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Author Topic: Berlin patient could still be infected??!!  (Read 6667 times)

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Offline Tadeys

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Berlin patient could still be infected??!!
« on: June 08, 2012, 07:32:33 PM »
Written by Alain LafeuIllade:
T he Weird Story of the Berlin Patient

The only case of a possible sterilizing HIV cure has been challenged today at the 2012 HIV resistance workshop: the patient, actually, could still be HIV infected!

 
 
Eveybody knows the story of the "Berlin patient" (see, in particular, several articles and an interview of him on this website).
As an American citizen he flew back to San Francisco 2 years ago after havin being "cured" in Berlin by Dr Hutter (first publication in the N Engl J Med in 2009, last one in Blood in December 2011).

But it came like a thunder this morning when Dr Steven Yukl presented his talk intitled "Challenges inherent in detecting HIV persistence during potentially curative interventions."
An enigmatic title that hides quite a weird story.
Note that coauthors of this abstract are:  T Chun, MC Strain, J Siliciano, E Eisele, R Buckheit, YC Ho, JK Wong, MP Busch, G Hütter, DD Richman, RF Siliciano, SG Deeks.
Actually, most of these people repeated several experiments on this patient blood plasma, blood cells and rectal biopsies...
And the facts are these:
"A large volume apheresis was performed and 9 billion PBMCs evaluated for the presence of replication-competent virus. All wells were negative for HIV p24, indicating that the frequency of replication-competent HIV was therefore estimated to less than one infected cell per 1.4 billion CD4+ T cells. A repeat experiment in a second laboratory confirmed these findings.  Using a variety of assays and approaches, very low levels of HIV RNA were intermittently detected in plasma, although sequence analysis of these variants were different from each other and different from those present before the transplant. Digital PCR for HIV DNA was negative for 1 copy per 2 million cells with a 95% confidence limit of less than 1.9 copies per million cells.  Collagenase-digested rectal biopsy-derived cells were positive for very low levels of HIV DNA but not RNA; no sequence for confirmatory studies could be obtained. HIV antibodies levels were low and declined over the course of approximately 18 months."
Data were also presented showing that although this patient remained HIV seronegative, he still has antibodies again HIV at a very low level of detection, below the cutoff of current commercially available ELISA tests.
These data raise several questions that are not addressed in the abstract conclusion: "Although the subject has had intermittent evidence for HIV persistence in some assays in some laboratories, the extremely low levels of virus which were detected, while pushing the limits of  sensitivity and specificity, and the inability to match sequence with the subject’s pre-therapy virus make it impossible to conclude that the subject remains HIV infected."
First, is that only a matter of PCR contamination or variability of tests at such low levels. I personnaly have serious doubts about this argument because Deeks group did not even mention this variability at last CROI when they reported that Disulfiram was able to exhibit an anti-latency effect, based on very small blips!
Second, it is strange to see that the different research teams do not have any data on soluble markers of inflammation, HIV in semen, or the tropism of the circulating strains...That is ongoing they answered.
Then, there are 2 other alternative explanations to this viral persistence during a "cure": one, the patient has never been cured and is a chimera, second, he has been cured but reinfected. In this direction goes the observation that rectal HIV DNA was negative when he was in Berlin, and now is positive. Also goes the fact that different circulating viral strains were isolated.

A terrific story if there is. It was amazing to see that Steve Yukl, the virologist who never met the patient was at the lectern without a bullet proof suit....and that Steve Deeks sat quietly at the back of the room, the one who currently follows the patient...

Offline younghopefulpoz

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Re: Berlin patient could still be infected??!!
« Reply #1 on: June 09, 2012, 05:12:20 AM »
Not good. :(, do they have proofs that the berlin patient is STILL infected?

Offline songs06

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Re: Berlin patient could still be infected??!!
« Reply #2 on: June 09, 2012, 05:25:10 AM »
come on! it doesn't matter if he is positive anymore because now he has bone marrow of someone who has CCR5 mutation. this means even he has the virus, it can't make any kind of disease. CCR5 is a surface protein that HIV uses to enter the target cells, and %1 of the european has its mutation that protects themself from getting infected. berlin patient might still have some traces of virus in his bloodstream but he will never turn to AIDS even without ART.

but bone marrow transplant is highly risky thing, i most likely do not want it if it will turn out only cure option in the future. you can live your normal lifespan with drugs, so why get a transplant and use drugs to supress your immune system for almost whole your life. it is not "less risky" than HIV infection.
18.03.2012 - infected.
14.04.2012 - first positive elisa - UD western blot
30.04.2012 - western blot confirmation positive
03.05.2012 - first lab- CD4: 256   VL: 2.3 M
01.06.2012 - sec lab- CD4: 390 (end of ARS)
01.07.2012 - third lab- CD4: 388 VL: 150.000
11.07.2012 - Started Truvada + Kaletra
04.08.2012 - CD4: 401 VL: 3800
30.09.2012 - CD4: 510 VL: 709
04.01.2013 - CD4: 650 VL: UD! (aka 20)
01.04.2013 - CD4: 460 VL: UD
09.2013 - CD4: 510
02.2014 - CD4: 490

Online mecch

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Re: Berlin patient could still be infected??!!
« Reply #3 on: June 09, 2012, 07:23:14 AM »
If he was rendered immune to infection, he might well be going bareback :)
“From each, according to his ability; to each, according to his need” 1875 K Marx

Offline Skydrake

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Re: Berlin patient could still be infected??!!
« Reply #4 on: June 09, 2012, 10:23:12 AM »
If he was rendered immune to infection, he might well be going bareback :)
.....but he isn't immune to X4-tropic strains. Viruses can also use the CXCR4 coreceptor, not only CCR5 coreceptor wich uses the CD4 to get into the cells. The X4-trophism is rare (<1%) but probably Timothy isn't so luckly.

Offline Tadeys

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Re: Berlin patient could still be infected??!!
« Reply #5 on: June 09, 2012, 10:30:58 AM »
1) perhaps the HIV DNA found in the rectum was due to cross-contamination in the lab.
2) or perhaps it was due to contamination in the bedroom (right mecch?).
3) and, if after further testing,  some virus IS found, well, the cure will just have a diferent last name: functional cure, and not sterilizing cure. But a cure non the less.

