What Do You Think About This Scary Study?

[JennyPenny]

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.

[FamosAmos]

This article (and especially the headline and conclusion) is wreckless.  If “cured,” the levels of virus in these people blood was undetectable, which also suggests the levels of virus in any bodily secretions (tears/saliva/semen/vaginal fluid) is also undetectable.  They extracted LYMPHOID CELLS from their lymph nodes, in an attempt to locate at least partially viable copies of the virus and culture them.  Interesting, but it cannot be concluded that these people are CONTAGIOUS.  You do not exchange lymphoid cells people when you have sex with them, so unless you are exchanging lymph nodes or organs with them, they would never be receiving your lymphoid cells inside their body.  They simply cannot reasonably conclude that “cured” individuals are still contagious and to say or even suggest it, is WRECKLESS.  They are purposely being provocative with the headline to increase readership of the “study.”

[flyinlynn]

The post and article are from 2009 the treatment at that time was interferon injections along with ribavirin and other medications so basically entirely not applicable to today’s new generation DAA medicines.

The 2009 data is not relevant to the current treatment regimens

[willing]

I finally got around to reading Marukian'08

http://www.ncbi.nlm.nih.gov/pubmed/19003912

which Zeuzem had referenced as challenging the conclusions of the MacParland paper that started this thread, and overall I think he may be right...As Goofydad has noted, it's all about penetration, or the extent thereof.  The approach in the two papers is similar, both set out to infect PBMCs derived from healthy patients, exposed the cells to virus and then checked for viral rna and proteins in  the infected cells. Both used standard techniques of limiting infection as controls.

MacParland was testing whether residual virus from SVRs could infect healthy cells. Marukian used a cell culture system widely used to study viral entry/replication:

http://www.ncbi.nlm.nih.gov/pubmed/15947137

to see if PBMCs could be infected.


However, the key difference is that Marukian tracked increases in the level of RNA in the infected cells - if the virus is really replicating the amount of RNA should increase. What she found was that in huh7 cells (a commonly used line of liver cancer cells)  RNA levels grew as a function of time, about 100 fold increase in 3 days, whereas in PBMCs the level of RNA remained flat, as it did if cells were treated with a drug, CMA, that blocked replication (Fig 2.)

They went on to show that viral entry in PBMCs (and broke these out into B/T lymphocytes, macrophages etc.) is very unlikely because of failure to express cell receptors needed for viral entry (over the past couple of years researchers have finally tracked down in detail how it gets into cells).

However, their results may also explain MacParland's data:

"We tested the ability of HCVcc to replicate in cmDCs (Fig. 2C), M (Fig. 2D), peripheral blood DCs (pbDCs) (Fig. 2E), monocytes (Fig. 2F), and T/natural killer (NK) cells (Fig. 2G). As for B cells, HCV RNA was detected in cultures of these cell types after 24 hours incubation with HCVcc and extensive washing. The level of HCV RNA associated with the cultures was <1% of the input; this declined or remained stable after washing away input viral RNA. In all PBMC subsets, the level of HCV RNA associated with cells and tissue culture supernatants was unaffected by 2CMA. Although no increase in HCV RNA was detected in PBMC subsets or in 2CMA-treated Huh-7.5 cells, viral RNA persisted for many days in culture. Longer cultures of up to 14 days also showed no increase in HCV RNA (data not shown)."

in other words, it seems virions are tightly and determinedly clinging to PBMCs but not necessarily getting in and replicating. Even when they introduced viral RNA directly into PBMCs no translation was detected, whereas it was in liver cells.

I assume MacParland's group has already checked whether infectivity of SVR-derived virus as measured by RNA increase, occurs in liver cells. It'll be interesting to hear the next installment.

