Genetica 95: 25-50, 1995
FACTOR VIII, HIV AND AIDS IN HAEMOPHILIACS: AN ANALYSIS OF THEIR
Eleni Papadopulos-Eleopulos,1 Valendar F.Turner,2
John M. Papadimitriou3 & David Causer1
1: Department of Medical Physics, 2: Department of
Emergency Medicine, Royal Perth Hospital, Perth, Western Australia;
3: Department of Pathology, University of Western Australia.
There are three steps in the revelation of any truth: in the
first, it is ridiculed; in the second, it is resisted; in the third,
it is considered self- evident.
In this review, the association between the Acquired Immune Deficiency
Syndrome (AIDS) and haemophilia has been carefully examined, especially
the data that have been interpreted as indicating transmission of
the human immunodeficiency virus (HIV) to the recipients of purportedly
contaminated factor VIII preparations. In our view, the published
data do not prove the hypothesis that such transmission occurs and
therefore HIV cannot account for AIDS in haemophiliacs.
Currently, it is accepted that many patients with haemophilia have
become HIV infected and/or developed the AID clinical syndrome as
a direct result of the transfusion of factor VIII preparations contaminated
with this particular virus. That this is indeed the case requires
1. of the existence of a unique, infectious retrovirus, HIV. (For
a critical discussion of this issue see Papadopulos-Eleopulos, 1988,
Papadopulos- Eleopulos et al., 1992, Papadopulos-Eleopulos et al.,
1993a, Papadopulos- Eleopulos et al., 1993b);
2. of the existence of HIV in factor VIII preparations;
3. of the existence of HIV in haemophiliacs;
4. that HIV is necessary and sufficient for the decrease in T4
cells observed in haemophiliacs;
5. that HIV and a decrease in T4 lymphocytes are necessary and
sufficient for the development of the clinical AID syndrome.
Factor VIII and HIV
Since factor VIII is made from plasma, as a first step in proving
contamination of this blood product with HIV, evidence must be presented
that infectious viral particles with morphological characteristics
attributed to HIV, are present in the plasma of "HIV infected"
individuals. Then it must be shown that HIV can survive (a) the
time between blood collection and freezing of plasma; (b) the freezing
and thawing itself; © the process of manufacturing factor VIII
from thawed plasma. In other words, as Jay Levy succinctly expressed
in 1989, it is "important to know whether retroviruses could
survive the preparation involved in producing Factor VIII concentrates.
Otherwise, AIDS in many haemophiliacs [a minority may have other
risk factors] could not be explained" (Levy, 1989).
HIV in plasma
To date, there is no evidence of the existence in human plasma
of particles with the morphological characteristics attributed to
HIV even though the plasma of at least some "HIV infected"
individuals is claimed to contain such particles. Thus Levy, whose
team reported most often on the relationship between HIV and factor
VIII wrote in 1988: "Human Immunodeficiency virus in plasma
or serum has been found in about 30% of specimens from seropositive
persons, generally at a concentration of less than 10 IP/mL12"
[IP=3Dinfectious particles] (Levy, 1988). Reference 12 cited by
Levy is a paper which he published in collaboration with Barbara
Michaelis but this paper does not contain a description of the method
used to show that (a) HIV seropositive (non-haemophiliac) plasma
was infected with "HIV particles"; (b) HIV was "present
in low titers"; (c ) the particles were "infectious".
Commenting on his and his colleagues' findings Levy wrote: "These
studies demonstrate further that not all seropositive individuals
have virus recoverable from their PMCs and that isolation from serum
is not a common event" [PMCs=peripheral blood mononuclear cells]
(Michaelis & Levy, 1987). "Thus, cell-free virus in body
fluids is unlikely to be a meaningful source of HIV transmission".
(Levy, 1988). At least one other eminent HIV/AIDS researcher is
also of the opinion that HIV cannot be transmitted through "...products
prepared from blood, such as albumin, plasma, protein fractions,
or hepatitis B vaccine" (Blattner, 1989). If HIV cannot be
transmitted through "cell-free" body fluids (plasma) because
it is not found in the plasma of 70% of seropositive individuals
and in the remaining 30% is "generally at a concentration of
less than 10 IP/mL", then it will be even less probable that
factor VIII prepared from plasma can be a "meaningful source
of HIV transmission" since, even if HIV were present in a plasma
collection, it would be diluted many times over during the process
of factor VIII manufacture. This is because factor VIII is made
by pooling plasma obtained from 2000 to 30,000 individuals amongst
whom at most, there will be only a few HIV seropositives. Since
factor VIII prepared from large batches of pooled plasma is ultimately
shared amongst many haemophiliacs, the load of HIV for each haemophiliac
will be substantially lower than 10 IP/ml.
Since 1989, detection of a 24,000 molecular weight protein (p24)
in cell cultures, (T cells from persons presumed to be infected),
or co-cultures, (of T cells from persons presumed to be infected,
with T cells from normal individuals), has been used to quantify
HIV in cells, "cellular viremia" (Masquelier et al., 1992).
Detection of p24 in cultures of T cells from normal individuals
with plasma from those presumed to be infected has been used to
quantify HIV in plasma, "plasma viremia" (Coombs et al.,
1989; Ho et al., 1989; Clark et al., 1991). There are many reasons
why p24 cannot be used to quantitate or even detect the presence
of "HIV infectious particles".
(a) there is ample evidence that the p24 protein is not HIV specific
(Papadopulos-Eleopulos et al., 1993a and see below);
(b) there is no relationship between plasma viraemia, cellular
viraemia (p24 in culture), and the titre of p24 in (uncultured)
plasma (HIV antigenaemia). "Only 45 percent of patients with
plasma viremia had HIV p24 antigen in either serum or plasma"
(Coombs et al., 1989). "Plasma p24-antigen titres before or
after acidification did not show any significant correlation with
quantification by tissue culture method" (Weber & Ariyoshi,
1992). Nor does correlation exist between the "most specific"
HIV antibody test, the Western blot (WB), and plasma p24. With methods
which have a reported lower limit of sensitivity of 10-50pg/ml,
p24 can be detected in only 12% of HIV positive sera (Jackson et
(c) "Much of the viral protein secreted from HIV-infected
cells is non-particulate, and the proportion of (for example) p24
in virions is a function of the viral genotype and the age of the
culture. In extreme cases, less than one percent of the total p24
and gp120 present is in virions" (McKeating & Moore, 1991).
[It must be pointed out that in the AIDS literature, the terms "HIV",
"HIV isolation", "pure particles", "virus
particles", "virions" and "infectious particles",
have a variety of meanings and include all of the following, but
most often without proof of the presence of a particle: (i) "RNA
wrapped in protein"; (ii) material from the cell culture supernatants
which passes through cell tight filters but through which organisms
such as mycoplasmas may pass; (iii) the pellet obtained by simple
ultracentrifugation of the culture supernatant; (iv) recently, very
often, detection in AIDS cultures of p24 (Papadopulos-Eleopulos
et al., 1993a)].