With a diferent immune sistem, can't seem to figure out how this ( IF TRUE) can happen. Think this is just BS.

Offline Tadeys

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Re: Berlin patient could still be infected??!!
« Reply #6 on: June 09, 2012, 10:46:13 AM »
To skydrake:

Before the transplant, around 2-3% of Brown's viral quasi species were X4-tropic. And if he WAS reinfected with X4, he would be plain positive ( and very stupid on his part), and not just for a few virus DNA up his ars.... :P

Online mecch

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Re: Berlin patient could still be infected??!!
« Reply #7 on: June 09, 2012, 12:26:38 PM »
The article is too complicated for me to understand anyway....
Also, this was never a model for us, for our cure, so, so what....
“From each, according to his ability; to each, according to his need” 1875 K Marx

Offline LM

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Re: Berlin patient could still be infected??!!
« Reply #8 on: June 09, 2012, 03:10:15 PM »
Doesn't matter much, as a sterilizing cure is very unlikely anyway, and scientists are seeking more a functional cure.

Offline freewillie99

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Re: Berlin patient could still be infected??!!
« Reply #9 on: June 09, 2012, 08:12:26 PM »
1) perhaps the HIV DNA found in the rectum was due to cross-contamination in the lab.
2) or perhaps it was due to contamination in the bedroom (right mecch?).
3) and, if after further testing,  some virus IS found, well, the cure will just have a diferent last name: functional cure, and not sterilizing cure. But a cure non the less.

With a diferent immune sistem, can't seem to figure out how this ( IF TRUE) can happen. Think this is just BS.

I'm calling bullshit as well.   There have been numerous articles regarding the ISHEID conference in Marseilles (where this presentation was held) and none mentioned this, if it was indeed true, earthshaking news.
Beware Romanians bearing strange gifts

Offline Tadeys

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Re: Berlin patient could still be infected??!!
« Reply #10 on: June 09, 2012, 11:10:53 PM »
To TM: Brown's cure was "sterilizing". A functional cure would mimic an elite controller: almost no floating virus in blood + high ( normal range) cd4s + no ART.

Offline LM

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Re: Berlin patient could still be infected??!!
« Reply #11 on: June 09, 2012, 11:59:45 PM »
To TM: Brown's cure was "sterilizing". A functional cure would mimic an elite controller: almost no floating virus in blood + high ( normal range) cd4s + no ART.

I think you meant me. And yes, I'm aware of that, but I believe a sterilizing cure is very unlikely to achieve for us, while a functional cure is way more reasonable. So who cares if he still has traces of HIV, that's just food for the media.

Offline Tadeys

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Re: Berlin patient could still be infected??!!
« Reply #12 on: June 10, 2012, 12:25:17 AM »
You are right. It will be more dificult. But it can be done. The stem cells that attack and kill Latent cells infected with HIV being developed at UCLA make me hopefull.  :D

Offline nursepoz24

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Re: Berlin patient could still be infected??!!
« Reply #13 on: June 10, 2012, 10:28:30 AM »
Hopes still up. never give up.

Offline freewillie99

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Re: Berlin patient could still be infected??!!
« Reply #14 on: June 10, 2012, 12:56:34 PM »
that's just food for the media.

LOL.  So true.
Beware Romanians bearing strange gifts

Offline Rev. Moon

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Re: Berlin patient could still be infected??!!
« Reply #15 on: June 10, 2012, 09:27:50 PM »
This has become lak mah favorite thread evah. 
"I have tried hard--but life is difficult, and I am a very useless person. I can hardly be said to have an independent existence. I was just a screw or a cog in the great machine I called life, and when I dropped out of it I found I was of no use anywhere else."

Offline Ann

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Re: Berlin patient could still be infected??!!
« Reply #16 on: June 11, 2012, 06:40:15 AM »
This has become lak mah favorite thread evah. 

You're easily pleased! ;D
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"...health will finally be seen not as a blessing to be wished for, but as a human right to be fought for." Kofi Annan

Nymphomaniac: a woman as obsessed with sex as an average man. Mignon McLaughlin

HIV is certainly character-building. It's made me see all of the shallow things we cling to, like ego and vanity. Of course, I'd rather have a few more T-cells and a little less character. Randy Shilts

Offline red_Dragon888

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OMG!!!


Is The Berlin Patient Reinfected with HIV?- New data to interpret with caution

http://survivinghiv.blogspot.com/2012/06/is-berlin-patient-reinfected-with-hiv.html


I have previously reported about Timothy Brown, aka "The Berlin Patient", the man who was cured of HIV after going through chemotherapy, radiation and two bone marrow transplants donated by a man genetically resistant to HIV (more articles here: articles about Timothy Brown )
Concerning data was presented by Dr Steven Yukl et al  in a talk intitled "Challenges inherent in detecting HIV persistence during potentially curative interventions" at the International Workshop on HIV & Hepatitis Virus Drug Resistance and Curative Strategies held at Sitges, Spain on June 5-9, 2012.

In this presentation, it was discussed that very small amount of proviral DNA (not fully formed virus) had been detected in rectal mucosa tissue obtained from Timothy Brown. This proviral DNA did not resemble that of the original HIV virus that Timothy had before he got cured. But caution about these results quickly emerged as activists in an HIV cure working group in which I am a member discussed the implications of making quick conclusions out of these data.  Unfortunately, controversy has now started.

Richard Jeffreys from Treatment Action Group wrote a great piece as a response to a blog post made by Alain Lafeuillade, who runs the biannual HIV Persistence Workshop. Lafeuillade had  hinted about the fact that Timothy Brown may have been reinfected with HIV. Lafeuillade's press release


It is important that the media understands that these new data do not conclude that Timothy has been reinfected so that no misleading news are spread before more discussions on this matter take place.
http://www.youtube.com/watch?feature=player_embedded&v=I3ba3lnFHik

“Neither look forward where there is doubt nor backward where there is regret. Look inward and ask not if there is anything o

Offline Rev. Moon

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"I have tried hard--but life is difficult, and I am a very useless person. I can hardly be said to have an independent existence. I was just a screw or a cog in the great machine I called life, and when I dropped out of it I found I was of no use anywhere else."

