Reprinted from Continuum Magazine Vol 3 No 5; Revised June 2004
Testing, Testing… Is a Positive Antibody Test Proof
of HIV Infection?
By Valendar Turner, MD
Department of Emergency Medicine,
Royal Perth Hospital, Perth, Western Australia
“An antibody test, even if repeated and found positive
a thousand times, does not prove the presence of a viral infection.
The failure to verify the antibody tests against the gold standard
of virus isolation is a serious omission of scientific method…”
What evidence authenticates a positive HIV antibody test as proof
of HIV infection? This question has greatly interested me since
those of us working in Emergency Medicine spend a considerable part
of our lives exposed to other people's blood and body fluids, a
circumstance that according to the experts places us under constant
threat of AIDS. If the experts are right, the life we save may cost
us our own. Given this ironic and sobering circumstance, some of
us have pursued the question of proving HIV infection to the very
From the early days of AIDS I was fortunate to collaborate with
Eleni Eleopulos, a Biophysicist at the Royal Perth Hospital, John
Papadimitriou, Professor of Pathology at the University of Western
Australia, and other colleagues. In one of our papers published
in June 1993 in the journal Bio/Technology , we were compelled
to confront many unsettling conclusions about the HIV antibody tests,
none of which accord with current wisdom, and some of which I share
in this article
The HIV antibody tests do not detect a virus. They test for any
antibodies that react with an assortment of proteins experts assure
us are unique to HIV. As almost everyone agrees, HIV is a retrovirus
and the cause of AIDS .
When you take an HIV antibody test, what happens is this: A sample
of blood serum is incubated with a mixture of these proteins in
a test called an ELISA, an acronym for Enzyme Linked Immunosorbent
Assay. The ELISA is positive if the serum solution changes colour.
A color change indicates a reaction between the proteins in the
test kit and the patient's antibodies. However, according to many
experts, the ELISA is not specific, meaning it may react in the
absence of HIV infection.
In response to this, testing authorities have developed strategies
such as repeat testing of all positive ELISAs and following up those
twice positive with a third but different antibody test known as
the Western Blot. In the Western Blot, the "HIV proteins,”
about ten of them, are located at discrete spots in a paper strip
rather like the one your doctor uses to perform multiple tests on
your urine. Serum is added and wherever there is a reaction a colour
change occurs which shows up as a dark band. The test is read by
noting which bands show up, in other words, which proteins react.
Certain combinations of bands are defined as a positive test.
It is enigmatic that the location and number of bands required for
a positive Western Blot varies around the world. They may even vary
between laboratories within the same city. In Australia, four bands
are required. In Canada and much of the United States, three bands
suffice, while in Africa, two will do. In the US Multicenter AIDS
Cohort prospective study involving several thousand gay men, just
one "strong band” was deemed sufficient to earn a positive
If each of the above criteria indicates HIV infection, then HIV
must cause different populations of antibodies to appear in different
places. I don't know about you, but to me that sounds very odd.
At the least it gives some Africans a way out. All an African has
to do is have a test in Australia because two bands would not be
considered positive here. Nevertheless, in spite of lack of standardization
and other problems such as reproducibility, the Western Blot is
accepted to be in excess of 99.9% specific and if positive is regarded
as synonymous with HIV infection. In some countries, similar claims
are now made for the HIV ELISA without “confirmation”
by Western Blot.
The rationale for the use of antibody tests is as follows: The immune
system has the ability to detect foreign agents and to respond by
producing antibodies that react with those agents. However, this
does not work in reverse. By that I mean the observation of an antibody
reaction with a particular agent is not automatic proof that the
antibody was produced in response to that agent.
The problem is that antibodies indulge in casual and indiscriminate
relationships. They are in fact promiscuous. Antibodies meant for
one agent may react with another agent, a perfect stranger. Or,
if you want it put technically, there is ample evidence (some of
the best in fact comes from the Pasteur Institute) that antibody
molecules, even the most pure (monoclonal antibodies) are not monospecific
and cross-react with other, non-immunising antigens.
Here are some examples to illustrate this most crucial fact. Firstly,
in a study of 1.2 million applicants for US military service ,
of the 1% or 12,000 who had first time positive HIV ELISAs, only
2000 were ultimately shown to be also WB positive and thus, according
to the authors, HIV infected. That left 10,000 positive ELISAs which
must have reacted for reasons other than "HIV antibodies",
a fitting testimonial to the problem caused by cross-reacting antibodies.
