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What Do You Think About This Scary Study?
Study: Patients With Resolved Hepatitis C Likely Still Contagious
Patients with chronic hepatitis C that has been resolved through therapy or immune response may still be able to infect others with the virus. That finding is from a new study in the May issue of Hepatology, a journal published by John Wiley & Sons on behalf of the American Association for the Study of Liver Diseases (AASLD). The article is also available online at Wiley Interscience (www.interscience.wiley.com).
About 170 million people worldwide are infected with hepatitis C virus, which can progress to chronic hepatitis, cirrhosis and even liver cancer. In some individuals, the infection seems to resolve, either spontaneously from the efforts of the immune system, or after treatment with interferon and ribavirin.
Patients who achieve a sustained viral response show no clinical or biochemical evidence of liver disease and standard tests can no longer detect the virus in their blood. However, more sensitive research tests are finding that such patients often still have miniscule amounts of the virus in their bodies. No one knows if these trace remainders are infectious.
Researchers led by Tomasz I. Michalak of Memorial University of Newfoundland, Canada examined this question using a system that allows for propagation of HCV in human T cells in vitro.
They began with nine patients with HCV who had achieved a sustained viral response that persisted for at least two years after treatment. HCV RNA was detectable in their blood only with the more sensitive tests.
The researchers set up twelve cultures of lymphoid cells from healthy donors, and exposed them to plasma or to supernatants of cultured circulating lymphoid cells from the HCV patients. Eleven of the cell cultures became HCV RNA positive. Furthermore, HCV from three of the nine patients was able to establish active HCV replication in the cultures.
“These findings provide in vitro evidence that trace quantities of HCV persisting in the circulation for a long time after therapeutically induced resolution of CHC can remain infectious,” the authors report.
Interestingly, HCV replication in the T cells was prevented after neutralization of the virus, and by treatment with interferon.
This study is the first to investigate the infectivity of HCV traces that remain when the infection is occult. It agrees with previous animal studies of the same question.
“Our present findings reveal that HCV circulating in some individuals with resolved hepatitis C is capable of inducing productive infection in vitro at doses of 20 to 50 copies,” the authors conclude. “This can be interpreted as a strong indication of potential virus infectivity in vivo.”
Article: “Hepatitis C Virus Persisting at Low Levels after Clinically Apparent Sustained Virological Reponse to Antiviral Therapy Retains Its Infectivity in Vitro.” MacParland, Sonya A.; Pham, Tram N.Q.; Guy, Clifford S.; Michalak, Tomasz I. Hepatology; May 2009.
Patients with chronic hepatitis C that has been resolved through therapy or immune response may still be able to infect others with the virus. That finding is from a new study in the May issue of Hepatology, a journal published by John Wiley & Sons on behalf of the American Association for the Study of Liver Diseases (AASLD). The article is also available online at Wiley Interscience (www.interscience.wiley.com).
About 170 million people worldwide are infected with hepatitis C virus, which can progress to chronic hepatitis, cirrhosis and even liver cancer. In some individuals, the infection seems to resolve, either spontaneously from the efforts of the immune system, or after treatment with interferon and ribavirin.
Patients who achieve a sustained viral response show no clinical or biochemical evidence of liver disease and standard tests can no longer detect the virus in their blood. However, more sensitive research tests are finding that such patients often still have miniscule amounts of the virus in their bodies. No one knows if these trace remainders are infectious.
Researchers led by Tomasz I. Michalak of Memorial University of Newfoundland, Canada examined this question using a system that allows for propagation of HCV in human T cells in vitro.
They began with nine patients with HCV who had achieved a sustained viral response that persisted for at least two years after treatment. HCV RNA was detectable in their blood only with the more sensitive tests.
The researchers set up twelve cultures of lymphoid cells from healthy donors, and exposed them to plasma or to supernatants of cultured circulating lymphoid cells from the HCV patients. Eleven of the cell cultures became HCV RNA positive. Furthermore, HCV from three of the nine patients was able to establish active HCV replication in the cultures.
