct value going from 11 to 37 really high ct value so not a lot of rna and 11 a low ct value quite a bit of rna so different ct values that they found in their patients the number of samples for each ct that were studied you can see in the middle they had a lot of samples and at the ends fewer and then the number of positive positive means infectious when they put these nasal washes or nasal swabs on cell cultures they could grow virus out of them 36 and 37 ct values they couldn't recover any infectious virus and cells so less rna less viruses recovered and then these lines indicate um the number of culture positives as well you can see 100 percent culture positive here at the low ct value and then it's going down and down as the ct values go up the ability to recover virus goes down to until it's zero at 36 and 37 so this is a study with thousands of patients again correlating ct value with infectivity and again the key is you can easily recover infectious virus from ct values from 11 up through 35-ish but then 36 and 37 there is no infectivity recovered but this tells you why i wasn't worried at ct of 34.5 all right so what happened next so i got this result on a friday night and so we rescheduled a new test i went in the following tuesday did the same procedure anterior nary sampling the sample was sent to the road the pcr was done in exactly the same way and that one came back negative so the moral of the story is first of all i had a false positive i wasn't at the beginning of an infection because if i were the second pcr would have shown an increase in in other words reflected by a lower ct value going 34.7 to something lower on the other hand it was not not likely that i had an asymptomatic infection because pcr result would not have turned negative in such a short period of time so the conclusion is that this was a false positive test random testing of people only works if it's done often one pcr test on its own is not informative especially in cases like mine when the ct value is quite high i hope that's useful for you to understand ct value pcr and the vagaries of testing in the ideal world everyone would be tested frequently but of course pcr is too expensive to do that and antigen tests the michael mina approach is the way to do that and i'll put a link to michael mina's twiv where you can hear him talk about that i'm vincent racquiniello i'm earth's virology professor and i will see you on the next video you

Channel: Vincent Racaniello

Views: 116,439

Rating: 4.8710551 out of 5

Keywords: virus, viruses, viral, virology, pandemic, COVID-19, coronavirus, SARS-CoV-2, PCR, cycle threshold, Ct, diagnostic test

Id: Lk64Zwcj3W8

Length: 14min 22sec (862 seconds)

Published: Thu Nov 05 2020

Reddit Comments

[u/Viewfromthe31stfloor]

Professor Raciniello does a thorough explanation of testing and why he’s not concerned.

His CT was 34.7. He explains that this means very little RNA and lower infectivity.