Aa
HR, Jim, mremeet, Goofy Dad, anyone that has knowledge of pcr testing?
Hi everyone,
Some of you have been following the saga of my "29" at week 12 as well as APK's. I do think/hope my 29 at week 12 is an "artifact" or false positive but for my own peace of mine, I want to comfirm that by getting the most sensitive and accurate pcr test available. Asked my doctor about it yesterday and the study can't provide one more sensitive than the one we've all been discussing but he told me I was welcome to get one on my own.
There's a lot of confusion in my mind surrounding these tests. Searched the archives and found something Goofy Dad posted in Nov.06
http://www.natap.org/2005/HCV/122805_04.htm
Conclusions: Among patients treated with combination therapy for chronic hepatitis C, the TMA test detects HCV RNA in all specimens that are Amplicor-positive, as well as an additional 21.6% that are Amplicor-negative. The increased sensitivity of the TMA test can be helpful in identifying patients with low levels of HCV RNA who are likely to relapse when therapy is stopped. Furthermore, among patients with EVR and a negative Amplicor test at W20, persistent detection of HCV with the TMA test during therapy predicts failure to achieve SVR.
*FDA-approved for detection of HCV RNA as evidence of active infection.
Can someone explain to me like I'm five years old what the above means. Also, HR posted on 11/12/06 the following:
(Continued below)
Some of you have been following the saga of my "29" at week 12 as well as APK's. I do think/hope my 29 at week 12 is an "artifact" or false positive but for my own peace of mine, I want to comfirm that by getting the most sensitive and accurate pcr test available. Asked my doctor about it yesterday and the study can't provide one more sensitive than the one we've all been discussing but he told me I was welcome to get one on my own.
There's a lot of confusion in my mind surrounding these tests. Searched the archives and found something Goofy Dad posted in Nov.06
http://www.natap.org/2005/HCV/122805_04.htm
Conclusions: Among patients treated with combination therapy for chronic hepatitis C, the TMA test detects HCV RNA in all specimens that are Amplicor-positive, as well as an additional 21.6% that are Amplicor-negative. The increased sensitivity of the TMA test can be helpful in identifying patients with low levels of HCV RNA who are likely to relapse when therapy is stopped. Furthermore, among patients with EVR and a negative Amplicor test at W20, persistent detection of HCV with the TMA test during therapy predicts failure to achieve SVR.
*FDA-approved for detection of HCV RNA as evidence of active infection.
Can someone explain to me like I'm five years old what the above means. Also, HR posted on 11/12/06 the following:
(Continued below)
MEDICAL PROFESSIONAL
First ; TMA = Transcription Mediated Amplification, an in vitro amplification method of small stretches of RNA that uses RNA Transcription rounds for its amplification cycles.
PCR = Polymerase chain reaction. It uses up to 55 rounds ( cycles) of a molecular biology reaction that starts with a small specific stretch of DNA e.g. 100 bases long that are part of the viral genome and duplicates that starting material in each round again and again so that in the end a relatively gigantic amount of that specific stretch the " amplicon" is made, still "proportional" to the starting amount, so by quantitating the end product so can quantitate the starting amount = the virus by comparison with a standard that you have equally amplified.
With HCV , a RNA virus, the RNA is first reverse transcribed into DNA, then PCR is used.
A qualitative test is nothing more than a TMA or PCR where:
1. a much larger amount of serum (starting material to investigate) is being prepared and
2. then the amplification process is driven to the extreme, to detect any copy of a virus present in the starting material. This way you obtain highest sensitivity, but you loose quantitation above the detection limit, because you literally "overamplify". Often even a single copy of the virus in the starting material of 1ml of serum will be detected.
Everything has to be optimized to achieve this result. This is called " Robust single copy PCR".
Sometimes HCV is so intensely lipidated, that the ultracentrifugation step used to pellet it down from the starting serum, despite lowering specific gravity of the serum,cannot cath this virion - it floats, because it contains too much fat. Thats why the sensitivity limit is "officially" 2 iU = 5 copies(virions)/ml.
In quantitative PCR we simply use much less starting material and less and less cycles to obtain amplification strength that allows quantitation in the desired range.
PCR = Polymerase chain reaction. It uses up to 55 rounds ( cycles) of a molecular biology reaction that starts with a small specific stretch of DNA e.g. 100 bases long that are part of the viral genome and duplicates that starting material in each round again and again so that in the end a relatively gigantic amount of that specific stretch the " amplicon" is made, still "proportional" to the starting amount, so by quantitating the end product so can quantitate the starting amount = the virus by comparison with a standard that you have equally amplified.
With HCV , a RNA virus, the RNA is first reverse transcribed into DNA, then PCR is used.
A qualitative test is nothing more than a TMA or PCR where:
1. a much larger amount of serum (starting material to investigate) is being prepared and
2. then the amplification process is driven to the extreme, to detect any copy of a virus present in the starting material. This way you obtain highest sensitivity, but you loose quantitation above the detection limit, because you literally "overamplify". Often even a single copy of the virus in the starting material of 1ml of serum will be detected.
