Why Viral Loads are Useful, Relevant and Important.

A commonly spread myth is that the HIV viral load tests are meaningless, non-specific and arbitrary. This myth is spread based on several lines of logic that are either flawed, misleading, or just downright lies.

Firstly a bit of background as to what a viral load test is. Simply put, a viral load is a measure of the amount of genetic material in the bloodstream, as judged by the relative amount of certain RNA molecules. In the context of HIV, viral load is used as a confirmatory test of infection, but more usually as a judge of how rapidly the disease is progressing. The higher the viral load, the faster the infection is likely to progress to AIDS, and ultimately death. The aim of anti-retroviral therapy is to reduce the viral load to below detection (less than 50 copies per ml with many modern tests).

The Dissidents do not like the test for several reasons, as discussed below.


1: “The VL test doesn’t look for actual virus, it only looks for RNA”.

This is true, but a molecular biologist would simply say “So what?” There is nothing wrong with this, no matter how many dissidents say there is or how loudly they say it. Ultimately unless you physically purify HIV from peripheral blood (see the email from Gelderblom as to why that isn’t likely with HIV) and look at it under EM, you are limited to using secondary detection measures. These will include looking for viral proteins or genetic material, or virus culture. It is a standard procedure for a great many other viruses, and increasingly for some bacteria. This argument is a bit like saying “There’s no proof I was at the scene of the crime, they’re just my fingerprints”.


2: “The VL is amplified many times then a number is back-calculated”

This isn’t really true. It is mind-numbingly obvious though that amplification HAS to be done. In order to visualize the RNA on a standard lab gel you would need to bleed 20 AIDS patients completely to get enough virus to barely see the RNA from all the virus in their bloodstreams (6.5ng). About 200 patients to make it believable at 65ng (a nanogram is a billionth of a gram) – there simply isn’t enough material. The “back calculation argument” though is very misleading. Due to the variability in this kind of amplification (called PCR – polymerase chain reaction) you must have some kind of controls. Typically these are internal controls, whereby samples of known copy-number are run at the same time in the same conditions. Then a graph can be drawn of all the “standards” and the experimental sample is then fitted to the graph. It is a common Dissident tactic to either purposefully (or through ignorance) leave out the fact that the proper controls are always done. It would be impossible to directly count the RNA strands after the amplification process has been done, which is what dissidents imply needs to be (and is) done. Instead, standard molecular techniques are used (where for example the concentration of RNA is shown by how absorbent the sample is for a certain color of light, compared to a control sample).


3. “You only look for a small part of the virus, not the entire genome”

This is true, but again the answer has to be “So what?” The big concern is that if you look at too small a sequence you might by accident discover an RNA molecule that looked similar to HIV in that small sequence. The HIV genome is about 10,000 bases long, and the viral load detects bands that are only a few hundred bases long. In order to get enough RNA to detect (actually DNA, since they copy the original RNA into DNA first because it’s far, far easier to copy and work with) many rounds of amplification have to be performed, around 20. Copying the entire 10,000 base genome would take around 20 minutes to perform per cycle, this means that with the rest of the protocol the entire process would take about 10 hours to complete! While not impossible, by limiting the process to only a few hundred bases it can be complete in an afternoon. However, by arguing that longer stretches need to be used the Dissidents only highlight their ignorance of a vital step in PCR – no matter how long your sequence is, the actual copying process is started by short stretches of DNA called oligos, which are typically 20 or so bases in length,

As such, it makes no difference whether you copy 100 or 10,000 bases, you’re going to have the same degree of accuracy, since the limiting step is the uniqueness of the oligos!

As for the specificity of the oligos, you can get a measure of how specific they are by how long they are. The chances of a single base matching are 1 in 4, since there are 4 different bases in RNA and DNA (A, C, G and T/U). The odds of two consecutive bases matching are 1 in 16. By the time you get to 20 bases you have a 1 in 1,099,511,627,776 chance of matching, or over 350 times more DNA than is in the entire human genome! Obviously certain sequences will be “conserved”, meaning their sequence is more likely to occur than pure chance would suggest. However, despite the existence of retrovirus-like sequences in the human genome (called endogenous retroviruses, HERVs), this is still very unlikely if you pick the right place to look. If you couple that with the fact that you are expecting a product of a certain size (say, 300 bases) you can be fairly sure that no other sequence of DNA, no matter how long, is going to bind to the oligos you use to produce a product of that size.