[jmjm530]

also want to make clear that I do not like to argue, or refute other peoples opinions.  So, Jim, if I sometimes seem argumentative, its only that I am stating my beliefs, and resisting being pushed into accepting either a dogma, or having to agree with the mainstream thinking, if I really have come to a different conclusion.  All in all, I appreciate everyones' viewpoints.
----------------
I certainly appreciate that statement and in the future will try and accept some of your opinions ( even though they are often directed at me for some reason LOL) as not being  argumentative or personal but rather as furthering the discussion. Hopefully, you will do the same with me. And while we obviously have some differences, we also agree on many core matters including that more work is needed in the area of post treatment side effects.

Be well,

-- Jim

[DoubleDose]

Thanks very much for your referenced article, and the comment on some of my past views.  I appreciate your understanding and open minded approach.  I certainly have never made comments just to be controversial, but only in response to personal observations, experiences, etc.  The family member issue that you referenced (and I am anxious to review the details of the article you cited) is an issue that I have observed, first hand, for years...all the while trying to convince myself that what I was seeing might NOT be connected to HCV.  I still continue to see specific symptoms in family members that reflect my past extrahepatic symptoms, AND they also seem to be increasingly apparent over the past five years!   I will copy and paste the quote from your post above:

"family members of patients with occult HCV infection show serological markers of HCV infection even more frequently than those of patients with chronic hepatitis C

I used to just think there might be some connection, but in the past few years I have come to be certain of it...even if it cannot be proven, or demonstrated by standard lab testing.  I am pretty sure that if they had testing done for HCV cellular immunity (different than serum antibody testing for HCV) that it would show up in all of my intimate contacts and family members.  I could go into  much detail on why I believe this, but it would still be only anecdotal.  I think that some very surprising things about HCV will be discovered in the future....not the least of which will be familial cellular immunity.  

I personally think the infection may exist in other states than a typical, full blown blood/liver infection...and the family member cellular immunity, and serological markers are the tip off that something is going on.  I see it from an experiential, and symptom based perspective....the researchers will discover the same, from a lab and cellular standpoint.

I also want to make clear that I do not like to argue, or refute other peoples opinions.  So, Jim, if I sometimes seem argumentative, its only that I am stating my beliefs, and resisting being pushed into accepting either a dogma, or having to agree with the mainstream thinking, if I really have come to a different conclusion.  All in all, I appreciate everyones' viewpoints.

Thanks for all of your references, and scientific interpretations of the research Willing.  You help keep us grounded in factual information.

Have a great day, all of you!!

DoubleDose

[jmjm530]

forgive the garble from my speech recognition software LOL the third sentence from the bottom should have read:

I don't think any of us are disregarding or belittling anything but simply formulating an opinion as we see it.

[jmjm530]

Willing:  I'm not sure any published paper has ever argued that occult is "a whole, virulent virus". It may in fact be, though I agree that seems unlikely....However it seems more honest and consistent to acknowledge this ignorance than to disregard available data and/or claim we already know the remnants to be benign....
--------------
As with many topics, I really don't think you and I are very far apart. I consider your statement above simply splitting hairs something that you and I tend to do with each other :) my opinions and to some extent the way they are expressed are consistent with most leading hepatologists as well as the quotes extracted from HR earlier in the thread. I don't think any of us are disregarding herbal little lameanything but simply formulating an opinion as we see it. if you the wording sounds a bit too certain given the data, that is your interpretation. Some of us feel that the speculation on the other side is also a bit much. Again, I don't think we're that far apart.

[willing]

DD: I know you haven't received much support here on this topic, but I think in some ways you've been ahead of your time. Hepatology's accepted articles/early view section includes yet another published  letter exchange on this topic ( the 3rd in a year). This one is a response by Carreno et al to the review by Welker/Zeuzem listed above along with Zeuzem's reply. Carreno references a forthcoming paper with the title (" Hepatitis C Virus Infection in the Family Setting of Patients with Occult Hepatitis C. ", J Med Virol. In press. Castillo et al '09) and states

"family members of patients with occult HCV infection show serological markers of HCV infection even more frequently than those of patients with chronic hepatitis C"

Zeuzem, of course, is skeptical

"These data may be interpreted in detail after full publication, but prima facie it seems difficult to understand why immune responses to HCV in family members of subjects with undetectable HCV-RNA in serum was more frequently than in family members of patients with detectable HCV-RNA."

we'll have to wait and see...