“These findings provide in vitro evidence that trace quantities of HCV persisting in the circulation for a long time after therapeutically induced resolution of CHC can remain infectious,” the authors report.
Interestingly, HCV replication in the T cells was prevented after neutralization of the virus, and by treatment with interferon.
This study is the first to investigate the infectivity of HCV traces that remain when the infection is occult. It agrees with previous animal studies of the same question.
“Our present findings reveal that HCV circulating in some individuals with resolved hepatitis C is capable of inducing productive infection in vitro at doses of 20 to 50 copies,” the authors conclude. “This can be interpreted as a strong indication of potential virus infectivity in vivo.”
Article: “Hepatitis C Virus Persisting at Low Levels after Clinically Apparent Sustained Virological Reponse to Antiviral Therapy Retains Its Infectivity in Vitro.” MacParland, Sonya A.; Pham, Tram N.Q.; Guy, Clifford S.; Michalak, Tomasz I. Hepatology; May 2009.
Jim I have never been stellar in mathematics but I do believe I have enough grasp of the subject to ascertain that the real world results do not jibe with the test tube results when extrapolated. We should all be up to our you-know-whats in medical literature detailing late relapses if the test tube results of occult research were anything close to an accurate description of real world conditions. We just don't see the results we would expect to see if so many people who have treated successfully (SVR) worldwide were actually still infected. Good to see you post. Hope all is well in your part of the world.
ML
ML
Thanks for the reply and the interesting notes. You know I can't let a nearly 10 year challenge fall at the hands of something like the Pradat study. ;) The obvious limitations of testing sensitivities, the adjunctive nature of the finding, and possible re-infection which you mentioned, could have all affected the results. The other study you linked to was indeed relapsers under immunosuppression therapy but even putting that aside they did not fall into my criteria as their relapse was earlier than 3 years--"During the long-term follow-up period, serum HCV-RNA was persistently undetectable in 32 of 34 (94%) patients with SVR. Two patients developed virological relapse at 8 and 15 months."
Here is a discussion of findings from Medscape concerning Pradat which details the reasons why Pradat does not rise to the challenge:
"Discussion
Among patients classified in 1996 as resolving acute hepatitis, 6% became HCV-RNA positive by PCR during the follow-up period. Similarly, among patients classified as sustained virological responders after antiviral therapy, 8% were found HCV-RNA-positive during follow up.
Because of progress in virological testing, one cannot exclude that some of these late "relapses" were not positive but were missed by the lower sensitivity of the HCV-RNA detection methods used 5-7 years earlier. It is indeed possible that if re-tested using current methods with higher sensitivity, some of the initial samples regarded as being negative for HCV-RNA, would turn out to be positive at very low levels. A recent study has shown that minimal residual viraemia could be detected by a transcription-mediated amplification (TMA) method.[13] The authors showed that among patients who were repeatedly HCV-RNA-negative by PCR at the end of therapy, 12.5% were found HCV-RNA-positive by TMA. Among these latter, 96% were relapsers after therapy withdrawal. In the present study, the possibility of re-infection cannot be ruled out because data on risk behaviour during follow up and concordance of genotypes were not available.
However, although the sensitivity of current conventional laboratory assays approved for HCV-RNA detection has substantially increased, it remains possible that low levels of HCV in serum or within white blood cells still escape detection. Two recent studies[14,15] have indeed shown evidence of the long-term persistence of HCV genomes in sera and circulating lymphoid cells in individuals considered to be clinically and serologically cleared of HCV infection. However, the clinical impact of such low levels of residual virus remains unknown but appears of limited significance".
I'm aware of the use of mitogen stimulation in vaccine development but I am not familiar with its use in that setting. The problem I have with this method is macrophages, for instance, make up some of the PBMC . Their job is to capture viral fragments from our sera, primarily. Cell division has been shown to 'leak' cytoplasm during the process which also allows for the release of any viral fragments including RNA genomic material which is non-infective and could have been harbored within the cell. Artificial stimulation to promote rapid cell division is a process that could result in the release of larger amounts of viral fragments, including genomic RNA. If you increase the amount of viral RNA fragments artificially to a sufficient amount to be amplified by PCR the suggested finding might not be accurate, in my view.