It is true that the virus RNA is only 1% of the RNA produced by an infected cell. By virus standards though, that it pretty high! That means you really only have to secure uniqueness to 99% of the RNA (if the ratio was the same as the length of genomes, you would need to show uniqueness to a 3 million-fold excess! It is exceedingly unlikely that any RNA detected would be from non-HIV sequences, and any that are would be very low levels. Normally RNA is not found outside the cell in appreciable numbers, whereas of course virus particles are definitely outside the cell! This further increases the likelihood of distinguishing HIV from any other sequence.


4. “The RNA doesn’t equate to numbers of infectious virus”

This is true, and contrary to what the Dissidents say is not assumed to be such by the scientists (an example of them putting words into our mouths). It is well recognized that 1 infectious virus equates to about 60,000 viral genomes per ml. It doesn’t matter, since we’re not trying to say that the virus in the bloodstream is causing AIDS. What?! I hear you cry, surely that’s what they say? No, that’s only what the Dissidents say the scientists say, another example of putting words into our mouths. Viral load is used as a marker because of very good reasons (detailed below) which do NOT include the assumption that the viral load is causing AIDS. In fact, it is very well recognized that the virus in the bloodstream is only a tiny portion of the virus in the body. The vast majority is to be found in lymph nodes, which is where AIDS really happens. Obviously a blood test is far easier than taking a lymph node biopsy, hence the reason why VL measurements are used instead of LN biopsies.

But how much more virus is there in lymph nodes? Far, far more than is in the bloodstream. Maybe 1 in 40 CD4 T cells in the bloodstream contain HIV in end-stage AIDS, far fewer in earlier stages of infection. In the lymph nodes maybe 1 in 4 T cells is infected! Since only 10% of T cells are in the bloodstream, it’s clear that only a small part of the virus replication goes on in the bloodstream. There are plenty of reports suggesting that LN biopsies are used as measurements of disease progression, at least there were in the days before viral load measurements!

On the other hand, it’s obvious that the relative amount of infectious RNA must equate to the viral load. Even with 1 in 60,000 being infectious, if a viral load increases 10-fold then the number of infectious virus must increase 10-fold. A small proportion does NOT equate to NO infectious virus! Having a low infectivity isn’t unusual for viruses (herpes is about 1 in 100), but HIV is admittedly worse than many. However, implying that every copy of the genome should be infectious isn’t right. This point also destroys another Dissident argument: that HIV cannot be found in infected people. Clearly if a ratio of 1 in 60,000 can be measured, is MUST be true that HIV can be cultured from patients! In fact, HIV can be cultured from all AIDS patients, and not from people without HIV antibodies.

In any case, it is known that non-infectious HIV can cause disease! The proteins contained in and on the virus have been shown to cause immunosuppression and cell dysfunction, as detailed in my refutation of Duesberg’s work.


5. “The Viral load is based on detecting RNA that hasn’t even been shown to be that of HIV”

This is blatantly false. Contrary to whatever people like the Perth Group say, HIV has been purified enough to know that the RNA molecule labeled as “HIV” is in fact that of a virus particle. The RNA encodes proteins that co-purify with it (and not cellular proteins), which to any virologist is practically 100% proof that it belongs to a virus. The fact that the Perth Group are not virologists is the only reason why they say the things they do. They quite simply have no idea what they are talking about.


6. “Anti-HIV medications reduce viral load by reducing levels of cellular RNA”

This is ludicrous. Viral loads can be reduced 100,000 fold by antiviral therapy. If cellular functions were reduced by that level you would die in very short order! Any “escapes” from therapy are correlated with mutations in the HIV proteins and RNA. This should not be happening if the effects were non-specific. Contrary to what the Dissidents say, the meds are very specific indeed. 100 to 10,000 more drug is needed to affect cells compared to virus, and that’s with the most toxic medications. The protease inhibitors can not affect cellular RNA or DNA production at all!