Jim: I'm not sure any published paper has ever argued that occult is "a whole, virulent virus". It may in fact be, though I agree that seems unlikely. The simple truth is that we know very little about whatever it is :

- it shares enough sequence similarity with wild-type that PCRs using standard 5'utr  primers detect it
- its proteins (at least ns5 and e2)  shares affinity for antibodies to standard viral proteins
- it is capable of infecting other cells in an artificial in-vitro setting
- it can trigger elevated cytokine levels in the host

However it seems more honest and consistent to acknowledge this ignorance than to disregard available data and/or claim we already know the remnants to be benign. Acknowledging  we don't yet understand why SVR remains durable in the presence of residual virus seems a good 1st step...

[DoubleDose]

Willing said:

" Overall, I've never understood why SVR durability is an argument against low-level viral persistence; the data seems to strongly support both. Every relapser who remains UND by the most sensitive tests for 36 weeks or more knows this... "


My comment:  I agree with this statement.  I have always felt that SVR is very highly durable, but, that there is still documented persistent HCV virus left in SVR's, that we don't fully understand.  The research pretty much confirms this in study after study.

What we do not know is just how dangerous this residual virus might be.  It does indeed appear, from several cited examples, that a few individuals have relapsed years after confirmed SVR's, after heavy immuno-suppressive therapy.  Yes Jim, I understand that not everyone who receives immuno-suppressive tx relapses, but the fact that some have relapsed, tells me that the exception might just be the rule...but only in extreme circumstance.  

What if we really ARE in a somewhat permanent state of viral remission, rather than complete eradication?  Are you absolutely certain that this is not the case Jim?  Because I do not believe that even the best researchers in the world are sure what is going on.  My point is just this:  I think we have some open questions and not clearly understood behaviors with this virus, and many researchers continue to try to hone in, and figure out what it all means.  

Of course we could all just ASSUME that the virus is completely gone, in spite of research findings, and we could just ASSUME that any possible residual infection, or remission behavior of this virus, is totally benign, and will not cause any future problems....but I personally do not believe that this is a scientifically sound choice to make.   I want to have clearer answers, and I really want to know whether a possible 'persistent infection', under the radar so to speak, might have consequences for our immune systems, out long term health, and even our level of infectivity, if there is any infectivity to be concerned about.

I prefer to believe that the real truth will become fully understood as time goes on, and further research is done.  I do not want to jump to conclusions on any aspect of this issue.  But I still do believe that a few documented cases of legitimate relapse more or less disprove the 'rule' that SVR equals eradication, no matter how rare, AND that these cases beckon us to further study this viral behavion, and the HCV persistence observations, so that we can all be better prepared to deal with the realities, or potential consequences.

Hopefully its all just smoke, and is totally benign, as you often claim....but I will feel much more comfortable after we get a much deeper understanding of what it all means.  I won't rehash past observations regarding immunosuppressed relapsers, and fluctuating positive PCR's after SVR, only to disappear again, etc. other than to state that much of what we read about, and see in these cases fit a model of 'remission' much better than eradication.

The virus might behave more like Herpes Zoster, and could possibly go away, only to cause different sorts of damage way down the road.  Maybe even just immune system disruption, cytokine storms, etc.  We just don't have all the answers yet, in spite of your certainty on the subject.  That is something I feel really comfortable betting on!  I respect your views nonetheless.

