Viral Load and the PCR
why they can’t be used to prove "HIV" infection
by Christine Johnson
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VOL. 4 No. 4
"Biotechnology’s version of the Xerox machine"— that’s what Forbes
magazine called the polymerase chain reaction (PCR). This revolutionary
technique enables a scientist to take a sample containing a minute amount
of DNA and replicate that DNA sequence until there are a million copies
instead of just one or two.
Kary Mullis, inventor of PCR, won a 1993 Nobel prize for his
billion-dollar invention, which has become indispensable to any genetics
lab. It is ironic that one of the first applications of PCR was to detect
HIV, considering that Mullis himself doesn’t believe his invention is
capable of this. Mullis states the problem is PCR is too efficient – it
will amplify whatever DNA is in the sample, regardless of whether that DNA
belongs to HIV or a contaminant. And how do you decide which part of the
amplified material could be HIV and which part the contaminant(s), if you
couldn’t detect HIV in the sample without using PCR?
One of the main arguments against the HIV/AIDS hypothesis is that, when
employing traditional methods of virus detection, HIV has never been
inferred in significant amounts in people with AIDS. Virus culture, for
instance, has been adequate to find other viruses, but not HIV. Why not?
When virus culture is employed to detect HIV, HIV is never seen or even
looked for in the cultures. Its presence is measured by very indirect
methods: assays for detection of reverse transcriptase or a p24 protein,
neither of which is specific for HIV. Indirect methods would not be
necessary if a significant amount of HIV were there to begin with. In
other words, if a meaningful amount of HIV were present, the time-honored
laboratory techniques should be able to find it. They can’t. Now we need
not only PCR, but continuous modifications and improvements on PCR, in
order to try to find HIV.
This is how the idea of "viral load" came about, inspired by two spates of
scientific papers that claimed HIV is busily replicating by the billions:
initially, papers claiming HIV was "hiding in the lymph nodes,"1,2 and
more recently, the Ho and Wei papers.3,4 The latter studies attempted to
measure "viral load" at a given point, after which "antiviral" drugs were
administered to the patient. The drugs were supposed to prevent
replication of any new HIV, and the viral load would decrease accordingly.
However, within a few days, the remaining virus would mutate into a form
resistant to the drugs, and in a few weeks the viral load would return to
its pre-treatment levels. Applying a mathematical formula to this dynamic,
the rate at which the virus replicates was allegedly determined.
Hence was born what I call "Dr. Ho’s kitchen sink theory". According to
Ho, billions of copies of HIV are being made every day, which infect
billions of T4-cells. These T-cells are destroyed not by HIV, but by the
immune system. They are replenished every day, but over the years, the
immune system loses ground and HIV finally wins. This process was likened
to a sink with the drain open, the water pouring in from a tap (new
T-cells being made) at a slightly lower rate than it drained away
(infected T-cells being destroyed).
It is most important to note that the viral load studies all rely
completely on PCR and related techniques. This article will discredit PCR
as an accurate method of determining HIV infection, which will in turn
cast doubt on any conclusions about HIV that have been made based on PCR
techniques.
SOME BASICS ON DNA
PCR takes advantage of certain fundamental properties of DNA. DNA (as well
as RNA) is a nucleic acid, and nucleic acids are composed of nucleotide
"building blocks". DNA exists as two complementary strands arranged in a
double helix formation (two intertwining spirals). These strands are made
up of many nucleotides hooked together to form a long chain of DNA.
The nucleotide molecule has three different parts: the phosphate and the
sugar (which form a backbone or a ribbon-like structure), and the base.
There are four types of bases: A, T, C, and G (adenine, thymine, cytosine,
and guanine). These bases are attached to the backbone, which is wound in
the familiar double helix.
The bases on one strand bind to the bases on the other strand, and this
gives DNA its stable double helix structure. (Think of the two strands as
forming a zipped-up zipper.) The distinct nature of an organism’s DNA code
depends on the order, or sequence, of the bases along the DNA chain.