Everything has to be optimized to achieve this result. This is called " Robust single copy PCR".
Sometimes HCV is so intensely lipidated, that the ultracentrifugation step used to pellet it down from the starting serum, despite lowering specific gravity of the serum,cannot cath this virion - it floats, because it contains too much fat. Thats why the sensitivity limit is "officially" 2 iU = 5 copies(virions)/ml.
In quantitative PCR we simply use much less starting material and less and less cycles to obtain amplification strength that allows quantitation in the desired range.
Sorry, I probably should have opened another thread for my question but since the discussion was about PCR testing I squeaked in here.
MEDICAL PROFESSIONAL
"The docs says UND is UND and that's what the research proves out, but we can have whatever test we want as far as he's concerned a PCR is a PCR. My insurance company says the same thing".
So like <600 iu has about the same meaning as < 2 iu ????
UND is UND ???
Obviously very, very wrong. It is nice to know that you have at least 300 times less virus in your blood when you are "UND" with the more sensitive test then with the crude test.
As I had posted before... ; A study presented at the Chicago AASLD (abstract # 2442 late breaker) conference comparing the predictive power of the then 400copy cutoff Amplicor with the 5 copy cutoff NGI test at week 4 gave 45% versus 92 % predictive power for later SVR..
Even with the very best test, if neg or non detected : It does not mean you do not have virus in your blood still circulating!It just means - with the very best test - that the one little Milliliter that we have tested did not contain more than 5 virions ( or 1). But you have 5000ml of plasma and much more interstitial fluid that still possibly contains virus.
If a patient has such a rapid decline that he is UND with a supersensitive test at week 4 then this means that the slope of the decline is steep and we EXTRAPOLATE from here on that by continuing treatment in the same fashion for 24 or 48 weeks, that the virus will further and further and further decline NOW ALL INVISIBLE AND UNTESTABLE but still real and deciding SVR or not.
If we had no more virus in the body once "UND" we could stop treatment right then.
So like <600 iu has about the same meaning as < 2 iu ????
UND is UND ???
Obviously very, very wrong. It is nice to know that you have at least 300 times less virus in your blood when you are "UND" with the more sensitive test then with the crude test.
As I had posted before... ; A study presented at the Chicago AASLD (abstract # 2442 late breaker) conference comparing the predictive power of the then 400copy cutoff Amplicor with the 5 copy cutoff NGI test at week 4 gave 45% versus 92 % predictive power for later SVR..
Even with the very best test, if neg or non detected : It does not mean you do not have virus in your blood still circulating!It just means - with the very best test - that the one little Milliliter that we have tested did not contain more than 5 virions ( or 1). But you have 5000ml of plasma and much more interstitial fluid that still possibly contains virus.
If a patient has such a rapid decline that he is UND with a supersensitive test at week 4 then this means that the slope of the decline is steep and we EXTRAPOLATE from here on that by continuing treatment in the same fashion for 24 or 48 weeks, that the virus will further and further and further decline NOW ALL INVISIBLE AND UNTESTABLE but still real and deciding SVR or not.
If we had no more virus in the body once "UND" we could stop treatment right then.
Quest probably has close to a dozen different tests for Hep C. Have no idea what test you might get if your doc simply checks "PCR". Not a great idea. However, if the word "HEPTIMAX" appears on the rx or requistion form, then there will be no confusion. There is only one Heptimax test.
Hope it all works out.
-- Jim
Hope it all works out.
-- Jim
MEDICAL PROFESSIONAL
Your above comment is mostly a good description of the viral kinetics. When it comes to the levels below UND we are somewhat in unknown territory.
""""
1-Initial quick drop in the first 24 hours of TX, this is due mostly to the initial kill of the virus directly and is due to the interferon mostly. Usually a 1 to 1.5 log reduction.""""
If you had a means to stop the production of new virus entirely you would get in the logarithmic y / linear x-time scale plot a straight line down to zero. Existing virus in the blood has a half life of 4 to 6 hours. So until de nove production becomes relevant again ( at a lower level) you are mainly seeing the exponential decay curve simply by removal of circulating virus ( this is not killing by interferon, simply removal as usual, as you had it before treatment). The de novo blockage at this level reflects the "direct antiviral effect of IFN", combined with intensified cytokine responses by the resident immune cells, thats mainly NK,Tcells and dendritic cells.
Once you have dropped to a level whereby the virus is still replenished by de novo production, the slope will turn to a more flat second line which is indeed reflecting the further reduction of viral production by eliminating infected hepatocytes.
""""
2-The next stage of viral reduction is usually represented by a logrithmic decay of the viral load and is typically ALSO linear along the log graph. This slope along with how long you treat will ultimately determine your chances of success. I think the slope of this line is due to your body's ability to kill the infected hypocrites (sp) and this can range from individual to individual.""""
Yes this second process will probably determine the ultimate outcome in each particular patient. What we do not know is if this process indeed proceeds to Zero or stops at a smaller amount of infected hepatozytes that have inside virus that has escaped by virue of epitope mutation or cytokine blocking mechanisms, but the memory cells remaining in the liver provide enough long term local "antiviral milieu" to block a new rise of the infected remnant population.