Offline freewillie99

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Re: Berlin patient could still be infected??!!
« Reply #19 on: June 11, 2012, 10:42:33 AM »
This one's for you, Reverend:

http://tagbasicscienceproject.typepad.com/tags_basic_science_vaccin/2012/06/new-data-on-the-berlin-patient-interpret-with-caution.html

New Data on the Berlin Patient: Interpret with Caution

On Friday at the International Workshop on HIV & Hepatitis Virus Drug Resistance and Curative Strategies in Sitges, Spain, Steve Yukl from UCSF presented new data on the case of Timothy Brown, the “Berlin Patient." Yukl described multiple experiments performed by several independent laboratories with the aim of searching intensively for any signs of residual HIV infection in plasma, peripheral blood mononuclear cells (PBMC) and biopsies from the gut and cerebrospinal fluid (CSF). The nature of these analyses is a testament to Brown’s extremely laudable willingness to undergo an array of unappealing procedures in order to advance research into curing HIV.

No infectious HIV was detectable in any sample (including samples containing huge numbers of cells). In most cases, no HIV RNA or DNA could be found either, but there were some exceptions: a minority of samples, analyzed by some labs, intermittently tested positive for extremely low levels of HIV RNA. A very small proportion of the rectal samples also tested positive for very low levels of HIV DNA. Genetic sequencing results were not available but the abstract indicates that the RNA positive samples did not show any relationship with each other or the original infecting HIV (a finding perhaps suggestive of PCR contamination). Levels of antibodies against HIV have continued to decline over 18 months of follow up, while CD4 and CD8 T cell counts have reached near normal levels. The researchers make it very clear that because the assays being used are at the limits of their sensitivity and specificity, it cannot and should not be concluded from these data that Brown is still infected. Although it is possible that there is some residual virus present and that Brown is a case of a “functional cure” rather than complete HIV eradication (or “sterilizing cure”), further work will be needed to explore that possibility. But it is far more likely that—as the study authors state—these new results are just evidence of the technological challenges associated with looking for miniscule amounts of viral genetic material.

Unfortunately, it is all too easy to envision the mainstream media picking up news of this presentation and wildly misinterpreting it (e.g. “Man Said Cured of HIV Still Infected!”). Alain Lafeuillade, who runs the biannual HIV Persistence Workshop and the HIV Reservoir Portal website, has not helped matters by writing a bizarrely misleading post on the study which suggests that the authors interpretation of the data is wrong and that Brown is either not cured, or—in an even stranger piece of speculation—that he may have been reinfected. The evidence supports neither claim.

In a related development, on June 7th the scientist Lawrence Petz held a press conference with Timothy Brown at a symposium in San Francisco on the use of cord blood to facilitate stem cell transplants. Petz revealed that around 100 cord blood donors homozygous for the CCR5 delta-32 deletion have been identified (out of 17,000 tested), and one HIV-positive individual in the Netherlands has recently received such a transplant as part of a course of treatment for another disease. Another similar transplant is to be performed soon for an HIV-positive individual in Madrid. These cases will be carefully followed to see if the beneficial outcome experienced by Timothy Brown can be duplicated.

International Workshop on HIV & Hepatitis Virus Drug Resistance and Curative Strategies, Sitges, Spain, June 5-9 2012.

Challenges inherent in detecting HIV persistence during potentially curative interventions.

SA Yukl1, T Chun2, MC Strain3, J Siliciano4, E Eisele4, R Buckheit4, YC Ho4, JK Wong1, MP Busch5, G Hütter6, DD Richman3, RF Siliciano4, SG Deeks1

1University of California, San Francisco, San Francisco, CA 2National Institute of Health, Bethesda, MD 3Unversity of California, San Diego, San Diego, CA 4Johns Hopkins University, Baltimore, MD 5Blood Systems Research Institute, San Francisco, CA 6Institute of Transfusion Medicine and Immunology, University Heidelberg, Mannheim, Germany

Background. The size of the HIV reservoir during long-term effective antiretroviral therapy and in "elite" controllers is close to the limit of detecting using standard assays. This imposes challenges for the design and assessment of potentially curative interventions. We applied a series of measurements of HIV persistence to the study of the "Berlin Patient", who underwent a hematopoietic stem cell transplant from a donor who was homozygous for the CCR5 delta-32 deletion and who had exhibited clinical evidence of being cured. Our objectives were to (1) determine if HIV had been fully eradicated as a consequence of the transplant and (2) define the potential role of various reservoir measurements in cure research.

Methods. The subject underwent a series of intensive virologic and immunologic studies beginning approximately five years after this transplantation. Replication-competent virus was measured in two laboratories, and HIV DNA and RNA levels (from blood and rectal mucosa) were measured in several laboratories using different approaches.

Results. A large volume apheresis was performed and 9 billion PBMCs evaluated for the presence of replication-competent virus. All wells were negative for HIV p24, indicating that the frequency of replication-competent HIV was therefore estimated to less than one infected cell per 1.4 billion CD4+ T cells. A repeat experiment in a second laboratory confirmed these findings. Using a variety of assays and approaches, very low levels of HIV RNA were intermittently detected in plasma, although sequence analysis of these variants were different from each other and different from those present before the transplant. Digital PCR for HIV DNA was negative for 1 copy per 2 million cells with a 95% confidence limit of less than 1.9 copies per million cells. Collagenase-digested rectal biopsy-derived cells were positive for very low levels of HIV DNA but not RNA; no sequence for confirmatory studies could be obtained. HIV antibodies levels were low and declined over the course of approximately 18 months.

Conclusion. Although the subject has had intermittent evidence for HIV persistence in some assays in some laboratories, the extremely low levels of virus which were detected, while pushing the limits of sensitivity and specificity, and the inability to match sequence with the subject's pre-therapy virus make it impossible to conclude that the subject remains HIV infected.
Beware Romanians bearing strange gifts

Offline Skydrake

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Re: Berlin patient could still be infected??!!
« Reply #20 on: June 11, 2012, 12:06:27 PM »

Conclusion. Although the subject has had intermittent evidence for HIV persistence in some assays in some laboratories, the extremely low levels of virus which were detected, while pushing the limits of sensitivity and specificity, and the inability to match sequence with the subject's pre-therapy virus make it impossible to conclude that the subject remains HIV infected.

In other words: they make a mistake in the lab, trying to use markers too sensitive. False alarm.