Secondly, there is the tantalising data reported in 1990 about dogs.
Writing in the journal Cancer Research, Strandstrom and colleagues
reported that 72/144 (50%) of dog blood samples "obtained from
the Veterinary Medical Teaching Hospital, University of California,
Davis" tested in commercial Western Blot assays, "reacted
with one or more HIV recombinant proteins (gp120--21.5%, gp41--23%,
p31--22%, p24-- 43%" . Assuming California dogs are not
infected with HIV (as did the authors), one must conclude these
data are further proof of antibody cross reactivity to many of the
What all this means is that you're not necessarily infected with
what your antibodies appear to tell you. This can be brought home
by two further examples. Firstly, some AIDS patients have antibody
reactions with laboratory chemicals but no one claims AIDS patients
are infected with laboratory chemicals. Secondly, as an example
removed from AIDS, the antibody test for glandular fever relies
on the fact that patients with glandular fever make antibodies that
react with the red blood cells of sheep and horses. But these patients
are not infected with animal blood and animal blood does not cause
glandular fever. Bearing all these examples in mind it is painfully
obvious we cannot pronounce someone infected with what is regarded
as a lethal human retrovirus merely because we observe an antibody
reaction. Before we pronounce any such reactions indicative of HIV
infection and long before we introduce the test into routine clinical
practice, we must exact solid proof of precisely why these reactions
take place. In doing we must not forget that biology is not mathematics
and despite our clever technology, in biology still we must stoop
to the relative ignominy of empirical proofs. Or, as Plato said,
"experiential data must always be interpreted in the light
of what Nature has revealed".
In science, we must constantly resist the temptation to stray beyond
our data, and in that spirit I put it to you there are only two
pieces of information that can be gleaned from an antibody test
(for mathematical purists, it's only one piece of information).
Either you see a reaction or you don't. That's all. You don't see
antibodies with labels attached saying what produced them. You cannot
construe the genesis of antibodies by observing changing colours
in a test-tube. The cardinal problem scientists face when ascribing
meaning to a set of antibody reactions is how can they tell whether
the reaction is caused by a real antibody or an imposter? One whose
proper partner is something else? In this context it is proper for
a disinterested scientist to allow for the possibility that there
are no real HIV antibodies whatsoever, that they're all pretenders.
When the only information is a reaction, and that reaction has more
than one possible cause as is the case with an antibody test, you
need extra information before you can ascribe a particular outcome.
So, if you want to claim an antibody reaction signals a particular
outcome such as HIV infection, first you have to prove it. And just
before we get to crunch time, consider this little morsel: AIDS
patients are exposed to many foreign agents are known to have antibodies
reacting with dozens of different substances and it makes perfect
sense that the more antibodies there are the more chance there will
be some that will spoil the test. What this means is that in the
very patients you suspect of harboring a virus there exists the
precise circumstances, lots of potentially cross reacting antibodies,
which make it imperative to sort out what is really going on.
What's the solution or, more to the point, what's the problem? The
problem is how do you know when you witness an antibody reaction,
that is, a positive test, that HIV itself is present, too? After
all, that's what you really want the test to tell you and the question
on the patient's lips is bound to be "Is HIV infection the
only cause of a positive test? If it’s something else I'd
rather have that, thank you very much". In technical terms
the patient's hopes are hanging on the specificity of the test.
Let me explain specificity and what is meant by 100% specificity.
One hundred per cent specificity means that positive tests only
occur in HIV infected people. That's the same as saying positive
tests never occur in uninfected people. And that's the same as saying
all uninfected people have a negative test. This leads us tothe
formal, mathematical definition of specificity. Specificity is the
number of negative tests in a large group of individuals who do
not have HIV infection. If 100% of one thousand people who do not
have HIV infection test seronegative, the specificity is 100%. If
one uninfected person tests seropositive, the specificity is reduced
to 999/1000 or 99.9% by virtue of a lone false positive. Thus, to
determine the specificity of an antibody test we need two pieces
of data. The numbers of persons with negative tests, and the numbers
of persons with no HIV infection. By the way, the false-positive
rate is (1-the specificity). An experiment to find the specificity
also gives the false positive rate and vice versa.
How should we design an experiment to discover this important data
Firstly, since the underlying problem is largely one of deciding
between bona fide antibodies and cross-reacting antibodies, we must
include in our sample persons who are likely to have a large repertoire
of antibodies to agents other than HIV. The more the merrier. Thus
we must include persons who are sick and who have diseases similar
to AIDS but do not have AIDS.