In the process of preparing plasma for factor VIII extraction,
great care is taken to exclude cells. Even if some cells are inadvertently
present, it would be most unlikely that they would constitute "a
meaningful source of HIV transmission" since, like "HIV
particles", "infected" cells present in the plasma
of a seropositive donor would be diluted many times over by the
plasma and cells from the manifold number of non-seropositive donors
from which factor VIII is made.
Furthermore, according to some of the best known HIV experts:
(a) "in early and intermediate stages of disease", (it
is unlikely that individuals with advanced disease, AIDS, would
be able to donate blood), the frequency of HIV infected cells in
the blood as determined by the polymerase chain reaction (PCR) is
1/10,000 in the early stages, and between 1/10,000 and 1/1000 in
the advanced stages (Pantaleo et al., 1993);
(b) with the PCR one does not detect viral particles or even the
whole viral genome, but only a small region, "a gene at best"
(c ) up to 99.9% of the "HIV genomes" in the plasma may
be defective (Sheppard et al., 1993) that is, one or several genes
Plasma processing and HIV
"Source plasma" obtained by plasmapheresis and fresh
frozen plasma from whole blood donation are best suited for the
preparation of factor VIII. The interval between collection and
freezing of plasma is approximately six hours (van Aken, 1991).
The plasma is kept frozen for lengthy periods, days to weeks, and
is then thawed for processing.
Researchers from the Laboratory of Molecular Retrovirology, Georgetown
University, took two blood samples from each of ten HIV seropositive
patients: "One sample from each individual was processed immediately
after phlebotomy to obtain plasma, and aliquots of this plasma were
used at once to infect PHA-stimulated donor PBMCs as described.
A second set of aliquots of this "immediately processed"
plasma was frozen at -70=F8C for 3 h, and then thawed and used to
infect the same donor cells. Five of the ten immediately processed/immediately
used plasma samples (50%) were positive for HIV-1 using the p24
antigen detection method, while all of the corresponding frozen
aliquots were negative (0%). The second blood sample from each of
the 10 patients was kept at room temperature for 3 h prior to plasma
separation. Again, after processing, one aliquot was used for the
infections while another was frozen and thawed before use. In this
experiment, only one of the ten samples (10%) was culture positive
after the 3 h delay and also after the one cycle of freezing and
thawing" (Dewar et al., 1992). Thus, although these workers
determined the optimum conditions for "HIV isolation"
prior to conducting the above experiments, they could "isolate"
(detect p24 in culture) HIV from only 10% of HIV+ plasmas which
were left at room temperature for three hours and from 1 of 20 (5%)
HIV+ plasmas which had been frozen for three hours. In 1984, Levy
himself, using "mouse retroviruses" reported that: "The
virus titre (108 IP) was not affected by mixing with cold (5=F8C)
plasma. In contrast, incubation of the virus with plasma at 37=F8C
for 30 min reduced its titre 100-fold. This finding accords with
the report of complement-mediated lysis of retrovirus by human serum"
(Levy et al., 1984). Other researchers have shown "freeze-drying
parameters commonly employed under commercial conditions for the
preservation of protein solutions are not favourable for survival
of viral suspensions" (Damjanovic, 1987). However, Levy and
his colleagues claim to have shown that a retrovirus "can survive
and remain infectious after procedures used in the preparation of
factor VIII (FVIII), cryoprecipitate or concentrates". According
to these researchers, when HIV was "added to human plasma (5=F8C),
no reduction in virus titre was observed". Cryoprecipitate
made from the plasma contained "a 10-fold reduced titre".
"Purification of cryoprecipitate by acid and glycine precipitation
and filtration to achieve a sterile FVIII filtrate resulted in a
further 10-fold reduction in virus titre". Lyophilisation of
factor VIII filtrate "lowered the infectious virus titre about
10-fold. When this lyophilised preparation was then heated, very
low titre virus was detectable after 10, 24 and 34 hours but not
after 48 hr of heating" at 68=F8C. They concluded "our
results indicate that lipid-enveloped retroviruses (both mouse and
human) if present in sufficient amount in plasma can be found in
infectious form in FVIII lyophilised products...heating lyophilised
FVIII for 72 hr at 68=F8C or the liquid product for 10 hr at 60=F8C
will eliminate infectious ARV [HIV] if it is not present in the
plasma at more than 106 infectious particles/ml." (Levy et
al., 1985). However:
1. Commenting on Levy and colleagues' findings Damjanovic wrote:
it is "surprising that HIV survived procedures used in the
preparation of Factor VIII before lyophilization";
2. Levy et al performed their experiments by "infecting"
plasma with 105IP/ml, while factor VIII which is administered to
haemophiliacs is made from plasma pooled from thousands of individuals
most of whom are not infected. Given that:
(a) the plasma from which factor VIII is prepared contains very
few or no particles per ml of plasma;
(b) the technique employed to prepare factor VIII reduced by a
thousand fold the concentration of any infectious particles present,
even before heat treatment; one would have to conclude that factor
VIII prepared before 1985 could not contain sufficient HIV particles
to be a "meaningful source of HIV transmission";
3. Levy and his colleagues detected and quantified HIV particles
"by induction of reverse transcriptase activity in the culture
fluids of normal human PMC maintained for up to 1 month after virus
inoculation" (Levy et al., 1985). In Levy's laboratory, PMCs
were cultured with interleukin-2, polybrene and phytohaemagglutinin
(PHA). To prove HIV infection, the activity of the enzyme reverse
transcriptase (RT) was determined using the primer-template An.dT15
(Levy et al., 1984). Detection of RT cannot be considered proof
of the presence of a retrovirus, certainly not HIV, and in fact,
the above template-primers can be copied by all cellular DNA polymerases
(see below). Because of this, reverse transcription of the primer-template
An.dT15 cannot be used specifically to quantify or even detect HIV
or any other retrovirus.
HIV in factor VIII
The belief, as Levy pointed out, that haemophiliacs develop AIDS
because they become infected with HIV by receiving contaminated
factor VIII can be entertained if and only if evidence exists which
1. factor VIII used to treat haemophiliacs is contaminated with
2. the particles are infectious.
A paper entitled "Detection, quantification and sequencing
of HIV-1 from the plasma of seropositive individuals and from factor
VIII concentrates" published in 1991, is the only paper to
claim proof of the existence of HIV in factor VIII used therapeutically
(Zhang et al., 1991). Using PCR the authors tested 8 batches of
factor VIII, all "unheated and prepared before the introduction
of donor screening for anti-HIV antibodies". Two batches "gave
positive results; in one case with the env primers, the other with
the pol primers". In sequencing their "HIV RNA" they
found that the sequences were "distinct from those of all published
HIV isolates and from any sequences obtained previously in our laboratory".