Offline Ann

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Re: Berlin patient could still be infected??!!
« Reply #21 on: June 11, 2012, 12:17:03 PM »
There's a reason why RNA and particularly DNA hiv tests are not normally used for diagnostic purposes - they can give false positive results. These results are usually "low levels" (or extremely low) just like these reports are mentioning. That's why I took the OP with a grain of salt.
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"...health will finally be seen not as a blessing to be wished for, but as a human right to be fought for." Kofi Annan

Nymphomaniac: a woman as obsessed with sex as an average man. Mignon McLaughlin

HIV is certainly character-building. It's made me see all of the shallow things we cling to, like ego and vanity. Of course, I'd rather have a few more T-cells and a little less character. Randy Shilts

Offline red_Dragon888

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Re: Berlin patient could still be infected??!!
« Reply #22 on: June 13, 2012, 12:43:14 AM »

http://news.sciencemag.org/scienceinsider/2012/06/evidence-that-man-cured-of-hiv.html

Evidence That Man Cured of HIV Harbors Viral Remnants Triggers Confusion

by Jon Cohen on 11 June 2012, 5:51 PM

Only one person ever has been cured of an HIV infection, and a presentation about the man at a scientific meeting in Sitges, Spain, last week has caused an uproar about the possibility that he's still infected.

Timothy Brown, initially referred to as "the Berlin patient" until he went public about his cure, received unusual blood transplants 5 years ago to treat his leukemia. The blood came from a donor who had mutant cells resistant to HIV. Following the procedure, Brown stopped taking antiretrovirals (ARVs), the virus never returned, and his doctors reported that he had been cured.

But new research presented on 8 June at the International Workshop on HIV & Hepatitis Virus "challenge these results," asserts Alain Lafeuillade of the General Hospital in Toulon, France, a well known HIV/AIDS cure researcher. Lafeuillade issued a press release, "The So Called HIV Cured 'Berlin' Patient Still Has Detectable HIV in His Body," that questions whether Brown was reinfected and may still be infectious to other people. Lafeuillade also posted a blog item, "The Weird Story of the Berlin Patient," raising similar questions.

The scientists who conducted the new study strongly object to Lafeuillade's interpretation of their results. "We weren't trying to say HIV was still there or he hadn't been cured," says virologist Steven Yukl of the University of California, San Francisco, who gave the talk.

Yukl, who works in Joseph Wong's lab, highlighted the difficulties that they and several labs they collaborated with have had determining if Brown truly had eradicated the virus from his body. "There are some signals of the virus and we don't know if they are real or contamination, and, at this point, we can't say for sure whether there's been complete eradication of HIV," says Yukl. "The point of the presentation was to raise the question of how do we define a cure and, at this level of detection, how do we know the signal is real?"

Using sensitive polymerase chain reaction (PCR) tests to scour through 9 billion of Brown's blood cells, Yukl and two other labs have found bits of viral RNA in his plasma. Although Yukl and others who plucked out positive signals believe they are real signs of HIV, Douglas Richman of the University of California, San Diego, another collaborator, found nothing and suspects his colleagues have found contaminants. "If you do enough cycles of PCR, you can get a signal in water for pink elephants," says Richman. Another lab also found HIV antibodies in Brown, though levels were low and declining.

Tae-Wook Chun, of the National Institute of Allergy and Infectious Diseases in Bethesda, Maryland, is one of the collaborators who found positive signals for HIV, but he stresses that no one isolated virus that could copy itself, and suggests Brown may simply harbor harmless, defective genetic pieces of the virus. "We're pushing the limits of detection," says Chun. "At the end of the day it's clear to us that he has some pretty low residual viremia. I don't really know what to make out of it, other than he's controlling viral replication or he doesn't have HIV that can restart the infection. It's a difficult case to talk about."

Ideally, the researchers would have shown that the viral pieces they found matched the virus that was previously in Brown. But no such evidence exists. Chun and another collaborator, Robert Siliciano of the Johns Hopkins University School of Medicine in Baltimore, Maryland, did sequence bits of virus they recovered, but the genetic codes did not match each other—or the virus found in Brown before his transplant. Yukl notes that unlike his own studies, these experiments cloned the virus and ran repeated PCRs. "There's a huge potential for contamination," Yukl says, stressing that would not invalidate his findings of a signal.

To Lafeuillade, the mismatched sequences suggested that Brown may have cleared the virus and then become reinfected. He also wondered why Brown still had any antibodies to the virus at all. "I am just asking scientific questions that nobody seems to be able/or to want to ask," says Lafeuillade in an e-mail.

Richman, who chaired the session at the meeting that included Yukl's talk, says Lafeuillade "completely misinterpreted" a thoughtful presentation and subsequent discussion—and that it in no way challenges whether Brown is cured. "[Brown's] been off ARVs for 5 years," says Richman. "That trumps all these assays."
« Last Edit: June 13, 2012, 12:48:36 AM by red_Dragon888 »
http://www.youtube.com/watch?feature=player_embedded&v=I3ba3lnFHik

“Neither look forward where there is doubt nor backward where there is regret. Look inward and ask not if there is anything o

Offline red_Dragon888

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Re: Berlin patient could still be infected??!!
« Reply #23 on: June 13, 2012, 12:53:30 AM »

Subject: NATAP: Berlin Patient-New Study at Resistance Workshop

The Berlin Patient: New Research Presented at the HIV Resistance Workshop in Sitges, Spain Two Days Ago
Reported by Jules Levin. See below discussion with study author, the Author's Summaries & Conclusions and Background & Methods

Abstract:

Challenges inherent in detecting HIV persistence during potentially curative interventions.

SA Yukl1, T Chun2, MC Strain3, J Siliciano4, E Eisele4, R Buckheit4, YC Ho4, JK Wong1, MP Busch5, G Hütter6, DD Richman3, RF Siliciano4, SG Deeks1

1University of California, San Francisco, San Francisco, CA 2National Institute of Health, Bethesda, MD 3Unversity of California, San Diego, San Diego, CA 4Johns Hopkins University, Baltimore, MD 5Blood Systems Research Institute, San Francisco, CA 6Institute of Transfusion Medicine and Immunology, University Heidelberg, Mannheim, Germany

Background. The size of the HIV reservoir during long-term effective antiretroviral therapy and in "elite" controllers is close to the limit of detecting using standard assays. This imposes challenges for the design and assessment of potentially curative interventions. We applied a series of measurements of HIV persistence to the study of the "Berlin Patient", who underwent a hematopoietic stem cell transplant from a donor who was homozygous for the CCR5 delta-32 deletion and who had exhibited clinical evidence of being cured. Our objectives were to (1) determine if HIV had been fully eradicated as a consequence of the transplant and (2) define the potential role of various reservoir measurements in cure research.