7. “False positive results are common”

There are several documented false-positive viral load cases. Some have led to mis-diagnoses. However, they are not as common as is suggested. Viral load is often mentioned in the same breath as p24 antigen testing, which is very clearly a bad test for diagnosis or monitoring of HIV infection. Even with the fact that viral load is a far better test, it is not used for routine diagnosis of infection. It is usually used for judgment of treatment initiation or change, as discussed below. How do these false positive results occur? The simplest explanation is that of accidentally binding to a close match of DNA in the reaction, or contamination in the lab. Due to the sensitivity of the test, detecting contamination is entirely possible. False-positives are usually low levels, around 1000 counts.


8. “HIV DNA detection is very non-specific, why should RNA be any different?”

HIV DNA detection can be non-specific, depending on the oligos used. Some cross-reaction with the endogenous viruses can occur. However, they do not produce RNA or virus particles, since they are usually inactive, so by removing the DNA and using only extra-cellular virus particles you reduce the likelihood of detecting these endogenous particles. I have seen it written that the sequences looked for: Gag and Env are “specific to HIV”. No-one with any experience with virology at all would say that: Gag and Env are simply names for genes that encode the structural proteins of a retrovirus. Every retrovirus in existence has its own version of Gag and Env: it is not surprising that depending on how you look you can confuse HIV Gag and HERV-K Gag, for example.


9. “Viral load improvements with therapy are only due to placebo effect”

Placebo means “it pleases me”. It is a psychological phenomenon. It cannot explain molecular measurements like viral load, unless they were measuring some kind of stress hormone. It also doesn’t explain why people with naturally low viral loads have strong anti-HIV immune responses, and why rising viral loads match with mutations that either escape the drugs or the immune response.

10. Viral load levels predict disease progression. This is the biggest anti-Dissident point of them all. In many studies, it is clear that the best predictor of how soon someone will progress to AIDS is how high their viral load is. This fact makes the level of viral load very important, even if it weren’t measuring infectious virus, or even HIV at all! This is undeniable. A viral load of over 30,000 equates to a 50% risk of progression to AIDS in 2 years, independent of CD4 count. Lowering viral load does correlate with reduced mortality and opportunistic infections. Lack of control of viral RNA correlates with worse outcomes. What more is there to say…?

Purifying HIV

Not too long ago I got in touch with Hanz Gelderblom, a renouned electron microscopist. The Perth Group have tried to use some of his review articles to state that HIV should be visible under EM of plasma samples. Since no such EM has been published, therefore HIV cannot exist (a ludicrous statement however you look at it). Hanz however has more specific things to say...

(Paraphrased at his request from the email)

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Despite being successful at getting EMs of HIV from cultures, in parallel experiments using plasma samples he could not get enough virus. This was because they only recieved two samples of less than 0.5ml of blood from late-stage patients, when ideally it should have been far more from the seroconversion stage when virus titre is higher. He also attempted to get virus from Africa but couldn't get samples. Collaborations with groups from Sweden and Italy also failed because no samples of sufficient titres could be obtained. From experience, he thinks he could have been successful with particle concentrations of 10^10 per ml, about 10,000 times higher than what we here [at the BMJ Rapid responses] thought would suffice. Having tried for some time on this path, he gave up attempting to isolate HIV from blood about 10 years ago.

There are several logistical problems associated with EM of HIV from blood samples as well. The best way to ensure particle stability "in the field" is to fix the sample in higher then 0.5% Glutaraldehyde (GA), to prevent shedding of gp120 knobs. However, this level of GA prevents immunolabelling which he wanted to do to characterise virally-incorporated cellular MHC molecules, but could also be used to label virion proteins. As such there is almost a mutually exclusive situation where you can either keep the virus intact, or analyse it using immunoEM. He did attempt to look at virus from pelleted samples (rather than the 2-drop method mentioned in the previously quoted article) which would have massively concentrated the sample, but even this failed due to low titres of virus (see my earlier postings for calculations of virus needed to fill a 1mm cube).