There are special rules about how bases form chemical bonds with other
bases: an A will only bind to a T, and a C will only bind to a G. A base
on one strand binding to a base on the other strand is called a
"complementary base pair". This rule of complementary base pairing is what
gives DNA its ability to replicate itself exactly.
Each time a cell divides, it has to make a copy of its DNA for the new
cell. The DNA double-strand first "unzips" itself into two separate
strands. Each single strand serves as a template, or pattern, from which
to make a new copy of its complementary strand. (So, strand #1 serves as a
pattern to make a new copy of strand #2, and vice versa.) The single
strand then incorporates new nucleotide building blocks from the
surrounding medium according to the rule of complementary base pairing. In
other words, an available A on the single strand will grab onto a T
nucleotide, a C will grab a G, and so on until the entire opposite strand
is duplicated. At the end of this process, the two original strands zip
themselves up again, and the two copied strands serve as DNA for a new
cell.
How PCR works
The theory of HIV says it, like other suggested retroviruses, contains RNA
but no DNA: when HIV is said to infect a cell, the reverse transcriptase
enzyme is thought to transform the RNA into complementary DNA, which is
then inserted into the host cell’s DNA.
Therefore, if PCR is used to analyse human tissue for the presence of HIV,
it would be looking for only a short segment out of the entire cellular
DNA strand. This short segment represents the genetic material proposed
for HIV, that in theory has been incorporated into the DNA of the cell.
(Viral load studies try to look for cell-free HIV. Even here, PCR is only
looking for part of HlV’s entire proposed genetic package, or genome, not
an entire virus.)
Obviously, it is necessary for the primers to be specific to HIV. Whether
the PCR will make an amplified product (a "positive PCR") depends on
whether the primers you add match part of the DNA in the target specimen.
Below, we will see that the specificity of the primers for HIV is in
doubt. Even if the primers were specific to HIV, if similar sequences are
present in the target, the primers, under lax conditions, will form
hybrids with (or bind) related sequences that are less than a perfect
match.
They will then prime the polymerase, which starts the amplification
procedure, even though no HIV was present to begin with.
PCR works in the following fashion:
Step 1: Heat the template
A long piece of DNA containing the smaller fragment to be copied is
heated. The two strands can be "melted" apart at elevated temperatures,
and will slowly come back together upon cooling ("annealing"). The two
separated strands are complementary to each other. They serve as templates
for the new strands.
Step 2: Add the primers
Something called a primer is necessary for the next step. Primers are
nucleotides that form a short sequence of new strand. Primers are designed
to be complementary to a known sequence which is part of a larger
sequence, and thus where the primers will bind (or hybridise) is known.
The primers attach to each end of the DNA segment that is to be copied
(the segment that represents HIV’s proposed genetic material). The primers
serve two purposes: a) to mark each end of the targeted segment so only
that segment will be amplified, and not the entire strand, and b) to get
the duplication process started. The new strands are built block by block
by the action of an enzyme called polymerase. The polymerase builds a new
DNA strand alongside an existing strand. The polymerase will not work
unless the old strand (the template) already has on it a few nucleotides
forming a short sequenceof new strand (the primer). (If you ever see a
reference to "template-primers," this is what they’re talking about.)
In other words, the polymerase can only form a new strand if the new
strand has already partially been formed. In nature, when your own DNA is
duplicating itself, other enzymes called DNA primases build the primer
onto the old strand.
Once the polymerase gets going, it crawls along the single DNA strand (the
template) adding to it the nucleotide building blocks one by one. The
primer ends up being part of the newly-made strand.
In nature, polymerases pull the DNA strands apart while they build the new
DNA strand. This is how duplicate copies of DNA are made so that cells
like blood and skin cells can divide into two new cells, a process
essential for life.
Step 3: Amplify
Once again, after melting and then annealing the primers, the polymerase
enzyme copies the DNA beginning at the primer, making a new copy of each
target segment. This process is repeated for as many as 30-40 rounds.