Despite this, they interpreted their signals as HIV and in fact
quantified the HIV and concluded: "the calculated amount of
HIV RNA in both batches of reconstituted factor VIII was only 2.5
copies per ml".
The minimum requirement for such an interpretation of a PCR signal
(or hybridisation in general), is prior proof that the PCR primers
and the hybridisation probes belong to a unique retrovirus, HIV,
and that the PCR and hybridisation reactions are HIV specific. Detailed
evidence had been presented elsewhere (Papadopulos-Eleopulos et
al., 1993a) that the specificity of the hybridisation signals in
general and of "HIV" PCR in particular have not been determined,
and that the finding of viral RNA or DNA, even if proven to belong
to a unique retrovirus HIV, is not proof of the presence of the
viral particle. Some additional points are:
1. Most, if not all probes used for hybridisation assays, including
the PCR probes and primers, are derived from the H9 (HUT78) or CEM
cell lines. The H9 cell line originated from a patient with T4 cell
leukaemia, a disease which Gallo claims is caused by a retrovirus
similar to HIV, HTLV-I (Gallo, 1986). Recently, a retrovirus has
been isolated from a non-HIV-infected CEM (SS) culture (Minassian
et al., 1993). Thus, the above cell lines contain at least one retrovirus,
if not more (see below), even when not infected with HIV. Since
even the well established method (Papadopulos-Eleopulos et al.,
1993a) for retroviral isolation (but which to date has never been
reported for HIV), cannot distinguish between retroviruses, one
cannot be confident that the "HIV" nucleic acid probes
and PCR primers are indeed specific for HIV;
2. The normal human genome contains HIV and HTLV-I sequences (Parmentier
et al., 1992; Schneider et al., 1993);
3. The specificity for HIV of hybridisation assays in general and
PCR in particular, can be determined only by the use of a gold standard.
However, according to one leading HIV/AIDS researcher, William Blattner,
"One difficulty in assaying the specificity and sensitivity
of human retroviruses [including HIV] is the absence of a final
'gold standard'" (Blattner, 1989). In addition to the above
mentioned problems there are many other difficulties associated
with the establishment of an HIV gold standard for PCR/hybridisation
studies (Papadopulos-Eleopulos et al., 1993a). One recently identified
problem is the fact that there are "striking differences"
between the proviral DNA and cDNA in one and the same PBMC sample
which "could not be explained by either an artefact of reverse
transcriptase efficiency or template selection bias" (Michael
et al., 1993).
4. The presently available evidence obtained without a gold standard
suggests that the "HIV hybridisation" is not HIV specific
(Papadopulos-Eleopulos et al., 1993a). Some additional evidence:
(a) to address the question whether the neuronal cells of patients
with AIDS dementia complex are infected with HIV, "the brains
from 10 patients with AIDS and neurological evidence of viral encephalitis
and the brains from 5 patients without HIV-1 infection" were
examined using an HIV gag probe. "The antisense riboprobe hybridized
to cells known to be infected with HIV-1. It hybridised to HIV-1
infected A3.O1 cells as well as splenic and renal lymphocytes obtained
at autopsies from patients known to have AIDS. The probe did not,
however, hybridize to neurones in the brain sections from 10 patients
with AIDS...Surprisingly, when we applied the control sense HIV-1
gag probe to the brain sections from patients with AIDS, we observed
specific hybridization to neuronal cells. Similarly, when brain
sections from five individuals not infected with HIV-1 were examined,
the HIV-1 sense probe detected transcripts in neuronal cells. Our
Northern blot analysis confirmed these results and demonstrated
the presence of a 9.0-kb polyadenylated transcript in brain tissues"
(Wu et al., 1993). Thus, either the positive hybridisation signals
obtained with the antisense probe are non-HIV- specific or, as the
authors concluded, there is a neurone-specific 9.0- kb transcript
that shows extensive homology with antisense gag HIV-1 sequences
and that this transcript is expressed in neuronal cells of both
HIV-1-infected and noninfected individuals;
(b) the finding of positive PCR in eosinophils has been interpreted
to "suggest that eosinophils may act as host cells for HIV-1"
(Conway et al., 1992). However, "Formaldehyde-fixed eosinophils
nonspecifically bind RNA probes [HIV RNA] despite digestion with
proteolytic enzymes and acetylation...When preparations are treated
with amounts of ribonuclease adequate to destroy viral RNA, the
eosinophilic binding remains" (Natoli et al., 1993);
(c ) negative controls and even buffers and reagents may give positive
HIV PCR signals (Conway, 1990);
One cannot but agree with Shoebridge et al that "until further
molecular biological and epidemiological studies are carried out,
it will be unclear as to what detection by PCR of proviral HIV-1
DNA, even when shown to be HIV-1, really means" (Shoebridge
et al., 1991).
Even if it is proven beyond reasonable doubt that factor VIII preparations
contain HIV particles, the particles could not be infectious. This
fact is of such pivotal significance it is essential to review the
mechanism of HIV infection as reported by leading HIV researchers.
1. Weber and Weiss, "The first step in any viral infection
is the binding of the virus particle to a component of the host
cell's membrane...For some time it has been known that the binding
takes place when CD4 interacts with an "envelope" protein
of the virus called gp120" (Weber & Weiss, 1988);
2. Moore et al, "HIV infection of CD4+ cells is initiated
by an interaction between its surface glycoprotein, g120, and the
cellular antigen CD4" (Moore & Nara, 1991);
3. Mortimer, "The gp120 surface protein interacts with CD4
receptors on T4 cells so that the viral RNA can be injected into
the cell" (Mortimer, 1989);
4. Matthews and Bolognesi, "First gp120 binds to the CD4 receptor
on an uninfected cell; then gp41 becomes anchored in the adjoining
membrane; next the two membranes begin to fuse, and the virus spills
its contents into the cell" (Matthews & Bolognesi, 1988);
5. Redfied and Burke, "Infection begins as a protein, gp120,
on the viral envelope binds tightly to a protein known as the CD4
receptor on the cell surface" (Redfield & Burke, 1988);
6. Rosenberg and Fauci, "The initial event in the life cycle
of HIV is the high-affinity binding of the HIV envelope glycoprotein
(gp120) to CD4 that is present on the surface of cells" (Rosenberg
& Fauci, 1990);
7. Montagnier et al, "The gp120 is responsible for binding
to the CD4 receptor" (Gougeon et al., 1993);
8. Haseltine and Wong-Staal: gp120 is "crucial to HIV's ability
to infect new cells" (Haseltine & Wong-Staal, 1988);
9. Callebaut et al, "The human immunodeficiency virus (HIV)
infects lymphocytes, monocytes, and macrophages by binding to its
principal receptor, the CD4 molecule, through the viral envelope
glycoprotein gp120. The V3 loop of gp120 is critical for HIV infection"
(Callebaut et al., 1993).