He also is of the opinion (and a highly experienced opinion it must be) that any attempts to purify or enrich HIV from plasma will induce artifacts, such as changes in virion fine structure, shedding of gp120, osmotic damage etc. Obtaining particles with HIV morphology might therefore be a practical impossibility (much like trying to observe the position and speed of a subatomic particle simultaneously).

He does however admit that maybe plasma-derived HIV is more stable than that of cell-culture derived HIV, and gives the example of Avian Leukosis Virus as a comparison. We simply do not know, because no-one has been able to do it. He feels that direct isolation of HIV from blood samples might well be possible to do, and should be done since it would give a more realistic research material to study (not, notice, in order to prove its existence!).

I am deeply grateful for Dr Gelderblom for taking the time to write
to me, and for giving me permission to quote him online.

***

Calculations performed regarding the size of HIV particles:

4/3*pi*r^3 for a 120nm diameter particle, into a 1mm cube gives a little over 1 trillion particles. Average blood volume taken to be 5 liters.

The size of the particle is such that 200 AIDS patients with viral loads of 1 million per ml would need to have their entire circulating blood volume concentrated to produce enough virus to fill a 1mm cube (a grain of salt). Viruses are small, even if they can be prolific. The total RNA purified from that (bearing in mind that each virus contains 2 copies of the genome) is about 65 nanograms, or only just enough to visualise on a standard ethidium gel. Asking for an EM from a virus that hasn't been cultured or RNA from a virus that hasn't been amplified by PCR (or culture) is simply setting up a straw-man argument.

Woohoo!

My first dissident arrives! Welcome, and stay silent. It's interesting to get the attention.

As soon as I organise some more ideas I'll get another refutation up, but for now I'll just enjoy having just a little bit of power over my tiny portion of the Web :o)

Anti-Dissident Site

I was recently put in touch with a vocal anti-Dissident, which is a refreshing change. All too often I find that the scientists are too quiet and those who don't know what they're talking about are too loud...

Check out DaveyBoy's link above or in the Links section (he was kind enough to link back here!). He's done some impressive work pulling a lot of sites together so deserves more than a little recognition. While not a scientist he has first-hand experience of HIV and is certainly aware of the problems that Dissidents can cause, most relevant being their ongoing attacks on orthodox support groups and information forums. How heinous can you get...

A refutation to some of Duesberg's Stuff

Prof Peter Duesberg is a retrovirologist who did some of the best work on Rous Sarcoma Virus. He mapped the src oncogene, a piece of work that was incredible considering the lack of molecular techniques available at the time.

However, he applies his understanding of simple retroviruses to HIV, a complex retrovirus (complex because it contains accessory genes) and concludes that HIV cannot cause AIDS. Moreover, he goes onto say that drug use causes AIDS.

Here is my personel rebuttal to Duesberg's anti-HIV argument.


Pharmac. & Ther. Vol. 55: 201-277, 1992
AIDS ACQUIRED BY DRUG CONSUMPTION AND OTHER NONCONTAGIOUS RISK FACTORS
PETER H. DUESBERG


3. Discrepancies Between AIDS and Infectious Disease

3.1.Criteria of Infectious and Noninfectious Disease

Based on common characteristics of all orthodox infectious diseases, infectious AIDS would be predicted to:

(1) Spread randomly between the sexes. This is just as true for venereal as for other infectious diseases (Judson et al., 1980; Haverkos, 1990).

******

Except a new infectious agent that spread into the Western world (which, lets face it, is all Duesberg is talking about) through the homosexual population. Spread to women and heterosexual men would be expected (and was observed) to be slow and incomplete.

HIV spread is equal among the sexes in the heterosexual epidemic in Africa.

