Dr. Jafar Rezeq
5:28PM Sep 16, 2020
polymerase chain reaction
But if I can, you know, as I will answer because I wasn't really sure what is this?
Yeah. So just so you know, I'm recording if you want something to be off the record, please tell me. But I'm happy to keep something off the record if you want. And something just so also you're aware. I also do a podcast for for the Oh, here he is. Hey, what's up?
Hey, as we were just about to get started. Okay, I'll mute myself. You're very capable and Dr. Resnick. Yeah. Are you
so just so you know, again, I, I'm recording and I do also have a podcast I don't always use every interview that I do for the podcast or the and I have a newsletter as well. But sometimes I do multitask. If you would rather not be on the podcast or if you would rather not be in a newsletter. Just let me know.
But Otherwise, I'm just going to assume that you're cool with it.
We get inquiries from readers often. And we don't check all of them. But we got one recently, where actually we got several recently about the the control thresholds in the state for the PCR test the RT PCR test. And and I was asked by my editor to look into it. What exactly does that mean? And there have been people who have said, there have been some I gather, there's some political implications behind this, that that that it's a question of, well, let me find the actual point. It was that if the if the control threshold is greater than 30 cycles, it means it's measuring the dead virus and therefore, false positives. And so I it's Sunday thing that that we haven't really reported on, I was asked to get a little education on the subject to see if it's something that is worth including in any of our coverage.
So, okay, now I get it because I wasn't sure what's the control cycle you're talking about. It's really not a controlled cycle. It's an uncontrolled threshold. It's a cycle a threshold CT value if that's what you are referring to. So, so, in a basic terms, PCR, which is the polymerase chain reaction is basically in a fundamental terms is is an amplification testing methodology, that for argument's sake, if, if in theory, you have one single viral particle in the patient's specimen, That single viral particle in this polymerase chain reaction technique is amplified into 10s of thousands and hundreds of thousands of particles for it to be detected by the polymerase chain reaction methodology. The way this technique was discovered years ago, and the individual who, you know, got it, won the Nobel Prize in Medicine years ago, basically uses temperature variations, cooling and heating, to generate cycles. And that's where this terminology cycle threshold. So in each cycle, basically you are you're, you're, you're amplifying the viral particle, you know, once so, you start with one and then they'd be too and the two particles each one will become too each and and and it just it just goes into that amplification. So, the most of the PCR detection methods that have been generated for the Coronavirus that we're talking about. Obviously, they have to receive the FDA clearance an authorization for that. And they provide their package insert to the FDA which means they determine the accuracy, the precision of the essay, and they determine the limit of detection, meaning what is the limit that these vital particles needed to be in a patient specimen for my essay To be able to detect it. No essay that I'm aware of is perfect. Some essays say okay, my limit of detection is 1000 particles there, one ml of patient specimen. This means that if a patient specimens has 1000 particles or more, in theory, that testing platform should be able to detect the viral particles. If the patient contains less than 1000 particles or less per ml, that doesn't mean that patients specimen is negative. That means that my testing platform is not sensitive enough to detect, for example, five or 10 or whatever in that patient specimen. So in order for these platforms to be able to go down that sensitivity level, obviously PCR plays an important role in that and They take a tiny particles, small number of particles and they amplifying to a larger number. And they do that through cycles. The cycles they have established and this is this is a science that needs to be verified probably with more robust data, but we our understanding is that the when they when they when the technology do does the cycling, the reason for the cycling to do more cycles is to be able to have enough viral particles to be detected by the one of the techniques is a fluorescence technology to have enough fluorescence lighting that will be able to be detected. when when when enough viral particles are being amplified.
Now, if the in theory, if you have too many particles already present in the patient specimen, the platform doesn't need to do too many cycles to be able to detect the presence of these viral particles 15 2025 cycles are enough to generate signals first and signals for that platform to capture the positive the signals and say this is a positive sample. However, if you are unable to collect enough lorrison signals by doing 20 or 25 or 30 cycles, it will continue up to 40 cycles and if you detect Any positivity in terms of any signatures that you capture for up to 40 cycles, they will tell you that in our platform that we have submitted approval from the FDA up to 40 cycles has been approved to detect enough cinamon fluorescent signal from this patient specimen to call it positive for coronavirus. So the theory is if we detect the viral particles at lower cytokines, that means the PCR did not, did not have to work hard to find these viral particles to be able to call it positive. It's It's It's available, plentiful. However, if the platform needs to go and do more cycles, that's a reflection of a viral load. However, it doesn't really matter at the end of the day, Whether it's positive because of a viral load or low viral load, there is some implications for contagiousness for infection control. But a positive result is a positive finding, and it has to be acted upon accordingly. Now, some platforms said that in our in our validation studies, we consider anything after the cycle 37 in our findings to be negative, meaning that, you know, we keep doing the cycles up until cycle number 37. Above that, you know, for us, we determined that the specimen should be declared negative. And as such, that's where they got the FDA authorization on or approval on other assays including the CDC, for example. It's, they say 40 cycles for us is because To be the cutoff cycle, above that, it's negative below that, we declare the specimen negative. Now, the the difference between polymerase chain reaction and the actual culture is that the polymerase chain reaction will pick that or live organisms. Culture on the other hand, if I have if I have to culture bacteria or viruses in the proper culturing modes, whatever grows is alive particles. If I have dead particles in the patient's specimen, they will not be able to grow. If I am doing a PCR the PCR is not looking for a live agent particles so dead particles will still be amplified will be detected as positive material in the patient's specimen. So that's a fundamental difference between a PCR testing methodology and a culture based methodology. Obviously, for viruses, because of the difficulty in doing culture, the vast majority of labs around the nation are dependent on PCR, some some major reference labs, including the CDC, would still use culturing for for the purposes of, you know, other other reasons that that needed catch up. methodologist.