During each cycle, the amount of segments doubles, so two segments become
four, four become eight, then 16, etc. By the end of the process,
approximately a million copies of the original segment have been made. Now
you have a whole lot of DNA, where originally you had only a minuscule
amount. This is why PCR is referred to as being able to find a "needle in
a haystack."
USING PCR TO FIND HIV
A problem for the HIV hypothesis was that, even with the use of standard
PCR, researchers could not find much, if any, HIV in persons with AIDS
diagnoses. To resolve this paradox, the authors of the new "viral load"
papers came up with two modifications of PCR, which they claimed were much
more efficient at finding HIV. These were the QC-PCR and the branched DNA
test (bDNA). And suddenly – eureka! – billions of copies of what was
believed to be HIV were found. The contradiction here seems to have
escaped the authors of these papers: Why would such powerful new tests be
needed at all to find a microbe that is present in the billions?
Traditional methods should suffice.
QC-PCR
This is the test used in the above-mentioned papers by Anthony Fauci
(Pantaleo) and Ashley Haase (Embretson), which claimed HIV was "hiding in
the Iymph nodes." These papers were accepted as fact, even though QC-PCR
was, and remains, an unvalidated technique.
Mark Craddock, of the University of Sydney (Australia), explained the
principles of and problems with QC-PCR as follows:8
"PCR mass produces fragments of DNA. You start with a small amount of DNA
and after each PCR cycle, the amount of DNA you have is between one and
two times the amount at the beginning of the cycle. Thus, the amount of
DNA you have to study increases exponentially. The fact that the PCR is an
exponential growth process means that experimental errors will also grow
exponentially, so you need to be very careful about what you do with the
process.
"A number of people have decided that it should be possible to estimate
the amount of DNA present in a sample by using PCR. This is the so-called
quantitative competitive PCR. The idea is to add to the sample to be
estimated a known amount of similar but distinguishable DNA and amplify
both together. The assumption is that the relative amounts of the two
products should stay the same, and hence you can work out the size of the
sample you started with by knowing the ratio of the two, determined by
observation when PCR has produced enough of both to measure, and how much
control DNA was added.
"What is absolutely crucial is that the relative amounts of the test DNA
and your known control must remain exactly equal. Close is not good
enough. The slightest variations will be magnified exponentially and can
produce massive errors in your estimate.
"The difficulties in using PCR quantitatively were pointed out by Luc
Raeymaekers in the journal Analytical Biochemistry in 1993. He noted
published papers on QC-PCR contain data that show that the fundamental
assumption that the relative sizes of the samples remain constant is not
met in practice. Despite this, HIV researchers continue to use PCR to
quantify viral load. There is simply no way of knowing whether a given
estimate is correct or is 100,000 times too high!"
Todd Miller calls QC-PCR the "latest fad in science" and agrees that if
the relative amounts of your test DNA and your known control are not
equal, there is one thing you can say for sure about the estimate of your
starting target (the amount of proposed HIV RNA in the patient’s blood
sample): It will be wrong.
How did QC-PCR, with all its flaws, become an acceptable HIV test? Miller
explains:
"The way this situation has manifested itself in modern science is like
this: First some people spend a lot of time trying to get this test to
work, and if they’re lucky, end up publishing papers about caveats in the
procedure. Second, others happen to get the test to give them an answer
that "makes sense" and publish their data as a significant contribution to
the field. Third, because of its relative newness and arcane nature, it
remains as quasi-accepted with many passive sceptics and a few users.
However, most who use it are more interested in their own pet phenomenon
than in the mechanics of the reaction."
bDNA
BRANCHED DNA PCR
This is the test used in Ho’s paper. Though it is not, strictly speaking,
PCR, it is referred to as such since it incorporates PCR-type technology.