******

(2) Cause primary disease within weeks or months after infection, because infectious agents multiply exponentially in susceptible hosts until stopped by immunity. They are self-replicating, and thus fast acting toxins. (Although "slow" viruses are thought to be pathogenic long after neutralization by antiviral immunity (Evans, 1989c), slow pathogenicity by a neutralized virus has never been experimentally proven (Section 6.1).)

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HIV causes an acute seroconversion illness in about 50% of those it infects. This is like most other viral illnesses, since the symptoms are not caused by the virus but instead by the release of cytokines such as IL-2 and the Interferons, which produce constitutional unwellness.

Also he is assuming that the presence of antibodies to HIV means that they are _neutralising_ antibodies, and yet he makes no effort to prove this. The fact that HIV can be detected even after an antibody response might argue against this, but doesn't take into account direct cell-to-cell spread. Many viruses persist despite antibody recognition, and antibodies do not have to be neutralising, some are in fact pathogenic. (Mitler and Hoffmann: Science 1989) Examples are HSV, EBV, VZV, Hep B. The herpes family maintain their genome in host cells, and their latency is less well understood than that of HIV. At least we know that T cell activation results in HIV expression (the same transcription factors, such as NF Kappa B are used) while for the herpes viruses we don't even have that handle. Hep B is a reversivirus, related to the retroviruses by its use of reverse transcriptase. It doesn't integate into the genome. However, it's persistance is also a mystery - there is a vigourous immune response, which results in the liver destruction which gives this virus its name (the virus is not in fact cytotoxic to liver cells). It also produces massive quantities of non-infectious particles, so much so that the viral particles have a special name (Dane particles) to distinguish them from the rest of the junk found in the serum. These may act as decoys for the virus against the immune response (ref for most of the above: Fields Virology, or any other virology textbook - Duesberg should have known all this).

Also, it is known that CD4 counts plummet during this acute stage, from ~1000 to ~500 (the lower end of normal) before picking up again as HIV-specifc immunity appears (Pantaleo et al: NEJM 1993 review). While these levels are not going to result in opportunistic infections appearing, they are a definite sign of things to come.

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(3) Coincide with a common, active and abundant microbe in all cases of the same disease. (Inactive microbes or microbes at low concentrations are harmless passengers, e.g. lysogenic bacteriophages, endogenous and latent retroviruses (Weiss et al., 1985), latent herpes virus or latent ubiquitous Pneumocystis and Candida infections (Freeman, 1979; Pifer, 1984; Williford Pifer et al., 1988). Hibernation is a proven microbial strategy of survival, which allows indefinite coexistence with the host without pathogenicity.)

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There is evidence of HIV virus particles in 100% of AIDS cases and over 99% of those with anti-HIV antibodies (Jackson et al: J Clinical Molecular Biology 1988 and 1990). The low level of HIV in the plasma of infected people (often to uncultureable, though not undetectable levels) after the first week/fortnight (Piatak: Science 1993, Pantaleo et al: NEJM 1993 review) suggests that the immune system is actively hindering HIV replication, especially since antibody titres to HIV go up reciprocally. As for abundant microbe - tetanus toxin from clostridium tetani bacteria can and does kill in quantities too low to mount an immune response to. This also feeds into the following claim...

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(4) Coincides with a microbe that lyses or renders nonfunctional more cells than the host can spare or regenerate.

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And/Or hinders the regeneration of those cells. HIV reduces the CD4 cell survival time and hinders replacement from the thymus. The effect of HIV is reversed by antiviral therapies. (Duoek el al: Nature 1998, Hellerstein et al Nature 1999) HIV is cytotoxic (Yelle et al: Archives of Virology 1994, Rasheed et al: Virology 1996, ), and may be immunosuppressive even in the absence of active infection (Diamond et al: J immunology 1988, Weinhold et a l: J immunology 1989, Daniel et al: Clinical Experimental Immunology 1993, Liegler and Stites: J AIDS 1994, Theodore et al: J AIDS 1994, Schols and De Clercq: J Virol 1996, etc - I haven't even started on the accessory proteins of HIV). The immune response to the virus will, of course, attack the immune system itself. As such the number of cells the virus actually kills by infection need not be the limit of the immune dysregulation. Direct in vivo evidence of this lack of correlation between cytotoxicity and immune suppression exists in chimps (Wantanabe et al: J Virology 1991).