Well, you did a great, you did a great job. Thank you very much and i i but so First of all, whose set it does the acceptable number of cycles change state to state or lab to lab? Or is it based on whoever develops to test? Like, if you use the Abbott Labs test? Do they decide? Oh, well, it's only it's only valid. You can only call it a positive test if you if it's 35 cycles, 37 cycles or 40 cycles, whatever they decide, or is it Connecticut? Is that your department? Is it your office that says, we are we're making the determination that it's 35 cycles as opposed to 40? Thank you, Jordan. This is a good question.
And it is really, it is not the state. It is not the lab who decides it's really the manufacturer of the essay. So let's say I am avid I am Thermo Fisher I am whatever. I am the researchers, the group, the developer of the essay based on the criteria of the chemistry of that essay, the decision about the number of cycles is being decided. And that information is submitted to the FDA for approval. And we in the lab, it depends on if it's a manual or automated, if it's a manual essay, then we have to abide by whatever the manufacturer's recommendation is. If it's an automated essay, then the manufacturer equipment will decide the 40 cycles or 37 cycles or whatever, and it will spit out the result based on that. So really, the This doesn't vary from state to state or it doesn't. It's really manufacturer dependent chemistry driven decision.
So and if you're if the PCR if the polymerase chain reaction is is not distinguishing between dead virus and live virus, I could see that that's positive and negative, right? Maybe Maybe, because on the one hand, if you find dead virus, right, it means that that person has been infected and therefore, that's a positive test. But it also doesn't necessarily mean that they're transmitting that virus anyway. So it could be considered a false positive. So it are both true is neither true. So,
so a false positive. If you ask a scientist or microbiologist, they will give you a different interpretation of all false positive or false negative from from what maybe the general would understand the false positive so I would I would say that the the the this advancement in the infectious disease diagnostics using the molecular biology essays like the PCR, really revolutionize the way we treat infectious diseases. If I tell you that using the polymerase chain reaction, you will be able to get results back to the healthcare provider within I would say, you know, minutes hours of specimen arrival in the laboratory culture. On the other hand, it may take days or weeks before you come back, a physician or healthcare provider and tell them by the way your specimen your patient specimen has such and such. So, so that's one thing. The other thing about detecting Dead or Alive particles. Remember, you know, patients are treated in totality, meaning a physician or healthcare provider, when they look at a patient, they do not only look at the patient, they ever work with the lab results, they look at the patient, current symptoms current status, they evaluate the overall totality of that patient. So, if a test result comes back on a patient that recovered from the infection, for example, and he or she is no longer symptomatic, and they passed the period of the incubation period or the the the infectious period, and a PCR test comes and say positive, that positive result is not a false positive from a microbiology sense, because the virus particles are there in the patient, sputum or respiratory tract or whatever. It's these viral particles are mostly likely to be dead particles with. So that means that case that that patient is no longer contagious, because that viral particles will not transmit. And this individual is not really it should not be worried about having these positive finding, you know, in his or her patient specimens. So, that's why, you know, the use of the PCR has its own algorithm, because a positive PCR can be positive for several weeks after an individual had recovered from the infection.
But, but then again, I mean, again, and doesn't that has implications when you're trying to gauge whether or not the disease is continuing to spread. I mean, you know, if you're, if you're unable to tell if, as you said, a person with dead virus, you know, in His body is not contagious anymore. If you can't distinguish between live and not live virus, doesn't that limit your understanding of whether or not the disease is spreading or even how the disease is it even how the disease is spreading from patient to patient was
it is but you know, it is it is it is a limitation of molecular techniques. But again, it has to be interpreted in the context of the patient presentation. And this is not really new to core Coronavirus. We have been doing molecular diagnostics for infectious diseases for you know, 2030 years or so, and physicians and health care providers are well trained on how to handle these molecular test results. You know, because of the of the series advantages that these molecular assays provide for patient management and for infection control practices. So, really, there is no good answer to that, other than to say that these test results. And we do, you know, when we report these positive findings, every lab have their disclaimers on these, you know, a negative test result does not preclude other infectious agents and a positive result. You know, we have all these disclaimers reported out, but the physician or a healthcare provider needs to look at these results. And, you know, put them in the proper context of the patient. So, you know, you know, in the absence of
you know, they needed to, if you have to look at it, I guess you have to look at it also in context of other cases. If you're getting into you know, the photos metric that the governor focuses on is percentage of tests that are positive. So if you're getting a higher percentage of tests, that is an indication of the spread of the disease, not necessarily one test being positive versus another test being positive.