Here is a study by Halfon that finds no such thing as infected PBMCs post SVR. Carreno et al fired a quick letter back to Halfon and then they were counter-punched by Halfon and colleagues with another letter back from them to Carreno. I like this competitive spirit between researchers - it can only be seen as a pos for those with HCV. This goes to your point of experts disagreeing on this subject.
Study - http://jcm.asm.org/cgi/content/full/46/6/2106
Letters - http://jcm.asm.org/cgi/content/full/46/10/3550
I'll try to remember to post about some studies involving +/- strand ratios later. I think it would be just another way to either bolster or diminish findings concerning the significance of the presence of neg strand for some researchers. I also have a study done with electron microscopy that I'll post. I have to leave right now.
I don't know where my '20' yr comment came from. And I may never know-lol
Thanks again for the reply and information.
ML
Here is a discussion of findings from Medscape concerning Pradat which details the reasons why Pradat does not rise to the challenge:
"Discussion
Among patients classified in 1996 as resolving acute hepatitis, 6% became HCV-RNA positive by PCR during the follow-up period. Similarly, among patients classified as sustained virological responders after antiviral therapy, 8% were found HCV-RNA-positive during follow up.
Because of progress in virological testing, one cannot exclude that some of these late "relapses" were not positive but were missed by the lower sensitivity of the HCV-RNA detection methods used 5-7 years earlier. It is indeed possible that if re-tested using current methods with higher sensitivity, some of the initial samples regarded as being negative for HCV-RNA, would turn out to be positive at very low levels. A recent study has shown that minimal residual viraemia could be detected by a transcription-mediated amplification (TMA) method.[13] The authors showed that among patients who were repeatedly HCV-RNA-negative by PCR at the end of therapy, 12.5% were found HCV-RNA-positive by TMA. Among these latter, 96% were relapsers after therapy withdrawal. In the present study, the possibility of re-infection cannot be ruled out because data on risk behaviour during follow up and concordance of genotypes were not available.
However, although the sensitivity of current conventional laboratory assays approved for HCV-RNA detection has substantially increased, it remains possible that low levels of HCV in serum or within white blood cells still escape detection. Two recent studies[14,15] have indeed shown evidence of the long-term persistence of HCV genomes in sera and circulating lymphoid cells in individuals considered to be clinically and serologically cleared of HCV infection. However, the clinical impact of such low levels of residual virus remains unknown but appears of limited significance".
I'm aware of the use of mitogen stimulation in vaccine development but I am not familiar with its use in that setting. The problem I have with this method is macrophages, for instance, make up some of the PBMC . Their job is to capture viral fragments from our sera, primarily. Cell division has been shown to 'leak' cytoplasm during the process which also allows for the release of any viral fragments including RNA genomic material which is non-infective and could have been harbored within the cell. Artificial stimulation to promote rapid cell division is a process that could result in the release of larger amounts of viral fragments, including genomic RNA. If you increase the amount of viral RNA fragments artificially to a sufficient amount to be amplified by PCR the suggested finding might not be accurate, in my view.
Here is a study by Halfon that finds no such thing as infected PBMCs post SVR. Carreno et al fired a quick letter back to Halfon and then they were counter-punched by Halfon and colleagues with another letter back from them to Carreno. I like this competitive spirit between researchers - it can only be seen as a pos for those with HCV. This goes to your point of experts disagreeing on this subject.
Study - http://jcm.asm.org/cgi/content/full/46/6/2106
Letters - http://jcm.asm.org/cgi/content/full/46/10/3550
I'll try to remember to post about some studies involving +/- strand ratios later. I think it would be just another way to either bolster or diminish findings concerning the significance of the presence of neg strand for some researchers. I also have a study done with electron microscopy that I'll post. I have to leave right now.
I don't know where my '20' yr comment came from. And I may never know-lol
Thanks again for the reply and information.
ML
A more germane quote per this thread title rom the Zeuzem study you referenced above, would be:
"In conclusion, HCV-infected patients with no detectable HCV RNA in serum 24 weeks after the end of treatment should remain to be considered noninfectious and cured of their infection..."