"""To get a sustain response you need to drop you viral count to nearly zero. NOT YOUR BLOOD tests which only measure a mere fraction of the the amount of plasma in your body, but you whole body."""
That is one - the "older" theory. With several studies finding remnant virus in SVR livers it might be more as i outlined abvove.
""""The amount of virus detected in your PCR represents a small fraction (1/10,000) of the viral copies in your total body. So say you have 9 copies (which is undetectable with a very sensitive test) you could have approx. 9 X 10,000 or 90,000 copies of virus in your system. That 90,000 needs to get pretty close to zero WE DO NOT KNOW HOW LOW IT HAS TO GOT to get a sustained response. You need another 4 to 5 log drop of viral load to be clear.
So hypothetically speaking, the sensitivity of a PCR test to actually tell you if you are clear of the virus would in theory be 0.00001 IU/ml.
So when we speak of a test that goes to 5, 10, 30, or even 50, all these tests are relatively the same number because they are within one log of each other.""""
IF YOU LOOK AT THE 4 COPY AND 400 COPY UND DIFFERENCE YOU HAVE A TWO LOG DIFFERENCE. That is enough of a difference to gauge the different steepness of the initial decline to allow meaningful extrapolation.
"""So to recap, somebody with a starting viral load of 1,000,000 would need an 11 log reduction to clear the virus. A (10 iu/ml)test would only measure a 5 log reduction, And a (50 iu/ml) test would only measure approx. a 4.75 log reduction. On a log scale these numbers are virtually the same. ""
I agree that the difference between 10 and 50 on the overall log scale becomes almost irrelevant. but between 615 and 2 the log difference and the reflection of the reductive power of the treatment is real and can be estimated with good predictive power.
""""
1-Initial quick drop in the first 24 hours of TX, this is due mostly to the initial kill of the virus directly and is due to the interferon mostly. Usually a 1 to 1.5 log reduction.""""
If you had a means to stop the production of new virus entirely you would get in the logarithmic y / linear x-time scale plot a straight line down to zero. Existing virus in the blood has a half life of 4 to 6 hours. So until de nove production becomes relevant again ( at a lower level) you are mainly seeing the exponential decay curve simply by removal of circulating virus ( this is not killing by interferon, simply removal as usual, as you had it before treatment). The de novo blockage at this level reflects the "direct antiviral effect of IFN", combined with intensified cytokine responses by the resident immune cells, thats mainly NK,Tcells and dendritic cells.
Once you have dropped to a level whereby the virus is still replenished by de novo production, the slope will turn to a more flat second line which is indeed reflecting the further reduction of viral production by eliminating infected hepatocytes.
""""
2-The next stage of viral reduction is usually represented by a logrithmic decay of the viral load and is typically ALSO linear along the log graph. This slope along with how long you treat will ultimately determine your chances of success. I think the slope of this line is due to your body's ability to kill the infected hypocrites (sp) and this can range from individual to individual.""""
Yes this second process will probably determine the ultimate outcome in each particular patient. What we do not know is if this process indeed proceeds to Zero or stops at a smaller amount of infected hepatozytes that have inside virus that has escaped by virue of epitope mutation or cytokine blocking mechanisms, but the memory cells remaining in the liver provide enough long term local "antiviral milieu" to block a new rise of the infected remnant population.
"""To get a sustain response you need to drop you viral count to nearly zero. NOT YOUR BLOOD tests which only measure a mere fraction of the the amount of plasma in your body, but you whole body."""
That is one - the "older" theory. With several studies finding remnant virus in SVR livers it might be more as i outlined abvove.
""""The amount of virus detected in your PCR represents a small fraction (1/10,000) of the viral copies in your total body. So say you have 9 copies (which is undetectable with a very sensitive test) you could have approx. 9 X 10,000 or 90,000 copies of virus in your system. That 90,000 needs to get pretty close to zero WE DO NOT KNOW HOW LOW IT HAS TO GOT to get a sustained response. You need another 4 to 5 log drop of viral load to be clear.
So hypothetically speaking, the sensitivity of a PCR test to actually tell you if you are clear of the virus would in theory be 0.00001 IU/ml.
So when we speak of a test that goes to 5, 10, 30, or even 50, all these tests are relatively the same number because they are within one log of each other.""""
IF YOU LOOK AT THE 4 COPY AND 400 COPY UND DIFFERENCE YOU HAVE A TWO LOG DIFFERENCE. That is enough of a difference to gauge the different steepness of the initial decline to allow meaningful extrapolation.
"""So to recap, somebody with a starting viral load of 1,000,000 would need an 11 log reduction to clear the virus. A (10 iu/ml)test would only measure a 5 log reduction, And a (50 iu/ml) test would only measure approx. a 4.75 log reduction. On a log scale these numbers are virtually the same. ""
I agree that the difference between 10 and 50 on the overall log scale becomes almost irrelevant. but between 615 and 2 the log difference and the reflection of the reductive power of the treatment is real and can be estimated with good predictive power.
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