DoubleDose

[jmjm530]

Overall, I've never understood why SVR durability is an argument against low-level viral persistence;
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because, if the low-level virus is found in the occult studies was truly a whole, virulent virus, then why wouldn't it keep replicating to the point where one would have a full-blown relapse. If the argument is that it's the immune system preventing this, then why doesn't this happen more often in immune compromised individuals? it seems to me at least that it's because what they're seeing is not the same kind of virus that gave us HCV in the 1st place. perhaps just some sort of lesser remnant.

[willing]

It's a bit hard to understand why a 36 month late relapse (after the 24w check period) is more significant than a 15 month late relapse, particularly when the additional note

" the reappearance of HCV RNA occurred 3 months after two treatments with steroid bolus therapy for severe rejection"

clearly suggests virus was present but maintained UND by a functioning immune system. Nevertheless, here's another:

"SUSTAINED VIROLOGIC RESPONSE AFTER PEGINTERFERON ALFA-2B AND RIBAVIRIN TREATMENT PREDICTS LONG-TERM CLEARANCE OF HCV AT 5-YEAR FOLLOW-UP"
abstract 804 by Manns et al from the 08 EASLD (available at J. of Hepatology site)
Of 366 SVRs from PEG/RBV tx followed for 5 years:

" Four SRs relapsed during the 5-year follow-up period (2 at year 1, 1 at year 2, and 1 at year 5)"

This is ref 150 in the Zeuzem survey cited above. Their table 2 gives a comprehensive list of data in the area. The criticisms of Pradat are legitimate but I believe are intrinsic to follow ups in the general population where it is essentially impossible to distinguish late relapse from re-infection, check for false positives, etc. Data that indicates causality between immuno-suppression and late relapse, as in the case above or a similar one:

http://www.ncbi.nlm.nih.gov/pubmed/18626242

argues pretty convincingly to my mind that SVR, notwithstanding undisputed durability, does not always equate to eradication.

Thanks for the link to the Hafon letter exchange - when I get a bit of time I want to go through the duelling PCR details - the discrepancies are frustrating! A similar exchange is a Pham Michalak  letter in response to the Bernandin study finding no HCV in PBMCs.

http://www.ncbi.nlm.nih.gov/pubmed/18537176

among other things they criticised the Bernandin authors for omission of mitogen stimulation:

"a brief culture of PBMCs with mitogens and cytokines that activated immune cells facilitated HCV RNA detection in as much as 75% of initially HCV-negative cases.[7] Further, in up to 20% of cases, the HCV genome can be found in stimulated PBMCs but not in parallel serum. The simultaneous detection of an HCV RNA replicative strand, HCV protein, and unique HCV variants served to authenticate active virus replication in PBMCs, irrespective of serum HCV RNA status, as our recent study reassured.[6] The above approach to HCV RNA identification was not employed by Bernardin and colleagues.[1] Thus, as they alluded, it remains a distinct possibility that ex vivo stimulation of PBMCs might have augmented HCV detection in their study."

tellingly, the authors in their response, don't dispute the point:

"We clearly acknowledged[1] that in chronically viremic subjects, PBMCs could be a site of HCV replication and that in vitro mitogen plus cytokine treatment might increase PBMC-associated HCV levels.[8-10]"

also the lymphocytes in the MacParland paper were paper were T cells, not macrophages. Mitogen stimulation is basically an augmentation technique which after all is what a PCR is; it may not reflect in-vivo conditions but enables detection of trace levels.

Overall, I've never understood why SVR durability is an argument against low-level viral persistence; the data seems to strongly support both. Every relapser who remains UND by the most sensitive tests for 36 weeks or more knows this...

[jmjm530]

the quote attributed above to ML, above, should have been abbreviated as such:

" 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." Fortunately, we live in the real world and not in a Petri dish.

[jmjm530]

That's an important point about test tube results versus real world relevance.

As technology advances, motivated scientists will be able to find and discover things unseen before.