Thus, there is general agreement that the HIV envelope protein
gp120 is crucial for HIV infection. However, agreement also exists
that "gp120 is easily shed by virus and virus-infected cells"
(Bolognesi, 1990). Gelderblom and his colleagues at the Koch-Institute
in Berlin who have conducted the most detailed electron-microscopy
studies of "HIV particles" have shown that the knobs on
the surface of the particles, where the gp120 is found, are only
present in immature (budding) particles, which are "very rarely
observed". "Mature", cell-free particles do not have
knobs, that is, gp120 (Hausmann et al., 1987).
Regarding infection by retroviruses, as far back as 1983 Gallo
pointed out that "the viral envelope which is required for
infectivity, is very fragile. It tends to come off when the virus
buds from infected cells, thus rendering the particles incapable
of infecting new cells". Because of this Gallo said, "cell-to-cell
contact may be required" for retroviral infection (Marx, 1983).
Since gp120 is "crucial to HIV's ability to infect new cells",
and since gp120 is not found in the cell free particles, even if
HIV particles are present in plasma or factor VIII preparations,
they will be non-infectious.
One must also consider the possibility that factor VIII is contaminated
with HIV-infected cells. Even if the plasma from which the factor
VIII is made contains cells, since preparation of factor VIII entails
(a) freezing and thawing which lyses cells; (b) sterilisation by
filtration which excludes cells and the majority, if not all, of
cellular fragments from the filtrate; it is most unlikely that factor
VIII would be contaminated with cells. Furthermore, even if the
filtrate were to contain some cellular fragments, they could not
be a source of HIV because the synthesis and assembly of type C
and type D particles, and Lentiviruses, require the presence of
an intact cell. In conclusion, the lack of evidence of HIV particles
in plasma, the use of non-specific methods to detect HIV in cultures,
the lack of gp120, considered to be crucial for HIV infection in
cell-free particles, the physical processes involved in processing
plasma into factor VIII even before heating was introduced, make
it impossible for factor VIII to be contaminated with infectious
retroviruses. It is not surprising therefore that, to date, nobody
has reported HIV particles in factor VIII preparations. Thus, on
the available evidence, HIV infected factor VIII cannot be the explanation
for AIDS in haemophiliacs. If factor VIII is not infected with HIV
then it is mandatory to explain the cause of the "HIV-related"
phenomena, that is, positive antibody tests, HIV isolation, T4 cell
decrease and AIDS observed in haemophiliacs.
HIV antibodies in haemophilia
By 1988 most, if not all haemophiliacs, (in the USA, Europe and
Australia), were tested for HIV antibodies and the vast majority
of those tested were reported as being positive. Based on the antibody
tests, as far back as 1984, the CDC concluded that "These serological
data, indicating a high risk of exposure to LAV from heavy users
of factor VIII concentrates, support the contention that LAV may
be transmitted by some blood products" (Ramsey et al., 1984).
However, the specificity of the HIV antibody tests for HIV infection
have never been determined. According to Philip Mortimer, director
of the Virus Reference Laboratory of the Public Health Laboratory
Service, London, UK: "Diagnosis of HIV infection is based almost
entirely on detection of antibodies to HIV, but there can be misleading
cross-reactions between HIV-1 antigens and antibodies formed against
other antigens, and these may lead to false-positive reactions.
Thus, it may be impossible to relate an antibody response specifically
to HIV-1 infection. In the presence of clinical and/or epidemiological
features of HIV-1 infection there is often little doubt, but anti-HIV-1
may still be due to infection with related retroviruses (e.g. HIV-2)
which, though also associated with AIDS, are different viruses"
[italics ours] (Mortimer, 1989).
The specificity of an antibody test, any antibody test, cannot
be determined by "clinical and/or epidemiological features".
In the case of the HIV tests, this practice may create several problems.
Given the fact that the vast majority of individuals who test positive
are asymptomatic, one must conclude that in these individuals, a
positive HIV antibody test is a false positive. Furthermore, the
1993 AIDS definition permits the diagnosis of AIDS solely on the
basis of a low T4 cell count and positive HIV serology. It has been
estimated that the new AIDS definition will treble the number of
AIDS cases compared to the 1987 AIDS definition (Brettle et al.,
1993), most of whom may be expected to be asymptomatic, and thus
a significant number of AIDS patients will have a false positive
HIV antibody test. Even if a patient did have one of the AIDS "indicator
diseases" (none of which is new and some of which are common),
(a) haemophiliacs are exposed to "an array of alloantigens
(and infectious agents)" (Levine, 1985);
(b) gay men and intravenous users are also subjected to a wide
variety of foreign antigens and infectious agents;
(c ) all these groups are known to possess a plethora of antibodies
directed against numerous non-HIV antigens; one would expect cross-reactivity
with "HIV antigens" to be the rule rather than the exception
and thus, in these groups, more than in any other, it will be difficult
to conclude that a positive HIV antibody test signifies HIV infection
and not cross-reactivity.
One cannot simultaneously use the presence of AIDS as proof of
HIV infection, and conversely, the presence of a positive HIV test
as proof that HIV is the cause of AIDS, as presently is the case.
The specificity of an antibody test requires the use of a gold standard.
A gold standard in an alternative, independent method of proving
the presence or absence of the condition for which the test in to
be employed and in the case of the HIV antibody tests the only admissible
gold standard is HIV itself. However, the use of such a gold standard
has never been reported and may not even be possible (Papadopulos-Eleopulos
et al., 1993a). This is a view shared by William Blattner: "One
difficulty in assessing the specificity and sensitivity of retrovirus
assays is the absence of a final 'gold standard'. In the absence
of gold standards for both HTLV-I and HIV-1, the true sensitivity
and specificity for the detection of viral antibodies remain imprecise"
(Blattner, 1989). In fact, at present, there is ample evidence which
suggests that the HIV antibody tests even in the high AIDS risk
groups (gay men, IV drug users, blacks and haemophiliacs), may not
be specific (Papadopulos-Eleopulos et al., 1993a). Some additional
data related to haemophilia are:
(a) haemophilia patients have hypergammaglobulinaemia and hypergammaglobulinaemia
correlates with HIV seropositivity (Brenner et al., 1991);
(b) haemophilia patients have anti-lymphocyte antibodies (Daniel
et al., 1989);
(c ) in one study, 12% of haemophiliacs were found to have HTLV-I
antibody, (the molecular weights of HTLV-I and HIV-1 proteins are
the same), 74% anti-cardiolipin antibodies, 28% anti-nuclear antibodies
and 85% immune complexes (Matsuda et al., 1993);
(d) HIV researchers accept that "antilymphocyte, antinuclear
and other autoantibodies" give rise to false positive HIV antibody
tests (Biggar, 1986);
(e) in haemophiliacs, hepatitis B virus seropositivity is a predictor
for HIV seropositivity (Brenner et al., 1991);
(f) at least one other group with chronic liver disease, alcoholics,
are known to have both false positive antibody tests and immune
deficiency (Mendenhall et al., 1986).