So yeah, context is everything as usual.
When my kids get strep throat,
um, you know, there's a fast test, you know, you take the swab and, and it's like, and then it's like, it's like, 80% accurate or something like that, versus the one where they actually have to send it to the lab. And this has happened a few times in our family, is we is and I always and now I ask always, can you send it to the lab to, you know, just to verify because that's like 99%? You know, cuz it's happened a couple of times. So, is that protocol when you're testing for coronavirus? Using the PCR Test. if, if, if the culture tests can detect it can be more delicate in in more specific in what they in the information that they get, but they're significantly slower. Do you send a proportion of the PCR tests of the slobs that you that you take to be tested in human culture technique as well, in order to get that kind of verification?
Yep, No, we don't. And so that's another good question. The FDA when the company or the manufacturer or the test developer submits their test for approval from the authorization from the FDA, they define how the positive and negative results are built to be interpreted. So they say, a positive result based on their studies. They say our positive result result is, is to be considered definitely Meaning there is no reason there's no need to confirm a positive finding. It's actionable you can act on our positive result. However, some platforms, they tell you, our negative results are to be taken as a presumptive. And if it clinically indicated, we recommend that you confirm with a PCR. Now, the reason why the PCR is is not at this point, confirmed by a culture for multiple reasons. Number one, the virus the CDC does not recommend any lab to do concerning for this because of its
there is that is because it's
the biosafety level that you need to work with this virus is a higher biosafety level and there is a concern and you know, laboratory acquired infections male care, so there is there is a safety risk for laboratory staff. So CDC At this point is not recommending any lab to do culturing for this virus. And, and the because of the sensitivity outcome of these PCR assays, really the, the the issue of Dead or Alive particles, you know, I'm not really and if you're if you ask, you know infectious disease experts and scientists, it's not really of a major concern because again, you know, this test has to be interpreted in the overall totality in the context of the patient. And so, for example, if I have just theory, I have a nursing home staff who tested positive and he or she is symptomatic, and he or she came forward and say, Well, you know what, I was in contact with X, Y, and Z individuals. And all of the x, y and z are asymptomatic now. And if x y and Z are tested with the PCR and X, Y and Z are positive. I'm going to act on this to consider it as a live viral particle.
And I am I'm
you know, I?
should be we should be. I mean, so just yeah, I have to run to but I do I have one more question and I'm so sorry. And I really appreciate your help. I, um, but if you because it just seems to me that if you if, if, if you test me and I'm positive, but I made symptomatic, right, I'm asymptomatic and I get a test because I'm going to college or something like that. And and I and the test comes back positive using the PCR, the test. I then will get quarantined and perhaps might have to actually leave school or, you know, even though I even though I'm unsung Nothing dramatic, but in the virus in the virus and and I may not be contagious anymore, because or maybe never was because, you know, like, because I've I've been, you know, I the only virus is is dead virus. So I mean doesn't that wouldn't that mean? I mean like Wouldn't it be helpful to know if I'm, if I'm consistent if I'm contagious isn't an indication of whether or not the virus and I keep going back to that idea that it's an indication of the contagiousness of any individual patient.
Yeah. Yeah. Obviously, in Taiwan, in theory, in theory, maybe that's a valid question. But if you think about, by the time I come back to you and tell you, oh, by the way, two weeks ago, I collected a specimen from you and that day showed that you had a live virus right? So, so really, you're talking about practicalities and what's the person? What is the possibility that you have? So if you come to me, you say, Okay, I, I had the symptoms, and I was tested two weeks ago and I was positive for the virus. And then three weeks later or two weeks later, I'm tested and my PCR is positive. I will tell you, most likely, what you have now is a dead viral particles. But I can say that on the test that has been done to you when you were symptomatic, or when during the time you develop the disease.
Right. Got it. Thank you.
The new technology that can work just as quickly maybe it's a valid question, but until then,
well, there is some I know that others are working on using the this molecular technology essays to be able to distinguish between Dead or Alive particles. So I think I think the work is being entertained. This is before Coronavirus. I mean people wanted to know whether we are dealing with the Dead or Alive infectious disease agents for other reasons, but we are not there yet.
Hmm. Thank you so much, sir. I really appreciate the education.
I'd like to thank you for reaching out.
Thank you Have a great day. Okay. Joe.