Secondly, we need a way of determining the presence or absence of
HIV infection. Obviously, this can't be the antibody test itself
because that's what we're trying to validate. When we measure specificity,
we are trying to find out how often reactions occur in individuals
who do NOT have HIV infection. Rather surprisingly, in the AIDS
literature, the specificity of the HIV antibody tests has been evaluated
by testing for reactions in healthy individuals such as blood donors.
These persons are chosen as de factos for the absence of HIV infection.
Under such circumstances, few if any positive reactions are found
but this is not necessarily, as the HIV/AIDS experts claim, because
the tests are highly specific. In fact, this is the wrong experiment
for two reasons.
Firstly, healthy people do not have large number or variety of antibodies
to react with the test. That goes with being healthy. That's why
we put them in the Army and let them donate blood. There are simply
not enough antibodies available to measure the propensity for unwanted
reactions. Secondly, good health cannot be used as a de facto for
the absence of HIV infection any more than good health can be used
as a de facto for the absence of gall stones, kidney stones, pregnancy,
hydatid cysts, deep vein thrombosis, cerebral aneurysms, pathogenic
bacteria or coronary artery disease.
The practice widely adopted by HIV/AIDS experts of appraising HIV
antibody tests by testing thousands of healthy blood donors also
creates an enormous dilemma: If healthy people are regarded as a
de facto gold standard for the absence of HIV infection, by what
criteria can similarly healthy people or even the same individuals
be regarded as infected at some future date?
But back to the problem of validation. We select our thousand people
who are sick. And let's make sure we include some who have diseases
similar to AIDS, a few healthy persons, and some cases of AIDS as
well—you never know, we might be in for a big surprise. We
might find some AIDS patients who are antibody positive in the absence
of HIV infection. In fact, if you read Gallo's May 1984 Science
papers that claims HIV was proven to be the cause of AIDS, HIV could
be "isolated" in less than half the AIDS cases.
Most of the people selected will have lots of antibodies and this
will give the test a fair run for its money. But hold on. If HIV
causes AIDS, and some of our patients have AIDS-like diseases—even
those who are healthy—how do we get past the sticky problem
of knowing who is or is not infected with HIV? We don't want to
include those who may actually be infected in our analysis because
we want to evaluate the test when there is no HIV infection.
By now, some of you will have arrived at the correct solution to
the problem of distinguishing who is and who is not infected. It's
rather obvious scientifically speaking—you have to use HIV
itself. You must divide your blood samples from each person in two:
One sample to test for the antibody reactions and the other to try
and isolate HIV. To know what the HIV antibody tests tell you about
HIV infection, you compare the reactions with what you are trying
to measure. The only way to distinguish between real reactions and
cross-reactions is to use HIV isolation as an independent yardstick
or gold standard.
What are the results of such an experiment? How many of an appropriately
chosen 1,000 patients from whom HIV cannot be isolated have an antibody
reaction? How many of the 1,000 from who HIV can be isolated have
no reaction? What does the medical literature tell us about this?
What does the data accompanying HIV antibody test kits say? I can't
tell you because—bizarre as it may seem—17 years since
the discovery of HIV and 15 years since the development of the HIV
antibody tests, this experiment has not been done. We still don't
know how many positive tests occur in the absence of HIV infection.
It might be none or it might be all. Nobody knows. There is no proof
of the specificity of the HIV antibody tests for HIV infection.
What if someone decided to do this experiment to prove the specificity
of HIV tests? Is it feasible? That's hard to say because it depends
on how much importance you place on the precision of defining HIV
infection. Ultimately, this can only be defined by the isolation
of a unique retrovirus. The word isolation comes from the Latin
word "insulatus" meaning "made into an island".
It refers to the act of separating an object from everything else
that is not that object, like solitary confinement.
The rules of retrovirus isolation are now old. All the HIV experts
should know them. They were developed in the several decades preceding
the beginning of the AIDS era in 1981 and were thoroughly discussed
at a meeting held at the Pasteur Institute in 1973 and attended
by now leading HIV/AIDS researchers including Barre-Sinoussi and
Chermann. These are a set of rules that credibly achieve the aim
of separateness. The problem is that no claim of HIV isolation yet
presented fulfils either the island concept or follows these rules.