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(5) Generate a predictable pattern of symptoms.

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Longitudinal studies shown that after ~15 years 90% of those with HIV infection with progress to a stage of gradual immune decline, with increased risk of opportunistic infections, a specific supression of cytotoxic immune responses with (usually) a simutaneous rise in antibodies.

********

Duesberg also moans that HIV cannot possibly be neurotoxic and a cause of AIDS dementia, since the retroviruses he worked on didn't infect non-dividing cells. He seems to ignore the fact that HIV does infect the non-dividing T cells, an ability later ascribed to the vpr protein found
in HIV, but not in the simple defective retroviruses he worked on. He also doesn't know that HIV's neurotoxicity is due to infection of the CD4+ astrocytes in the brain, and subsequent loss of neuronal support leading to cell death. Again - a non-direct method of disease.


There is plenty more of course, but based on this logic the argument that HIV cannot be the cause of AIDS seems untenable to me.

AZT, not toxic enough

One very key part of the AIDS dissent is concerning the antiretroviral drugs. In particular, due to an early series of papers by Prof Peter Duesberg, AZT (Azidothymidine) has been targetted. It is said to be an anti-cancer drug that was canned due to being too toxic. This is false for lots of reasons.

The original inventor of AZT, Dr Richard Beltz, is quoted as saying that AZT was in fact inactive against cancer cells in the lab and in mice. This quote comes from an email to an AIDS dissident (see link) - what better source can you imagine?

"It proved to be completely inactive in all of the test systems [Dr Sartorelli] employed. In my laboratory I found AZT incapable of inhibiting the growth of Jensen sarcoma cells in vitro at very high concentrations. Thus, AZT showed no activity as a potential anticancer drug at that time."

As regards various quotes that have Dr Beltz saying that "AZT was shelved for two reasons: My studies showed that it caused cancer at any dose and it was too toxic even for short term use":

"Now let me say that I am aware of the existence of certain quotes attributed to me on the Internet, such as the one you mentioned in your letter. Such quotes are completely untrue!"

Duesberg also makes an assumption that AZT was stopped due to toxicity. From his book "Inventing the AIDS virus".

"However, when he tested the compound on cancer­ ridden mice, it failed to cure the cancer. Horowitz was so disappointed he never bothered publishing the experiment and eventually abandoned that line of research. The drug MUST HAVE killed the tumors, which contain dividing cells, but it so effectively destroyed healthy growing tissues that the mice died of the extreme toxicity." [Emphasis my own]

Duesberg also says that even with enzyme specificity there is so little viral DNA compared to human DNA that it must kill many more healthy cells than virally-infected cells. The evidence doesn't bear this out, with up to 100-10,000 times more drug needed to kill cells compared to virus. [refs 1-4 below]

Some have said that AZT is currently used in cancer therapy. A drug that has the same abbreviation, Azathioprine is used in treating leukaemias, but it is not Azidothymidine.

All in all, far from being a toxic anti-cancer drug that was abandoned due to toxic effects, it doesn't kill enough cells. It is, unarguably, a nasty drug. Many (perhaps a quarter to a third) of patients require blood transfusions while on it, at least they did on the older doses which were twice those of today. However, it is quite clearly not, and never was, an anti-cancer drug.

Refs:
(1) J Virol 2002, 76(12):5966-73

(2) J Biol Chem 1989, 264:6127-33

(3) Antimicrobial Agents and Chemotherapy 1990, 34:637-641

(4) NEJM 1987, 317:192-197

First Post!

How original, title wise, but what the hey.

Since 1998 I've been involved with discussing the HIV/AIDS debate, that is the actual debate over whether or not HIV causes AIDS. Believe it or not, such a debate has existed in various forms since 1983.

From what I've seen, aside from conventional websites, nearly nothing has been done to directly address the various arguments used by the AIDS Dissidents.

This is my attempt to redress that.