"In conclusion, HCV-infected patients with no detectable HCV RNA in serum 24 weeks after the end of treatment should remain to be considered noninfectious and cured of their infection..."
apache:
> So once again, researchers take patients that are not SVR (by current
> available vl tests)
as far as I know the current dogma is that SVR only comes in one flavor: if the 12w post-eot test is UND you are SVR, more or less for life, with all the privileges appurtenant thereto. Sensitivity of the post-eot test is irrelevant insofar as, if there is a class of relapsers with VL consistently below detectable levels, it has not yet been detected/reported (apart from the occult papers). These pts repeatedly measured VL UND and have normal enzymes. If they're not SVR how would you describe them?
The twist to the paper I hadn't caught until you brought up more more sensitive testing is that even the Amplicor v2, *if it performs as advertised*, should have detected VL in patient 6 but didn't. Clearly one of those two PCR tests is lying.
ml:
>show me one documented case of relapse beyond 3 years SVR
> which is verifiable and has been peer-reviewed by a
> reputable scientific body and published
among long-term follow ups in the general population there's
Pradat'07 which found a 7% late relapse rate
"Ninety-three per cent of the HCV patients who had cleared the virus (spontaneously or after antiviral therapy) remained HCV-RNA-negative during follow up"
( from http://www.ncbi.nlm.nih.gov/pubmed/17650289 )
and among potentially immunocompromised OLT recipients there's
"Two patients with SVR developed late virological relapse. "
(from http://www.ncbi.nlm.nih.gov/pubmed/15996238 )
However, there's no way of distinguishing late-relapse from re-infection so the rate is probably even lower than what is reported.
On the plus side, experts in the field are as confused on this topic as we are. A careful compilation of all available data was published by Welker and Zeuzem in Feb:
"Occult hepatitis C: how convincing are the current data?"
http://www.ncbi.nlm.nih.gov/pubmed/19105211
which includes tables distinguishing different classes of late relapse. This triggered a prompt response from Carreno et al, citing MacParland's paper among others. Zeuzem ( reply pending publication) is still not convinced
" If clinical significant replication of HCV takes place in patient with “occult” HCV infection and sustained virologic response (SVR) after interferon-alfa (IFN) based antiviral therapy for chronic HCV infection, high late virologic relapse rates may be expected. However, late virologic relapse rates are overall very low in patients with SVR, irrespective whether their immune system is impaired or not (1)."
his strongest bit of ammunition, IMHO, is a recent careful study, Marukian'09, which goes directly against the MacParland/Michalak results.
http://www.ncbi.nlm.nih.gov/pubmed/19003912
These guys were flat out unable to get HCV to infect PBMCs, even when they transfected the cells with HCV RNA. Only one of MacParland and Marukian can be right... and they both seem to have done very careful work.
As for your points about the methods in the MacParland paper (and I believe Pham's first paper was only '04), the relative ratio of +/- strands is unlikely to be reliable because (a) errors in quantification of very low RNA levels (b) ignorance about the quantitative details of viral replication. We know one, unsheathed, genomic (+) strand yields one antigenomic (-) strand which is then multiply transcribed to yield progeny virions, but as far as I know this is very poorly characterised (eg see Bartenschalger'04
http://www.ncbi.nlm.nih.gov/pubed/15530561)
Also mitogen stimulation is a routine technique in immunology.
It's worth noting that MacParland's paper uses multiple forms of evidence, presumably in part to deflect the old 'contamination' objections. Along with the RNA pcrs shown in Figs 1 and 3, they include fluorescence microscopy-based evidence of HCV protein NS5 detection in Fig 3, sequencing results in Fig 4, and electron-microscopy evidence of binding to viral particles via HCV E2 protein in Fig 7.
jim:
>some sort of partial/impotent imposter.
aye, the old evil twin plot twist... is this really different from the broken down has-been hypothesis? But we now know the impostor can infect (and can be prevented from infecting). Also, comparing top and bottom lines in Fig 4 suggests the impostor looks an awful lot like real HCV (at least in this snippet, which admittedly is in the most conserved part of the viral genome).