Methods. The subject underwent a series of intensive virologic and immunologic studies beginning approximately five years after this transplantation. Replication-competent virus was measured in two laboratories, and HIV DNA and RNA levels (from blood and rectal mucosa) were measured in several laboratories using different approaches.

Results. A large volume apheresis was performed and 9 billion PBMCs evaluated for the presence of replication-competent virus. All wells were negative for HIV p24, indicating that the frequency of replication-competent HIV was therefore estimated to less than one infected cell per 1.4 billion CD4+ T cells. A repeat experiment in a second laboratory confirmed these findings. Using a variety of assays and approaches, very low levels of HIV RNA were intermittently detected in plasma, although sequence analysis of these variants were different from each other and different from those present before the transplant. Digital PCR for HIV DNA was negative for 1 copy per 2 million cells with a 95% confidence limit of less than 1.9 copies per million cells. Collagenase-digested rectal biopsy-derived cells were positive for very low levels of HIV DNA but not RNA; no sequence for confirmatory studies could be obtained. HIV antibodies levels were low and declined over the course of approximately 18 months.

Conclusion. Although the subject has had intermittent evidence for HIV persistence in some assays in some laboratories, the extremely low levels of virus which were detected, while pushing the limits of sensitivity and specificity, and the inability to match sequence with the subject's pre-therapy virus make it impossible to conclude that the subject remains HIV infected.

---------------------------
from Discussion with study author:

The presentation at the Sitges Resist Wksp is getting attention and the interpretation by some appears to be controversial, so this report is intended to capture the facts.

"We applied a series of measurements of HIV persistence to the study of the “Berlin Patient,” who has achieved an apparent cure after a hematopoietic stem cell transplant from a donor who was homozygous for the CCR5 delta-32 deletion. Our objectives were: (1) to determine if HIV had been fully eradicated as a consequence of the transplant; and (2) to define the potential role of various reservoir measurements in cure research."


The study results reported, as with all studies, are open to interpretation by anyone, so you might hear other interpretations, some of which may get the facts of the studies correct but interpret the findings as they choose, while others may get the facts incorrect. Some observers  may interpret the results as false positives (due to contamination), while others may believe they are true positives. However, this study is the first planned in a series to try to understand exactly what is the case with the Berlin patient. The purposes of this study, as told to me by one of the investigators, Steven Yukl, were to see if they could detect any virus and to better understand the roles and limits of various measurements in evaluating potentially curative interventions. The Berlin Patient could have noninfectious HIV, which would be a "sterilizing cure", or it could be that some of it is infectious but his immune system is resistant to infection and/or controls the virus.   The Berlin patient had 97% CCR5-dependent virus before the transplant & the the donor cells do not produce CCR5, so even if he has some residual virus, most of it would be unable to infect his donor immune system even if it were "infectious" to another person.  Yukl said it's also possible there was contamination associated with the assays and that there is no HIV present; the findings reported from this study are inconclusive. But these researchers plan to continue studying this to try to find out if the Berlin patient received a sterilizing cure or if the immune system he has is controlling HIV, which we would be called a "functional cure".
The facts are that 3 labs were able to detect HIV sequence from 2 different sites (plasma and gut) at 4 different time points, but no sequence data are available from any of these samples.  some or all of them could be real, or they could all be false positives from contamination.  if "real," i think that the virus is more likely to be noninfectious, as only a small percentage of HIV is fully infectious, and 2 labs were not able to detect any infectious virus in co-cultures from blood cells. however, it is worth noting that the co-cultures assess for infectious virus from blood cells (where HIV was not detected in the quantitative PCR assays) rather than plasma or gut (the places where HIV was detected in the PCR assays), and that the  culture assays to detect infectious virus are much less sensitive than the quantitative PCR assays.  he could certainly have intermittent production of very low levels of virus, and it could even be infectious R5 virus, which would not be expected to able to infect his donor immune system, or (much less likely) infectious X4 virus that is controlled by his immune system before it has a chance to spread. the bottom line is that we can't be certain that the detectable HIV is real, and we don't know whether all of it is noninfectious or whether some of it could be relication-competent.  additional studies are needed.  however, he is still able to control virus without medicines ("functional cure"), and he may still have had a "sterilizing cure" (no infectious virus), so i think he is still an example of a very successful treatment.

from poster: Results (Summary)

•CD4+ T cell counts remained fairly stable and within the normal range (Figure 2).

•In plasma, HIV RNA was detected in 2 (of 4) labs from 3 different time points (Table 1).

•No HIV DNA or RNA could be detected in PBMC (4 and 3 labs, respectively; Table 2-3).

•No replication-competent virus could be detected in 2 labs with extensive experience with co-cultures, even with 1.4 billion CD4+T cells from leukapheresis (Table 4).

•HIV DNA was detected in the rectum in 1 lab (Table 5).

•No HIV could be detected in CSF (2 labs) or in cells circulating in CSF (1 lab) (Table 6).

•Three different labs detected HIV (2 from plasma, 1 from gut) at 4 different  time points.  Levels were lower than typical ART-suppressed patients (Table 7) and close to the limit of the most sensitive assays.   Sequence data are not available from plasma or gut.

•Two labs cloned and sequenced HIV from PBMC. However, these sequences bear little resemblance to each other or the pre-transplant variant, and one of the two sequences exhibits near perfect homology to a common lab strain, suggesting contamination.

•HIV-specific Ab levels were readily detectable but tended to decrease over time (Figure 3).


Summary from slides

1)  Most assays for HIV were negative:

–No HIV DNA or RNA was detected in PBMC
–No infectious virus was isolated from peripheral CD4+T cells
–No HIV was detected in CSF fluid or cells

2)  However, 3 different labs were able to detect HIV (2   from plasma, 1 from gut) at 4 different time points:

–Levels were lower than typical ART-suppressed patients and close to the limit of the most sensitive assays. 
–Sequence data are not available from plasma or gut.