As has been already noted, by 1988, most haemophiliacs had already
been found to be HIV seropositive. However, the test utilised by
many researchers including Gallo, Blattner, Weiss, Montagnier and
Chermann in papers published as late as 1990, was the ELISA (Melbye
et al., 1984; Allain, 1986; Eyster et al., 1987; Goedert et al.,
1989; Wagner et al., 1990). Although before 1988 some researchers
used WB to confirm the ELISA, the criteria used then to define a
positive WB would not satisfy even the "least stringent"
criteria presently used to define a positive WB result (Lundberg,
1988). A few examples will suffice to illustrate this point:
1. "Serological reactions with any combination of 18kd, 25kd
and 41kd proteins of LAV were scored as positive" (Jason et
2. "A positive Western blot test was defined as the presence
of at least one band characteristic of antibody against an envelope
protein (gp41, gp120, or gp160) and at least one other HIV-1 characteristic
band" (Jackson et al., 1988);
3. "Serological reactions were scored as positive if there
was reactivity with the 41-kD protein of HIV or reactivity with
the 24-kD protein together with any one of several other HIV-associated
proteins (18kD, 31kD, 51kD, 55kD, 65kD or 110kD)" (Lawrence
et al., 1989).
Thus, it is a distinct possibility that if haemophiliacs who have
been tested using only ELISA, or even ELISA and WB prior to 1988,
were reappraised, a significant proportion may no longer be classified
as HIV seropositive.
In 1984, a number of researchers from the USA, including the well
known retrovirologist Myron Essex, reported the finding of HIV antibodies
in haemophiliacs and concluded: "The present results suggest
that exposure to HTVL-III is widespread in asymptomatic haemophiliacs",
but also added "However, it is possible that a significant
proportion of asymptomatic haemophiliacs might be exposed only to
inactivated HTLV-III rather than to the virus, owing to the manufacturing
process involved in the preparation of commercial factor VIII concentrate"
(Kitchen et al., 1984). But the mere fact that some HIV antibody
positive haemophiliacs have symptoms is not proof that they are
infected with the virus. (As we have already mentioned, one cannot
simultaneously use the presence of AIDS as proof of HIV infection,
and conversely, the presence of a positive HIV test as proof that
HIV is the cause of AIDS).
Later, the finding that haemophiliacs who received only heat treated
factor VIII (van den Berg et al., 1986; CDC, 1987) also became HIV
seropositive, was interpreted as evidence that these patients "may
not have been infected but rather immunized by preserved viral proteins"
(Damjanovic, 1989; Jackson, 1989). As far back as 1985 researchers
from the CDC wrote:
"It is possible that antibody to LAV is acquired passively
from immunoglobulins found in factor VIII concentrates...Likewise,
it is possible that seropositivity is caused not by infectious virus
but by immunization with noninfectious LAV or LAV proteins derived
from virus disrupted during the processing of plasma into factor
VIII concentrate" (Evatt et al., 1985). Thus a positive HIV
antibody test cannot be considered proof of HIV infection. Nonetheless,
"Because the virus has been isolated from the lymphocytes of
about 30% of antibody-positive asymptomatic haemophiliacs and because
immune dysfunction has been progressive in other patients, it is
believed that antibody positivity is indicative of infection instead
of immunization in most, if not all, of the antibody-positive haemophiliacs"
(Brettler et al., 1988). According to other authors, "Strictly
speaking, detection of the virus is therefore necessary for diagnosis
of an HIV infection in HIV-seropositive haemophiliacs" (Schneweis
et al., 1989). In conclusion, the presently available evidence does
not prove that a positive HIV antibody test in haemophiliacs is
proof of HIV infection.
In a paper published in the Lancet in 1984 entitled "Isolation
of a New Lymphotropic Retrovirus from two Siblings with Haemophilia
B, one with AIDS", Montagnier and his associates were the first
to describe "isolation of HIV" from haemophiliacs. The
T lymphocytes of the two children, one symptomatic and the other
healthy, were cultured with amongst other chemical agents, PHA,
IL-2, polybrene and anti-human alpha-interferon. >From the symptomatic
sibling they reported the following findings:
1. In the culture, retrovirus-like particles;
2. In the material from the cultures which in density gradients
banded at 1.16 gm/ml:
(a) proteins which using the ELISA reacted with sera from a gay
man with lymphadenopathy and several specimens of the patient's
serum collected prior to the blood used for "HIV isolation".
No serological data is given regarding the blood from which the
HIV was isolated. However, the serum collected after treatment and
clinical improvement was non-reactive. In WB analysis a p24/25 protein
which banded at 1.16 gm/ml was found to react with the patient sera
as well as with the serum from the gay man with lymphadenopathy.
The same sera did not react with goat antiserum specific for the
p24/25 of HTLV-I;
(b) RT activity which "showed a preference for poly-A-oligo-dT12-18
and poly-C-oligo-dG12-18 over poly-dA-oligo-dT12-18, a feature which
usually distinguishes retroviral enzymes from cellular DNA polymerases.
The maximum activity was obtained with Mg2+ over Mn2+ with poly-A-oligo-dT
as template primer as previously described for human retrovirus
such as HTLV or LAV". They also reported the finding of "viral"
particles and RT in the culture from the second sibling. In the
ELISA his serum reacted with LAV and IDAY2 (immunodeficiency-associated
virus=F0the material from the culture of the first patient which
banded at 1.16 gm/ml). In the WB, the p24 of IDAV2 was recognised
by his serum. Montagnier and his colleagues concluded: "Our
findings are consistent with the hypothesis that retroviruses such
as that found in our patients can be transmitted by way of plasma
products" (Vilmer et al., 1984).
Using similar methods researchers from the CDC and the Children's
Hospital of Los Angeles reported in 1985 the isolation of HIV from
6 of 19 healthy seropositive haemophiliacs (Gomperts et al., 1985).