The difference is that bDNA amplifies the signal, not the target. That is,
regular PCR makes more of the target so you can find it, whereas bDNA sort
of shines a bright spotlight on it so you can see it better. Project
Inform was kind enough to send me the following explanation of how bDNA
works:9
"Copies of a DNA probe are attached to the wall of a small laboratory
vessel; then the sample is put in. [A DNA probe is a small piece of DNA
complementary to the target DNA sequence.] This probe binds to a certain
part of HIV RNA, if it is found in the sample, holding the RNA in the
vessel. Then another DNA probe is put in; one end of this attaches to
another part of the HIV RNA. The other end of the second probe has many
branches and each branch ends with a "reporter" chemical that, under
certain conditions, will produce light, which can be detected by
laboratory equipment. Each molecule of HIV RNA can attach to one of these
branching structures and hold on to a small number of light sources, not
just one. In this way, very small amounts of the target RNA can be
detected, without the need for PCR amplification."
In his initial paper, Ho gave no data on the protocols for this test or
whether it was reliable. The reader was referred to two other papers that
were "in press". So, no data was available at that time to anyone who
wanted to verify this method. The data obtained from bDNA was confirmed by
QC-PCR, the details of QC-PCR being set out in a reference authored by
four co-authors of the Wei study, hardly what you might call independent
or objective researchers. In the tradition of HIV research, unproven
theories and faulty studies are accepted without question and incorporated
into the "conventional wisdom" before being properly validated. By then,
the damage is done, and if subsequent flaws are discovered it hardly
matters.
The mechanics of bDNA are complex: Five different hybridisation reactions
are going on. Hybridisation is a standard technique wherein a DNA probe is
put into a sample and will bind to any complementary segments it finds.
It’s another indirect test, and it has a lot of problems. According to
molecular biologist Bryan Ellison, "The only time molecular biology works
is if you purify things first. There’s always the possibility of
cross-reactions, especially when you put your probes into a big soup of
proteins" (which is exactly what the target blood sample is).
Duesberg pointed out the following: After making the appropriate
adjustments to his calculations, Ho himself later found that more than
10,000 viruses inferred by the bDNA assay used in his Nature paper would
actually correspond to less than one infectious virus, leading one to
wonder what it is that is actually being measured on these tests.10 Yet
these speculative and unvalidated papers have been accepted as gospel
truth!
In Ellison’s mind, Ho’s study is "Pure fantasy. There’s never been a paper
that shows viral load."
The Problems with PCR
THE ACCURACY OF PCR HAS NEVER BEEN VERIFIED BY A PROPER GOLD STANDARD
To find out if any diagnostic test for HIV infection actually works, it is
necessary to verify the test with an independent gold standard. The only
proper gold standard for this purpose is HIV itself. In other words, the
results of your experimental test, whether it’s PCR or anything else, must
be compared to the results of virus isolation in each sample tested. If
virus is actually found in each patient with a positive PCR, and no virus
is found in each patient with a negative PCR, then you could say PCR is
extremely accurate for detecting HIV.
The concept of virus isolation as a gold standard is particularly
important in the case of HIV, since HIV has been extremely difficult, if
not impossible, to define in genetic or molecular terms. Even if anyone
had ever accomplished virus isolation for HIV11, it has never been used as
a gold standard for any HIV diagnostic test, including PCR. As it stands
right now, bDNA uses QC-PCR as a gold standard; QC-PCR uses regular PCR as
a gold standard; regular PCR uses antibody tests as a gold standard, and
antibody tests use each other. I have noticed time after time that studies
which are "verifying" an HIV antibody test will invariably state that they
evaluated the performance of their test on samples which were known to be
TRUE-POSITIVE or TRUE-NEGATIVE. How did they know this? It’s simple:
Without a gold standard, they didn’t.
It is sometimes argued that "studies have shown" these tests to agree with
each other or confirm each other’s findings, and therefore they must be
correct. This is not rigorous scientific thinking. Sometimes you can get
the results of different tests to agree with each other, but that does not
prove anything – no more than it would prove if five criminals all agreed
that they were somewhere else when the bank was being robbed.