compiled by n John Clarkson
A Load of what?
A clever process used to solve murders and
work out family ties between long-dead Egyptian mummies is used to
determine how much HIV is in our blood. But what exactly is viral load?
Words Jeffrey Williams
Image Russell Plows
When you receive your CD4 count, your doctor gives the result of a lab test
where someone counted the CD4 cells in a small quantity of your blood. So
how do labs measure your viral load?
Most people know this much: blood is taken from your arm and a couple of weeks
later your doctor tells you your viral load is either ‘undetectable’
or gives you a large number. But a large number of what?
A revolutionary process
Viral load is a measure of the amount of HIV in a quantity of your blood.
The measurement relies on a chemical process called the ‘polymerase
chain reaction’ or PCR. PCR has revolutionized biology in the last 15
years and earned its discoverer a Nobel Prize.
PCR is useful because the genetic material (DNA and RNA) of every living thing
(from humans to HIV) possesses sequences of chemical building blocks that
are combined together in a unique way.
PCR exploits the ability of certain chemical catalysts present in all living
organisms to make exact copies of genetic material. It copies the process
which happens when genetic material is transferred from one generation of
an organism to the next.
Molecular photocopying
Sometimes referred to as ‘molecular photocopying’, PCR can characterize,
analyze, and synthesize any specific piece of genetic material. It even works
on extremely complicated mixtures; finding, identifying, and duplicating a
particular bit of genetic material from blood, hair, or tissue specimens,
from microbes, animals, or plants, some of them many thousands of years old.
HIV, like all living organisms, contains genetic material that it passes on
to future generations when it reproduces. The chemical composition of this
genetic material is now well known but even in someone with advanced disease,
the amount of virus in the blood is difficult to measure. This is where the
PCR process comes into its own. It is able to amplify the amount of genetic
material to such a degree that it becomes quantifiable.
Tagging
To begin the PCR a technician will add a chemical ‘label’ to a
sample of your blood, which contains some HIV and hence some HIV genetic material.
This ‘tag’ is designed to only combine with the genetic material
of HIV. A tagged piece of HIV genetic material is called the ‘template’.
It is this template which is copied. Because the building blocks of the genetic
material only combine together following specific rules, they will only combine
together in the presence of the template to make copies of the original labeled
genetic material. In this way, the original minute quantity of genetic material
in the blood can be amplified to make it measurable.
From one to a million
PCR requires three steps. First, the genetic material is removed from the
virus. Second, the specific tag is attached to HIV’s genetic material.
And, finally, comes chemical amplification. At each stage of amplification
the number of original pieces of labelled genetic material doubles. So starting
from one piece of genetic material, one can make, two, four, eight, 16, 32...
and so on.
All the PCR requires is a reaction tube, the chemicals that are the building
blocks of the genetic material being investigated and a source of heat. Different
temperatures produce best results at each step, so machines now control these
temperature variations automatically. Each cycle of amplification takes only
a few minutes, and repeating the process for just a couple of hours can generate
millions of copies of genetic material. Although the amplification process
is quite rapid, amplification of the genetic material of HIV is run over a
couple of days. So, while a CD4 count in your blood takes a few minutes in
the lab and results are available quickly, viral load measurements take a
lot longer.
Margins of error
As in all laboratory measurements, technical limitations apply to PCR. The
most important is contamination of a patient’s sample with unwanted
genetic material that could also be amplified and generate numerous copies
of irrelevant genetic material. Contaminated amplification can sometimes lead
to wrong conclusions. Labs take special precautions against the accidental
introduction of even the tiniest amounts of a contaminant, especially amplified
genetic material from previous measurement. Preventing
contamination is a special challenge in medicine, where identification of
future illness and decisions about changes of therapy are important.Because
there is potential for contamination in the PCR process, there is a high threshold
of uncertainty in viral load measurements. This is not a criticism of lab
eequipment, but merely a statement of the present state of rapidly evolving
technology.
Uncertainty
Final measurement of viral load is given in ‘copies’ because what
is measured is additional ‘copies’ of HIV’s genetic material.
This is why doctors will talk of a viral load of 100,000 copies. If your viral
load is undetectable it means there is so little HIV present in your blood
that, even with amplification, the PCR reaction cannot produce a measurable
quantity. Undetectable does not mean there is no HIV present, only that your
treatment is working and the amount of virus is very small.
Remember: the uncertainty of viral load measurement is high. Uncertainty on
the final measurement can be as high as plus or minus 50 per cent. So, for
example, if your viral load is 100,000 copies, the actual value could be as
high as 150,000 or as low as 50,000. So when you compare two successive measurements;
for example, 200,000 and 150,000, is there a significant difference between
them? If each has an uncertainty of plus and minus 50 per cent, we are considering
two measurements which lie between 300,000-100,000 and 225,000-75,000. Any
statistician will tell you that it’s very difficult to see any ‘clear
daylight’ between these two measurements with such overlapping margins
of error.
Only together
It’s important to only consider a viral load together with your CD4
count; if one is changing, the other measurement should give confirmation.
Of course, if one viral load measurement is 200,000 and the next is 20,000,
there is clearly a difference and the anti-HIV therapy is working.
Not only has the PCR process made it possible to measure the amount of HIV
in the blood, it’s now an essential tool in forensic science, the so-called
‘genetic fingerprint’. PCR is routinely used to determine the
paternity of a child and has been used to follow the family
relations of ancient Egyptian mummies. Even if the Pharaoh has been dead 3,000
years, there is still enough genetic material present in the embalmed body
to establish if they are related to the mummy in the tomb next door.