None of these claims even fulfils the initial and most basic of
these rules, the requirement to obtain an electron micrograph of
the material that is present at a sucrose density gradient of 1.16
In fact, no claim of HIV isolation is isolation. All such claims
are based on a set of phenomena ranging from so-called HIV proteins
such as p24, to reverse transcriptase enzyme activity, "HIV
particles,” or “HIV PCR” that are detected in
cultures of tissues of AIDS patients, none of which is even specific
for retroviruses. And without isolation, who can say whether the
proteins used in the HIV antibody tests are unique to HIV? As Philip
Mortimer and his colleagues from the UK Public Health Laboratory
Service noted: "Experience has shown that neither HIV culture
nor tests for p24 antigen are of much value in diagnostic testing.
They may be insensitive and/or non-specific".
Yes, I know that we have all been shown pictures of something called
HIV, but in the extensive retrovirology literature, retrovirus-like
particles are commonplace. For a start, insects, reptiles, fish
and tapeworms have them. Retroviruses are also found in the majority
of healthy human placentas. And while it is true that electron microscopy
reveals retroviral-like particles in 90% of enlarged lymph nodes
from AIDS patients, the identical particles can also be found in
90% of enlarged lymph nodes from patients who do not have AIDS and
who are not at risk for developing AIDS . If the particles seen
in lymph nodes from AIDS patients are HIV (as the AIDS experts assure
us), what are the particles seen in the lymph nodes of patients
who are not at risk for AIDS and what is their relationship to the
plethora of other particles seen tissue cultures from AIDS patients?
Wait, some may ask, what about the PCR test (the polymerase chain
reaction test or “viral load”)? For those who don't
know, PCR is a new and very sensitive technique for finding genetic
blueprints. But can PCR validate the antibody tests? No, I'm afraid
To perform the PCR you need to begin with a piece of RNA or DNA
that you can say for certain belongs to the HIV genome. To obtain
the HIV genome, you first need to isolate an HIV particle. That's
where the HIV genome comes from and that is the only way to know
the RNA or DNA that actually belongs to the virus. Even the most
charitable interpretation of the data available to date does not
show that a unique retrovirus, HIV, has been isolated. Furthermore,
even if one assumes that the process of selecting the RNA and DNA
molecules (molecular probes) used in the PCR are from the HIV genome,
there are still many problems with the use of the PCR to prove HIV
At best, the PCR detects single genes and most often, only bits
of genes. If your PCR finds two or three genetic fragments out of
a possible dozen complete genes is this proof that you have all
the genes? The whole genome? No it is not, and in fact HIV experts
admit that the majority of HIV genomes studied are defective. This
means they are incomplete and could never orchestrate the synthesis
of a viral particle. Even if all genomes were complete, let's not
imagine for a moment having the blue prints means you've built the
house. Basic retrovirology long teaches us you can carry a whole
retroviral genome around inside your cells all your life without
ever making a viral particle. And in 1992, in the only study of
its type, French researchers found the HIV PCR non-reproducible
and the agreement between the PCR and the HIV Western Blot was found
to vary between 40-100% and was especially poor when fragments of
more than one gene were sought . In this study, there were several
PCR negative/HIV positive as well as several PCR positive/HIV negative
samples. In other words, the two tests don't match. As far as which
test proves HIV infection, you pay your money and take your pick.
Finally, a specificity in excess of 99.9% sounds pretty convincing,
but is it? What if you were found to have a positive HIV antibody
test? What is your chance of being truly HIV infected, and not a
To answer this, let's imagine a population of one million people
where somehow, by authentic isolation studies, we know 1/1000 persons
are HIV infected. Let's also assume that there is definite proof
measured against a viral isolation gold standard that the HIV antibody
tests are 99.9% specific for HIV infection. If the test is also
100% sensitive, it will detect all of the 1,000 infected people.
However, 0.1% (1-specificity) of the 999,000 non-infected remainder
will also be seropositive. That's another 999 people making a total
of 1,999 positive tests, 1,000 who are infected and 999 who aren't.
In this case, if you were randomly selected and found to be antibody
positive there is only a 50/50 chance you are actually infected.
The test will be wrong half the time. But for most of you, we can
probably do better than this because most of you are arguably somewhat
removed from the risk groups that dominate the statistics. If your
odds are only 1/2000 of being infected, and if we drop the specificity
of the test slightly to a mere 99.6%, a positive test will be wrong
in 89% of cases, or in other words, almost all of the time.
Where does all this leave HIV/AIDS patients?