In 1987, another group of American researchers reported the isolation
of HIV from 16 of 66 (24%) haemophiliacs seropositive for HIV, but
not from any of the six without HIV antibody. For this, patients'
PBMC were co-cultured with cells from healthy seronegative donors
that had been stimulated with PHA. To the co-cultures they also
added IL-2 and polybrene. The findings in the culture of:
(a) RT, "An assay count of 104 cpm/mL (after subtraction of
cellular polymerase activity) was considered positive for virus",
using An.dT12-18 as template-primer;
(b) cells positive for viral RNA by cytoplasmic dot blot hybridisation;
were considered proof of HIV isolation (Andrews et al., 1987).
Using the same co-culture techniques and conditions as the above
authors, in 1988 Jackson et al tested "75 unselected hemophiliacs
to determine whether patients positive for HIV-1 antibody are actively
infected rather than immunized by viral proteins in non-heat-treated
factor VIII or IX concentrates". An "ELISA kit that primarily
detects the core p24 antigen of HIV-1" was used to test the
culture. The finding of two serial supernatant fluid samplings as
positive, "with the later sampling showing greater reactivity",
was considered synonymous with HIV isolation. They reported HIV
isolation from "55 (98%) of 56" haemophiliacs seropositive
for HIV and concluded "that antibody-positive hemophiliacs
have been actively infected by HIV-1" (Jackson et al., 1988).
In 1989 Schneweis et al reported that between 1986 and 1988 they
were able to "isolate HIV" from 70 of 211 (33%) of haemophiliacs
who were seropositive for HIV. "After March 1988 an increase
in sensitivity of virus isolation was attained by testing the supernatants
of the culture for the presence of p24 antigen (p24Ag) instead of
reverse transcriptase (RT)" (Schneweis et al., 1989). One year
later the same authors "isolated" HIV from 29 of 46 haemophiliacs
(63%) (Wagner et al., 1990). As can be seen, by HIV isolation is
meant detection of one or more of the following phenomena: rarely,
virus-like particles and positive hybridisation signals for "viral"
RNA, and most often, RT and p24. Elsewhere we have presented evidence
that detection of these phenomena cannot be considered synonymous
with isolation. They can only be used for viral detection, even
then if, and only if, they are first shown to be specific for HIV.
The above phenomena have been discussed in detail (Papadopulos-Eleopulos
et al., 1993a) and it has been shown that none is specific for HIV
or even for retroviruses. Below some additional points regarding
virus-like particles, RT and p24 will be considered, (additional
points regarding hybridisation have been presented above).
Although the origin and role of "retrovirus particles"
are not known, they are considered ubiquitous and this is especially
the case in cell cultures and in pathological tissue. In 1969, Chopra
et al, noticing that "Viruslike particles resembling the C-type
[some classify HIV as a C-type] particles associated with mouse
leukemia have been reported in human leukemic tissues by a number
of investigators" reported that: "These particles have
been observed in the density gradient purified fractions of milk
samples obtained from women having breast cancer and from milk of
a normal woman with a family history of breast cancer. A few particles
have also been detected in tissue-culture of a breast cancer biopsy"
(Chopra & Feller, 1969). Levine et al examined (blindly) plasma
of leukaemic and healthy individuals "A specimen was considered
positive if there were numerous double-membraned particles with
dense nucleoid which were about 100uu in diameter and comparable
to the type C particles described by Porter and Dalton. A specimen
was designated as suspicious if particles were found which were
morphologically similar to those in positive specimens, but were
very few in number. Specimens with numerous but less typical particles
and "empty" particles were also considered suspicious.
All other specimens were classified as negative...In this study
the problems of false positives was largely eliminated by using
ultrathin sections of high speed plasma pellets". They reported
that "Of 45 patients with "myelocytic leukemia, five with
acute and four with chronic myelocytic leukemia showed multiple
virus-like particles. Seven additional patients had similar particles
in lesser numbers or particles devoid of the dense nucleoid. In
these 16 patients the particles were detected when the disease was
untreated or not responding to therapy. Three patients with acute
myelocytic leukemia and numerous virus-like particles in the florid
leukemic phase showed no particles while in complete or partial
remission. Numerous particles were found in the plasma of one patient
with acute lymphocytic leukemia but none were found in samples from
14 patients with chronic lymphocytic leukemia. One suspicious sample
was obtained from a patient with infectious mononucleosis but 14
other nonleukemic samples were negative" (Levine et al., 1967).
In 1972, virus-like particles with morphological characteristics
similar to those ascribed to HIV by some researchers (Lentiviruses),
were reported in cultures of human brain cells (Hooks et al., 1972).
By 1974, researchers from the Koch-Institute in Germany including
Gelderblom reported virus-like particles in HeLa cells, and Canadian
researchers reported the same particles in cultures of marrow cells
from leukaemic patients (Bauer et al., 1974; Mak et al., 1974; Watson
et al., 1974). In conclusion, particles with morphological characteristics
ascribed to HIV are not specific to this virus.
Although at present some of the best known AIDS researchers consider
RT as being the "sine qua non" of retroviruses, and regard
the detection of reverse transcription in lymphocyte cultures from
AIDS patients not only as proof of the presence of such viruses
but of HIV itself, according to some of the best known retrovirologists
including the discoverers of RT, reverse transcription is a property
of all cells, and is by no means confined to retroviruses (Temin
& Baltimore, 1972; Varmus, 1987).
"Reverse transcriptase (RT) was first discovered as an essential
catalyst in the biological cycle of retroviruses. However, in the
past years, evidence has accumulated showing that RTs are involved
in a surprisingly large number of RNA-mediated transcriptional events
that include both viral and nonviral genetic entities...the possibility
that reverse transcription first took place in the early Archean"
is supported by a number of facts and "the hypothesis that
RNA preceded DNA as cellular genetic material" (Lazcano et
As has already been stated, when the HIV researchers Andrews and
colleagues used RT for proving HIV isolation from haemophiliacs
"An assay count of 104 cpm/mL (after subtraction of cellular
polymerase activity) was considered positive for virus". However,
the demonstration of higher levels of reverse transcription from
the cells of haemophiliacs is not proof that the activity is due
to HIV. How does one know that the higher activity of these cells
in not due to:
(a) activation "of cellular polymerase activity" by factor
VIII itself or the many contaminants present in Factor VIII preparations
to which haemophiliacs are exposed?
(b) the many factors (PHA,IL-2, polybrene) to which the haemophiliacs
cultures are exposed?
Even if RT were a property only of viruses, it is not specific
to retroviruses. According to Varmus: "Reverse transcription
was assigned a central role in the replication of other viruses
[hepatitis B and cauliflower mosaic viruses] and in the transposition
and generation of other kinds of eukaryotic DNA" (Varmus, 1988).