Who knows what remnants the human body harbors when you get beyond the cellular level and keep looking and looking.  It may turn out that everybody has some remnant of HCV (or most every other disease) if you magnify and extrapolate enough. But again, what is the clinical relevance?

What we do know is what you just said " 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. Fortunately, we live in the real world and not in a Petri dish." This is not to diminish the importance of these studies, just a comment on their relevancy in terms of the real world concerns many have expressed in this thread.

Hope all is good  in your part of the world as well.

- Jim

[Mr Liver]

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

[Mr Liver]

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

[jmjm530]

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..."

[willing]

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).

[JennyPenny]

Or, infecting a cell in a laboratory setting, is different than infecting a cell in a human body.

[Rockerforlife]

We are seeing one badly beaten monster thats afraid of rearing its ugly head.

[jmjm530]

ML: -just 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. So far no one has been able to do so. This, I'm afraid is the big elephant in Pham's lab.
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Yes, because if the virus is not truly gone like Pham suggests, then it must be the immune system keeping an SVR from relapse. Yet, we don't see relapses in SVRs even in immune compromised individuals. So what's keeping the virus "under the radar"? One explanation is that what they are seeing really isn't the replicative/infectious virus that is known as HCV but some sort of partial/impotent imposter.

[Mr Liver]

I think this topic has sure had legs. I'm willing to bet Pham would happily concur since he's been making a living trying to prove something unsuccessfully for twenty years.

I've wondered for some time why his research seems to avoid employing techniques
that would bolster his findings. Now, I may have missed something and its been awhile but the following are problem points I have with his studies.

He has hung his hat for a long time on the proposition that the neg strand is a sign of replication, ergo, its existence indicates ongoing viral infectivity.  To my knowledge its presence is an indication of replication but its discovery is meaningless by itself. This presence of the neg strand does not indicate completion of the replicative process.  It is exactly what it is called---an intermediate step (necessary for replication). The pos strand is also an intermediate necessary for the neg strand to do its part in the process. By measuring the presence of both pos and neg strands a ratio of +/- can be estimated and compared to known ratios. I don't believe I've seen this in his studies.

Macrophages (part of the family of white cells known as PBMC) collect viral debris from our blood. By collecting these macrophages and stimulating them to divide at a much faster rate  poses some problems for me. Cell division can result in the release of some cell contents each and every time it divides. Along with the crushed cells done by centrifuge one can artificially create a scenario that only exists in vitro and not in nature. The released contents of these cells includes RNA fragments which alone have no ability to infect. However with a large enough sample it can then be amplified by RT-PCR whereby it would detect the presence of the RNA indicating active virus.

I've offered the same challenge for nearly ten years now---just 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. So far no one has been able to do so. This, I'm afraid is the big elephant in Pham's lab.

If I have misinterpeted, misread, or misunderstood, I am prepared to modify my views. I haven'tlooked over any of these studies for some time and I'm working with less than optimum memory lately so its a distinct possibility.
ML

[apache1]

willing,
We hashed this over before,
remember 8 of 9pateints were over 40vl...and  you wrote:
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" Furthemore, VL in patient 6 (59/F) with a VL of 1600 vge should have been picked up even the Roche Amplicor v2. This discussion and the paper's  lack of comment on the discrepancy in VL tests is making me
***see the paper in a new light:***
apart from the main result being reported, the paper is a shot across the bow of all the sensitive VL testing technology. "
=============================================================

Has some new study caused you to  'lose the light'  on this willing ?

So once again, researchers take patients that are not SVR (by current available vl tests), and show that they have virus that can replicate when stimulated in a laboratory test tube.
Is this a big surprise, or even a new finding ???

apache

[JennyPenny]

Thanks Jim.

[jmjm530]

Full-text is online here for free:

http://www3.interscience.wiley.com/cgi-bin/fulltext/121581244/HTMLSTART

[JennyPenny]

I've been edited. PM me if you want the whole study. I have it. This place has more rules than a trafffic cop.