Firstly, the only evidence that HIV is the cause of AIDS is the
perception by the AIDS experts of a correlation between antibody
reactions and the presence of AIDS-defining diseases. However, for
AIDS patients who have had antibody tests and have been diagnosed
HIV infected solely on the basis of these tests, we can argue that
there is no proof that even one such patient is infected with a
virus called HIV. Secondly, in these cases, the tests provide no
justification for the administration of potentially toxic drugs
like AZT on the basis of a perceived anti-viral activity. Certainly
the HIV antibody tests confirm that certain diseases are AIDS rather
than just those diseases but this can be construed as an artefact
The only scientific conclusion we are permitted to make is that
in some but not all well defined at-risk individuals, there is a
correlation between antibody reactions, whatever their raison d'etre,
and the propensity to develop and die from certain diseases. On
the other hand, if you're HIV positive but not in a risk group and
especially if you're healthy, any pronouncements on your likely
outcome will be severely confounded by knowing you are positive,
a situation we might describe as 20th century bone pointing*. And
your health may suffer further from the use of medications administered
in good faith to kill a virus you may not have. The failure to verify
the antibody tests against the gold standard of virus isolation
is a serious omission of scientific method. In the absence of such
validation, these tests should not be used to diagnose HIV infection.
In the entire AIDS literature there is only one study, that of Colonel
Donald Burke and his colleagues  from the Walter Reed Army Institute,
that is widely regarded as the definitive proof of the specificity
of the HIV Western Blot.
Over an eighteen month period, Burke and his colleagues tested 1.2
million applicants for US military service. Burke's testing procedure
was a progression through two ELISAs and two Western Blots. From
these data, the HIV seroprevalence was found to be 1.48/1000. Burke
then retrospectively investigated a highly selected sample of this
population in which the HIV seroprevalence was 1/10th that of the
1.2 million. This group comprised 135,187 persons aged 17-18 years
who resided in rural areas where the cumulative incidence of AIDS
was low. Many would assume this group to be no different from healthy
blood donors and would regard all HIV positives found among
them as false positives, but Burke and his colleagues' premises
were the opposite.
Assuming there were true positives among these healthy, rural American
youth and wishing to evaluate the false positive rate and specificity
of the Western Blot, Burke needed to define HIV infection. This
was done by performing a panel of four more antibody tests on sera
from the 15 out of 135,187 applicants who had already been found
twice ELISA and twice Western Blot positive.
Two of the extra four tests were other Western Blots and two were
similar tests. Any individual found positive in all four of the
extra tests, thereby making a total of eight positive antibody tests,
was deemed HIV infected. Those who failed any of the extra four
tests were deemed non-HIV infected. Of the 15, one failed to react
on all four tests and thus Burke conceded only one, not 15, false-positives.
From these data, Burke calculated the specificity of the HIV Western
Blot to be in excess of 99.9%.
There are many flaws in this study and they are outlined in reference
1. Here I wish to draw to your attention to the fact that an antibody
test, even if repeated and found positive a thousand times, does
not prove the presence of a viral infection.
* Bone pointing is a traditional, ritualistic punishment practiced
by Australian aborigines. A bone is pointed at an individual as
a method of retribution. That individual soon becomes sick and death
within weeks or months is an invariable consequence.
1. Eleopulos-Papadopulos E, Turner VF, Papadimitriou JM. 1993. Is
a positive Western blot proof of HIV infection? Bio/Technology 11:696-707.
2. Eleopulos-Papadopulos E, Turner VF, Papadimitriou JM. 1993. Has
Gallo proven the role of HIV in AIDS? Emergency Medicine [Australia]
3. Burke DS, Brundage, JF, Redfield, RR et al. 1988. Measurement
of the false positive rate in a screening program for human immunodeficiency
virus infections. NEJM 319: 961-964.
4. Strandstrom HV, Higgins JR, Mossie K, et al. Studies with canine
sera that contain antibodies which recognize human immunodeficiency
virus structural proteins. Cancer Res 1990; 50: 5628s-5630s.
5. Mortimer P, Codd A, Connolly J, et al. Towards error free HIV
diagnosis: notes on laboratory practice. PHLS Microbiol Digest 1992;
6. O'Hara CJ, Groopmen JE, Federman M. 1988. The ultrastructural
and immunohistochemical demonstration of viral particles in lymph
nodes from human immunodeficiency virus-related lymphadenopathy
syndromes. Human Pathology 19:545-549.
7. Defer C, Agut H, Garbarg-Chenon A. 1992. Multicentre quality
control of polymerase chain reaction for detection of HIV DNA. AIDS