"The hepatitis B viruses (HBVs) are small DNA viruses that
produce persistent hepatic infections in a variety of animal hosts
and replicate their DNA genomes via reverse transcription of an
RNA intermediate. All members of this family contain an open reading
frame (ORF), "P" (for pol), which is homologous to retroviral
pol genes" [pol=3Dpolymerase] (Chang et al., 1989). "Hepatitis
B virus (HBV) resembles retroviruses, including HIV, in several
respects. In particular, both viruses contain reverse transcriptase,
and replicate through an RNA intermediate". Because of this,
it has been suggested that hepatitis B infection should be treated
with the same antiretroviral agents as HIV infection (Mitsuya &
Broder, 1989). At present, evidence exists which shows that although
the major target organ for hepatitis B virus is the liver, cells
other than hepatocytes "including peripheral blood lymphocytes
and monocytes, may become infected with HBV" (Neurath et al.,
1992). Lymphocyte stimulation in general and PHA stimulation in
particular is associated with production of hepatitis B virus from
peripheral blood lymphocytes in patients infected with HBV including
"viral replication in chronic hepatitis B infection of childhood"
(Vegnente et al., 1991; Sarria et al., 1993). It is of pivotal significance
to note that 98% of HIV seropositive patients with haemophilia are
infected with hepatitis B virus (Brenner et al., 1991). It is also
of interest to note that AIDS patients suffer frequently from bacterial
infections and that "bacteria too, have reverse transcriptases"
In 1989 Blattner wrote: "Assays for reverse transcriptase,
the unique viral enzyme, employ special oligonucleotide templates
in the presence of magnesium. A characteristic profile of enzyme
activity suggests the presence of a retrovirus, but false positivity
arising from cellular enzyme activity or false negativity because
of low reverse transcriptase level make this technique too unreliable
for epidemiologic application" (Blattner, 1989). However, there
is no "characteristic profile of enzyme activity" in the
haemophilia cultures/co-cultures and no "special oligonucleotide
templates" are used. To prove HIV infection, all researchers
use the template-primer poly-A-oligo-dT12-18 (An.dT15).
However, this template-primer is not specific to retroviral RTs.
As far back as 1972 Gallo and his colleagues showed that reverse
transcription of the template-primer An.dT15 can be achieved with
material obtained from cultures of "PHA stimulated (but not
unstimulated) normal human blood lymphocytes", which in sucrose
density gradients bands at 1.16 gm/ml (Gallo et al., 1973). Not
only is this template-primer not specific to retroviral RT, but
all the cellular DNA polymerases, =E0, =E1 and y, can copy An.dT15
(Sarngadharan et al., 1978). In fact, in 1975, an International
Conference on Eukaryotic DNA polymerases (Weissbach et al., 1975)
defined DNA polymerase y, "a component of normal cells"
(Robert-Guroff et al., 1977), "found to be widespread in occurrence"
(Sarngadharan et al., 1978), whose activity can be increased by
many factors including PHA stimulation (Lewis et al., 1974), as:
the enzyme which "copies An.dT15 with high efficiency but does
not copy DNA well" (Weissbach et al., 1975). Thus, reverse
transcription including that of the primer-template An.dT15 cannot
be considered specific to HIV or even to retroviruses.
The p24 protein
The p24 protein is considered to be coded by the HIV gag gene,
that is, by the gene which codes the group specific antigens of
retroviruses. As far back as 1974 Gelderblom and his colleagues
wrote "While the virus envelope antigens are primarily virus-strain
specific, the bulk of internal proteins of the virion with molecular
weight (mw) between 10,000 d and 30,000 d are group-specific (gs)
for viruses originating in a given animal species (gs-spec. antigens).
The major protein constituent of mammalian C- type oncornaviruses
with a molecular weight in the range of 30,000 d was found to possess,
besides gs spec. antigen, an antigenic determinant that is shared
by C-type viruses of many mammalian species including monkeys and
was thus termed gs interspecies (gs-interspec.) antigen" (Bauer
et al., 1974). As late as 1989 Blattner stated: "It may be
feasible to use viral antigen probes to look for cross-reactive
antibodies, since certain viral proteins, particularly the polymerase
and gag proteins may be highly conserved between subtypes of virus"
(Blattner, 1989). Thus, even if p24 were to be specific to retroviruses,
it cannot be HIV specific. Indeed, apart from a joint publication
with Montagnier in 1988 (Gallo & Montagnier, 1988) where it
is claimed that p24 is unique to HIV, Gallo and his colleagues have
repeatedly stated that the p24 of HIV and of two other human retroviruses,
HTLV-I and HTLV-II, which Gallo claims to have isolated from humans,
immunologically cross-react (Wong-Staal & Gallo, 1985).
The whole blood cultures of 49/60 (82%) of "presumably uninfected
but serologically indeterminate" individuals and 5/5 seronegative
blood donors" were found positive for p24 (Schupbach et al.,
1992). The "HIV proteins (p17, p24)" appear in the blood
of patients (previously negative for all HIV markers) following
"transfusions of HIV-negative blood and UV-irradiation of the
autoblood" (Kozhemiakin & Bondarenko, 1992). p24 is detected
in a significant number (up to 36% of patients with systemic lupus
erythematosus (Barthel & Wallace, 1993). Detection of p24 has
been also reported in organ transplant recipients. In one kidney
recipient (the donor was negative for p24 antigen) who, 3 days following
transplantation developed fever, weakness, myalgias, cough and diarrhoea,
all "Bacteriological, parasitological and virological samples
remained negative [including HIV PCR]. The only positive result
was antigenaemia p24, positive with Abbot antigen kits in very high
titers of 1000pg/ml for polyclonal and 41pg/ml for monoclonal assays.
This antigenaemia was totally neutalizable with Abbot antiserum
anti-p24...2 months after transplantation, all assays for p24-antigen
became negative, without appearance of antibodies against HIV. Five
months after transplantation our patient remains asymptomatic, renal
function is excellent, p24 antigenaemia still negative and HIV antibodies
still negative" (Vincent et al., 1993). Using two kits, the
Abbot and Diagnostic Pasteur, in one study, p24 was detected transiently
in 12/14 kidney recipients. Peak titres ranged from 850 to 200 000
pg/ml 7-27 days post- transplantation. Two heart and 5/7 bone marrow
recipients were also positive, although the titres were lower and
ranged from 140-750 pg/ml. Disappearance of p24 took longer in kidney
(approximately 6 months) than in bone-marrow (approximately 4-6
weeks) recipients. According to the authors: "This may be related
to differences in immunosuppression therapy". Discussing their
findings they wrote: "The observation of a 25- 30kD protein
[the French researchers report p24 as p25] binding to polyclonal
anti-HIV human sera after immunoblots with reactive sera raises
several questions. This protein could be related to a host immune
response to grafts or transplants...Its early detection after transplantation
might indicate the implications of immunosuppression therapy...The
25-30kD protein could therefore be compared with the p28 antigen
recently described with human T-cell-related virus lymphotropic-endogenous
sequence...The characterization of this 25-30kD protein may represent
an important contribution to the detection of HIV-1-related endogenous
retroviruses" (Agbalika et al., 1992).