3)  HIV-specific Ab levels were readily detectable, but detuned assays tended to show a decrease in Ab over time.



from Poster:

Conclusions

•Reconstitution with an R5-resistant but effective immune system has led to viral control ("functional cure") and possibly eradication of replication-competent HIV ("sterilizing cure"), although the intermittent  detection of low-level virus raises the possibility that there has not been complete eradication.

•The collective data suggest alternative possibilities:  (1) the 4 positive samples may be false positives due to PCR contamination at the extreme limits of assay sensitivity;  or (2) HIV DNA may persist in tissue if not blood cells, and the infected cells may make HIV RNA that can be intermittently detected in plasma.

•Given the nature of the transplant, it is theoretically possible that any persistent virus will be found in non-T cell populations.

•The gradual decline in HIV-specific Ab suggests a slow loss of host Ab-producing cells and lack of progressive infection of the R5-resistant donor immune system.

•The combination of plasma, blood, and mucosal measures of HIV nucleic acid may not be sufficient to prove or disprove cure.  Other measures, including HIV antibody levels, may be required.

Conclusions from slides:

1) Reconstitution with an R5-resistant immune system has led to viral control ("functional cure") and possibly eradication of infectious HIV ("sterilizing cure").  However, the intermittent  detection of low-level virus makes it impossible to conclude definitively that a full cure was achieved.


2) The collective data suggest alternative possibilities:

  A.  The 4 positive samples may be false positives due to PCR contamination at the limits of assay sensitivity

  B.  HIV DNA may persist in tissue if not blood cells, and these infected cells may make HIV RNA that can be intermittently detected in plasma. 

    Further corroboration requires sequencing.


3) Given the nature of the transplant, it is theoretically possible that any persistent virus will be found in non-T cell populations. 



BACKGROUND from poster:

Background

We applied a series of measurements of HIV persistence to the study of the “Berlin Patient,” who has achieved an apparent cure after a hematopoietic stem cell transplant from a donor who was homozygous for the CCR5 delta-32 deletion. Our objectives were: (1) to determine if HIV had been fully eradicated as a consequence of the transplant; and (2) to define the potential role of various reservoir measurements in cure research.


Case Report (Figure 1)


Pre-transplant

•Heterozygous for CCR5Δ32
•HIV for ≥10yrs (on ART for 4 yrs) prior  to diagnosis of AML (FAB M4).  2.9% X4 or dual-tropic

Transplant (Hutter et al NEJM 2009):

•Feb 2007: Treated with chemo, total body irradiation (TBI), conditioning (ATG, cyclosporin, mycophenolate), and allogeneic stem cell transplant (SCT) (day 0) from HLA-matched, homozygous CCR5Δ32 donor.   
•Achieved 100% chimerism.
•ART stopped at time of transplant
•AML relapsed; treated with chemo, TBI, and second allogeneic SCT (March 2008) from the same donor

Post-transplant studies (Hutter et al NEJM 2009; Allers et al Blood 2011):

•HIV RNA undetectable in plasma (45mo) and CSF (17 mo)
•HIV DNA undetectable  in PBMC (45mo), bone marrow (40mo), brain (17mo), and colon (29 mo)
•Loss of HIV-specific T cell responses
•Loss of Ab to Pol and Gag but not Env
•Increase  in peripheral and colonic CD4+T cell numbers to ≥ normal (24mo)
•No CCR5 expression or wild type gene in colonic CD4+T cells (24, 29mo)
•CCR5 detected on colonic macrophages at 5.5 mo but not at 24-29mo.
•No CCR5 expressed in liver (12mo) or brain (17mo)

Study Rationale and Hypotheses


Several theoretical reasons suggest HIV might persist or recur, including:

•Presence of X4 variant prior to transplant
•After transplant, his CD4+ T cells could be infected ex vivo with X4 (but not R5) virus
•Presence of CCR5+ target cells post-transplant (5.5 mo)
•Possibility of long-lived HIV reservoirs or CCR5+ cells not susceptible to conditioning

Hypotheses:

•HIV may persist in rare long-lived cells in tissues that are more resistant to chemo, radiation, and graft-replacement.
•Latent R5 virus may be intermittently expressed at low levels, but will likely not be able to establish a spreading infection.
•If X4 virus persists, it may eventually emerge and spread.

Methods

Procedures to obtain specimens included multiple large volume blood draws, leukapheresis, flexible sigmoidoscopy with biopsies from rectum (30 biopsies),  and a lumbar puncture.

Samples (plasma, PBMC, gut, CSF) were sent to 6 different labs with expertise in detecting extremely low levels of virus: (1) Blood Systems Research Institute (Michael Busch), (2) NIH (Tae-Wook Chun), (3) Solna, Sweden (Sarah Palmer), (4) UCSD/VASD (Doug Richman), (5) Johns Hopkins (Robert Siliciano), and (6) UCSF/SFVAMC (Joseph Wong).


Cell-associated HIV DNA in PBMC was measured in 4 labs using qPCR or digital droplet PCR.
Plasma HIV RNA was measured in 4 different labs using different techniques: (1) Roche AmpliPrep assay, (2) Single Copy Assay (SCA), (3) Transcription-Mediated Amplification (TMA), and (4)  modified Abbott assay that involves pelleting virus from 30ml of plasma.

Cell-associated HIV RNA in PBMC was measured in 3 labs using qRT-PCR or TMA.

Latently-infected peripheral CD4+ T cells (in infectious units per million cells, IUPM) were measured in 2 labs by co-culture.

Cell-associated HIV DNA and RNA were measured from both bulk rectal biopsies and collagenase-digested rectal cells in 1 lab using qPCR and qRT-PCR.