> So once again, researchers take patients that are not SVR (by current
> available vl tests)
as far as I know the current dogma is that SVR only comes in one flavor: if the 12w post-eot test is UND you are SVR, more or less for life, with all the privileges appurtenant thereto. Sensitivity of the post-eot test is irrelevant insofar as, if there is a class of relapsers with VL consistently below detectable levels, it has not yet been detected/reported (apart from the occult papers). These pts repeatedly measured VL UND and have normal enzymes. If they're not SVR how would you describe them?
The twist to the paper I hadn't caught until you brought up more more sensitive testing is that even the Amplicor v2, *if it performs as advertised*, should have detected VL in patient 6 but didn't. Clearly one of those two PCR tests is lying.
ml:
>show me one documented case of relapse beyond 3 years SVR
> which is verifiable and has been peer-reviewed by a
> reputable scientific body and published
among long-term follow ups in the general population there's
Pradat'07 which found a 7% late relapse rate
"Ninety-three per cent of the HCV patients who had cleared the virus (spontaneously or after antiviral therapy) remained HCV-RNA-negative during follow up"
( from http://www.ncbi.nlm.nih.gov/pubmed/17650289 )
and among potentially immunocompromised OLT recipients there's
"Two patients with SVR developed late virological relapse. "
(from http://www.ncbi.nlm.nih.gov/pubmed/15996238 )
However, there's no way of distinguishing late-relapse from re-infection so the rate is probably even lower than what is reported.
On the plus side, experts in the field are as confused on this topic as we are. A careful compilation of all available data was published by Welker and Zeuzem in Feb:
"Occult hepatitis C: how convincing are the current data?"
http://www.ncbi.nlm.nih.gov/pubmed/19105211
which includes tables distinguishing different classes of late relapse. This triggered a prompt response from Carreno et al, citing MacParland's paper among others. Zeuzem ( reply pending publication) is still not convinced
" If clinical significant replication of HCV takes place in patient with “occult” HCV infection and sustained virologic response (SVR) after interferon-alfa (IFN) based antiviral therapy for chronic HCV infection, high late virologic relapse rates may be expected. However, late virologic relapse rates are overall very low in patients with SVR, irrespective whether their immune system is impaired or not (1)."
his strongest bit of ammunition, IMHO, is a recent careful study, Marukian'09, which goes directly against the MacParland/Michalak results.
http://www.ncbi.nlm.nih.gov/pubmed/19003912
These guys were flat out unable to get HCV to infect PBMCs, even when they transfected the cells with HCV RNA. Only one of MacParland and Marukian can be right... and they both seem to have done very careful work.
As for your points about the methods in the MacParland paper (and I believe Pham's first paper was only '04), the relative ratio of +/- strands is unlikely to be reliable because (a) errors in quantification of very low RNA levels (b) ignorance about the quantitative details of viral replication. We know one, unsheathed, genomic (+) strand yields one antigenomic (-) strand which is then multiply transcribed to yield progeny virions, but as far as I know this is very poorly characterised (eg see Bartenschalger'04
http://www.ncbi.nlm.nih.gov/pubed/15530561)
Also mitogen stimulation is a routine technique in immunology.
It's worth noting that MacParland's paper uses multiple forms of evidence, presumably in part to deflect the old 'contamination' objections. Along with the RNA pcrs shown in Figs 1 and 3, they include fluorescence microscopy-based evidence of HCV protein NS5 detection in Fig 3, sequencing results in Fig 4, and electron-microscopy evidence of binding to viral particles via HCV E2 protein in Fig 7.
jim:
>some sort of partial/impotent imposter.
aye, the old evil twin plot twist... is this really different from the broken down has-been hypothesis? But we now know the impostor can infect (and can be prevented from infecting). Also, comparing top and bottom lines in Fig 4 suggests the impostor looks an awful lot like real HCV (at least in this snippet, which admittedly is in the most conserved part of the viral genome).
Or, infecting a cell in a laboratory setting, is different than infecting a cell in a human body.
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