Cell-free HIV RNA in CSF was measured in 2 labs; 1 lab also measured HIV DNA in CSF cells

Blood and gut cells were also sent for various immunology studies, including CD4/CD8 T cell counts, markers of T cell activation, coreceptor usage, HIV-specific T cell responses, and levels of HIV-specific antibodies.   
http://www.youtube.com/watch?feature=player_embedded&v=I3ba3lnFHik

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Offline red_Dragon888

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Re: Berlin patient could still be infected??!!
« Reply #24 on: June 13, 2012, 06:45:03 AM »
The Berlin Patient: New Research Presented at the HIV Resistance Workshop in Sitges, Spain Two Days Ago - (06/12/12)
Traces Of Virus In Man Cured Of HIV Trigger Scientific Debate

http://www.natap.org/2012/HIV/061212_04.htm

NPR (blog) - ‎1 hour ago‎ June 13 2012
by Richard Knox Timothy Ray Brown, widely known in research circles as the Berlin patient, was cured of his HIV infection by bone marrow transplants. Now scientists are trying to make sense of the traces of HIV they've found in some cells of his body.

Traces Of Virus In Man Cured Of HIV Trigger Scientific Debate

by Richard Knox
Audio for this story from Morning Edition will be available at approx. 9:00 a.m. ET
04:41 am
June 13, 2012
 

Richard Knox/NPR
Timothy Ray Brown, widely known in research circles as the Berlin patient, was cured of his HIV infection by bone marrow transplants. Now scientists are trying to make sense of the traces of HIV they've found in some cells of his body.
Top AIDS scientists are scratching their heads about new data from the most famous HIV patient in the world – at least to people in the AIDS community.
Timothy Ray Brown, known as the Berlin patient, is thought to be the first patient ever to be cured of HIV infection.
Brown, 45, had two bone marrow transplants in Berlin in 2007 and 2008 to treat leukemia that is apparently unrelated to his HIV infection. The blood cells for the transplants came from a donor with a genetic mutation that makes his cells immune to HIV — they lack receptors the virus needs to gain entry to cells. Details about his case were published in the New England Journal of Medicine.
The transplants appear to have snuffed out Brown's HIV infection. After an initial spike, the stubborn virus disappeared from his system — even though he is no longer taking anti-HIV drugs.
But new data presented last week in Spain raise a question about whether there are minute traces of HIV in some tissues — not whole virus capable of replicating, but pieces of viral genes.
 
Researchers have combed through 9 billion of Brown's cells, retrieved from his blood, lymph nodes, spinal fluid and intestinal tract. Four different labs could find no trace of HIV in his blood cells. But three groups, using tests at the very limit of detectability, think they have identified bits of HIV genetic material — two from blood plasma and one from intestinal cells.
It could be a false reading, due to laboratory contamination, scientists say. For one thing, the fragments of viral genes don't completely match those of the HIV Brown harbored before his transplants.
"Although the subject has had intermittent evidence for HIV persistence in some assays in some laboratories," the researchers wrote in a summary, "the extremely low levels of virus which were detected, while pushing the limits of sensitivity and specificity ... make it impossible to conclude that the subject remains HIV-infected."
Dr Douglas Richman of the University of California, San Diego, who found no hint of HIV in Brown's cells, thinks the signals are due to laboratory contaminants. If you do enough runs of these ultra-sensitive tests, Richman told Science magazine's ScienceInsider blog, "you can get a signal in water for pink elephants."
Scientists at the University of California, San Francisco, who are following Brown most closely, declined to comment publicly, citing his confidentiality as a patient and research subject.
But AIDS researchers — and Brown himself, in an interview with Shots — stress that even if the new findings constitute real evidence of HIV in his system, they don't mean he's not cured. Although, it's clear the findings do raise questions about what sort of cure he has.
Scientists hoped Brown had a so-called sterilizing cure — that is, the HIV has been completely eradicated from every cell in his body.
But long and bitter experience with HIV has shown that the virus can hide out in the genes of very long-lived resting immune cells. As these latently infected cells get activated over the years, HIV might reappear in the form of the whole virus or perhaps pieces of its genes.
But if that is happening in Brown, there is no evidence that the virus is actively replicating. To do that, it would need to infect other cells and hijack their genetic machinery to crank out more virus. Since Brown's replacement immune system (from the bone marrow donor) doesn't have the entry portal HIV needs, these new viruses (if they exist in his case) can't spark a new viral conflagration.
Therefore, he may be functionally cured, even if he's not totally free of HIV.
That's what Brown himself thinks may be going on, from his discussions with researchers who have been poking and prodding him for the past five years.
"With a sterilizing cure, they have to be sure that a patient is completely clear of HIV — that they've looked everywhere and can't find any," Brown says. "In my case, I still have the dead virus and it's still showing up in some ways and so I've got a functional cure."
To him, that's just as good.
But Brown is distressed at the suggestion by some bloggers — in particular a French AIDS researcher named Alain Lafeuillade of the General Hospital in Toulon — that he's not truly cured.
"It's not the case, but people are spreading it," Brown says. "That concerns me because I've been told by many people that I give them hope — people who have HIV. And that's what I want to do. I want to be able to continue spreading my message and be able to do that without having conflicts of people who are misinterpreting the truth."
Brown is particularly upset at suggestions that he has become reinfected with HIV through unsafe sex. "That is not the case," he says. "It's very difficult for me to listen to those things and read those things."
The latest findings are sure to be debated among AIDS researchers and advocates. Their main significance is to show how tricky it will be to determine exactly what constitutes a cure, as researchers devise various tricks to cure AIDS with less drastic means than bone marrow transplants. The question is, when can they be reasonably sure a cure has occurred?
For his part, Brown says he'll continue to be a guinea pig for as long as it takes if he can help resolve that big question.
"Hopefully one day I won't have to do it any more," he says, because he'll just be one of many cured patients. "That would be nice."




_______________________________________________
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« Last Edit: June 14, 2012, 10:10:45 AM by red_Dragon888 »
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Offline misterprice

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Re: Berlin patient could still be infected??!!
« Reply #25 on: June 13, 2012, 07:16:41 AM »
.....but he isn't immune to X4-tropic strains. Viruses can also use the CXCR4 coreceptor, not only CCR5 coreceptor wich uses the CD4 to get into the cells. The X4-trophism is rare (<1%) but probably Timothy isn't so luckly.

X4-trophism is not rare. Having this mutation does not mean that the virus disappear from his blood, so he will always be infected. However, the virus can't infect his cells due to the mutation so the virus can't deplete his CD4+ T cells.

Offline Solo_LTSurvivor

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Re: Berlin patient could still be infected??!!
« Reply #26 on: June 13, 2012, 12:24:56 PM »
Subject: NATAP: Berlin Patient-New Study at Resistance Workshop

from poster: Results (Summary)

•CD4+ T cell counts remained fairly stable and within the normal range (Figure 2).

•In plasma, HIV RNA was detected in 2 (of 4) labs from 3 different time points (Table 1).

•No HIV DNA or RNA could be detected in PBMC (4 and 3 labs, respectively; Table 2-3).

•No replication-competent virus could be detected in 2 labs with extensive experience with co-cultures, even with 1.4 billion CD4+T cells from leukapheresis (Table 4).

•HIV DNA was detected in the rectum in 1 lab (Table 5).

•No HIV could be detected in CSF (2 labs) or in cells circulating in CSF (1 lab) (Table 6).

•Three different labs detected HIV (2 from plasma, 1 from gut) at 4 different  time points.  Levels were lower than typical ART-suppressed patients (Table 7) and close to the limit of the most sensitive assays.   Sequence data are not available from plasma or gut.

•Two labs cloned and sequenced HIV from PBMC. However, these sequences bear little resemblance to each other or the pre-transplant variant, and one of the two sequences exhibits near perfect homology to a common lab strain, suggesting contamination.

•HIV-specific Ab levels were readily detectable but tended to decrease over time (Figure 3).

Is it possible to provide a link to this study so we can see the missing data tables and figures that accompanied this? Otherwise it makes absolutely no sense to post this information, if it's incomplete.
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____________________________

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Current regimen: Atripla. 
Last regimen:  Epzicom, Sustiva (since its inception with NO adverse side effects: no vivid dreams and NONE of the problems people who can't tolerate this drug may experience: color me lucky ::))
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Offline freewillie99

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Re: Berlin patient could still be infected??!!
« Reply #27 on: June 13, 2012, 08:16:24 PM »
he will always be infected.

Really?  So a few potential pieces of non replicating viral fragments constitute infection?  I had chicken pox as a kid and I'd betcha there are a few floaters left over somewhere inside me.  Does that make me infected as well?
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Offline Rev. Moon

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Re: Berlin patient could still be infected??!!
« Reply #28 on: June 13, 2012, 08:40:41 PM »
Really?  So a few potential pieces of non replicating viral fragments constitute infection?  I had chicken pox as a kid and I'd betcha there are a few floaters left over somewhere inside me.  Does that make me infected as well?

Chile please.  You're wasting your time and keystrokes.  The copypasta queens and the curesters cannot be persuaded.  It is a futile endeavor. 
"I have tried hard--but life is difficult, and I am a very useless person. I can hardly be said to have an independent existence. I was just a screw or a cog in the great machine I called life, and when I dropped out of it I found I was of no use anywhere else."

Offline freewillie99

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Re: Berlin patient could still be infected??!!
« Reply #29 on: June 13, 2012, 08:57:50 PM »
Chile please.  You're wasting your time and keystrokes.  The copypasta queens and the curesters cannot be persuaded.  It is a futile endeavor.

lol...I hear you.  Btw, like that song.  have you seen this clip?  Love it:

http://www.youtube.com/watch?v=lEsPhTbJhuo

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Offline Rev. Moon

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Re: Berlin patient could still be infected??!!
« Reply #30 on: June 13, 2012, 09:35:30 PM »
lol...I hear you.  Btw, like that song.  have you seen this clip?  Love it:

http://www.youtube.com/watch?v=lEsPhTbJhuo


Yeah.  Not a bad version :p.   That single is lak mah favourite choon this year. 
"I have tried hard--but life is difficult, and I am a very useless person. I can hardly be said to have an independent existence. I was just a screw or a cog in the great machine I called life, and when I dropped out of it I found I was of no use anywhere else."

Offline Ann

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Re: Berlin patient could still be infected??!!
« Reply #31 on: June 14, 2012, 07:04:22 AM »
Is it possible to provide a link to this study so we can see the missing data tables and figures that accompanied this? Otherwise it makes absolutely no sense to post this information, if it's incomplete.

Absolutely. In fact, in this forum's welcome thread - which people are supposed to read before posting in here - it explicitly states that links are to be included when copypastaing.

I've posted more about this in the recent "A Request" thread. I suggest everyone reads it - I've also stickied the thread - and takes heed of what I have to say on this matter.
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Offline red_Dragon888

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Re: Berlin patient could still be infected??!!
« Reply #32 on: June 14, 2012, 10:13:56 AM »
Is it possible to provide a link to this study so we can see the missing data tables and figures that accompanied this? Otherwise it makes absolutely no sense to post this information, if it's incomplete.
Done.  I mistook the email as a link.
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Offline Solo_LTSurvivor

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Re: Berlin patient could still be infected??!!
« Reply #33 on: June 14, 2012, 05:42:32 PM »
Done.  I mistook the email as a link.

See post #23. No link?
don't equate intelligence with lack of masculinity
Jim Phelps, Mission Impossible
____________________________

Seroconverted: Early 80s
Tested & confirmed what I already knew: early 90s

Current regimen: Atripla. 
Last regimen:  Epzicom, Sustiva (since its inception with NO adverse side effects: no vivid dreams and NONE of the problems people who can't tolerate this drug may experience: color me lucky ::))
Past regimens
Fun stuff (in the past):  HAV/HBV, crypto, shingles, AIDS, PCP

Jan 2012: 818/21%
Apr 2012: 964/22%
Jul. 2012: 890/21%
Oct. 2012: 920/23%

Still UD after all these years

Offline red_Dragon888

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Re: Berlin patient could still be infected??!!
« Reply #34 on: June 16, 2012, 07:09:35 PM »
See post #23. No link?
That is from a yahoo "Pozhealth" group.  Unless you are a member you cannot see the article, but here you are:

http://health.groups.yahoo.com/group/PozHealth/message/37850
http://www.youtube.com/watch?feature=player_embedded&v=I3ba3lnFHik

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Offline buginme2

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Re: Berlin patient could still be infected??!!
« Reply #35 on: June 16, 2012, 07:56:08 PM »
Paul Sax wrote about this in his blog and his take on the whole story is that it is "unlikely to be of much clinical significance."

http://www.thebodypro.com/content/67537/questions-about-hiv-cure-and-a-very-funny-quote.html



 


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