There are many reasons why the p24 detected in the sera and cultures
of haemophiliacs, like the p24 detected in organ recipients may
not be the protein of an exogenous retrovirus, HIV, but either a
non-viral-protein or the protein of an endogenous retrovirus:
1. Like transplant recipients, haemophiliacs receive material derived
from other humans;
2. Like organ transplant recipients, haemophiliacs are immunosuppressed
3. HIV cannot be "isolated" unless the cultures are mitogenically
4. The normal human genome contains many copies of endogenous retroviral
sequences (proviruses), "including a complex family of HIV-1
related sequences" (Horwitz et al., 1992), a "large fraction"
of which "may exist within a host cell as defective genomic
fragments. The process of recombination however may allow for their
expression as either particle or synthesis of a new protein(s)"
(Weiss et al., 1982; Varmus & Brown, 1989; Cohen, 1993; Lower
& Lower, 1993; Minassian et al., 1993). Varmus describes as
follows the genetic behaviour of retroviruses: "During the
virus life cycle, several interesting genetic and quasi-genetic
phenomena may occur, especially if cells are infected by more than
one virus: production of heterozygotic dimeric genomes, formation
of pseudotypes at high frequencies (particles with core proteins
and genome provided by one virus and envelope proteins by another),
frequent deletions and nucleotide substitutions, and recombination
between related, coinfecting viruses. [Recombination between retroviruses
is surprisingly efficient but its mechanistic basis has not been
resolved]" (Varmus, 1988).
5. Cultivation of normal "non-virus" producing cells
leads to retroviral production (expression), "the failure to
isolate endogenous viruses from certain species may reflect the
limitations of in vitro cocultivation techniques" (Todaro et
al., 1976). The expression can be accelerated and the yield increased
by exposing the cultures to mitogens, mutagens or carcinogens, co-cultivation
techniques and cultivation of cells with supernatant from non-virus
producing cultures (Toyoshima & Vogt, 1969; Aaronson et al.,
1971; Hirsch et al., 1972). For HIV isolation, in most instances,
all the above techniques are employed. Thus, even if "true"
(Popovic et al., 1984) retroviral isolation can be achieved from
the cultures/co-cultures of tissues from haemophiliacs, it would
be difficult if not impossible to be certain that the retrovirus
in question is an exogenous retrovirus which is acquired through
factor VIII administration. For such evidence to be accepted as
proof of the existence of HIV, the activation of an endogenous provirus
or a provirus assembled by recombination of endogenous retroviral
and cellular sequences would need to be rigorously excluded.
Thus, although AIDS researchers acknowledge that:
(a) plasma is "unlikely to be a meaningful source of HIV infection";
(b) cell free particles in plasma lack the gp120 protein which
is "crucial to HIV's ability to infect new cells";
(c ) factor VIII preparations are cell free;
(d) the physical processes employed in the manufacture of factor
VIII even in the absence of heating, destroy both cells and viruses;
AIDS researchers claimed and continue to claim that "HIV"
has been "isolated" from haemophiliacs. However, and in
spite of this affirmation, the above data strongly signifies that
the HIV phenomena (particles, RT, antibody-antigen reactions (WB),
HIV-PCR-hybridisation) observed in patients with haemophilia, whatever
they represent, are inconsistent with the parenteral acquisition
of an exogenous retrovirus.
Lastly, "HIV" has been "isolated" from children
(a) who had no other risk factors other than haemophilia;
(b) where each plasma unit from which factor VIII was made "had
been tested for HIV antibody, hepatitis B surface antigen and alanine
aminotransferase, usually within 2 days after collection" (Remis
et al., 1990);
(c ) where factor VIII was heat treated at 60=F8C for 30 hours
(according to some authors HIV is "completely inactivated in
the samples within a few minutes", of heating (Hilfenhaus et
(d) where the source plasmas from which the lots of factor VIII
were made were retested "within several months after donating
factor VIII, and were found negative" (Neumann et al., 1990;
Remis et al., 1990).
This is as close a proof as one can get that what has been called
HIV infection in haemophiliacs is not caused by an exogenous retrovirus
to which haemophiliacs have been exposed by the administration of
factor VIII preparations.
It is generally accepted that in patients with haemophilia, HIV
destroys T4 lymphocytes leading to acquired immune deficiency. Although
this view has prevailed for ten years, at least one well known group
of researchers of AIDS in haemophiliacs, that from the University
of Bonn, questioned the above relationship between HIV and T4 cells
as recently as 1990. "It is not clear whether the virus-host
interrelationship in HIV infection is regulated primarily by the
virus or by the host; i.e., are CD4+ cells depleted by non- viral
mechanisms and does the virus adjust itself to the weakened defense?
Or is the depletion of CD+ cells the consequence of the spread and
cytopathogenicity of virulent viral variants, which developed at
random from avirulent precursors?" (Schneweis et al., 1990).
Discussing their data a year earlier they concluded "The results
suggest that reactivation of HIV occurs when immune deficiency has
become manifest" (Schneweis et al., 1989). The question whether
HIV leads to T4 cell depletion or conversely whether T4 cell depletion
leads to "HIV infection" (particles, RT in cultures, antigen/antibody
reactions, "HIV-PCR"), can only be resolved by having
direct evidence that HIV destroys the T4 cells of haemophiliacs.
No such evidence exists. An indirect method of resolution is the
examination of the chronological sequence of HIV infection and T4
cell depletion. Numerous reports from many well known researchers
of AIDS in haemophiliacs have shown that T4 cell depletion precedes
1. Mortimer and his colleagues state, "The OKT4 subset was
reduced in both seropositive (p<0.01) and seronegative (p<0.05)
haemophiliacs but there was no difference between seropositive and
seronegative patients" (Moffat et al., 1985);
2. Weiss and colleagues report, "We have thus been able to
compare lymphocyte subset data before and after infection with HTLV-III.
It is commonly assumed that the reduction in T-helper-cell numbers
is a result of the HTLV-III virus being tropic for T-helper-cells.
Our finding in this study that T-helper-cell numbers and the helper/suppressor
ratio did not change after infection supports our previous conclusion
that the abnormal T-lymphocyte subsets are a result of the intravenous
infusion of factor VIII concentrates per se, not HTLV-III infection"
(Ludlam et al., 1985);
3. Kessler and colleagues found, "Repeated exposure to many
blood products can be associated with development of T4/T8 abnormalities"
including "significantly reduced mean T4/T8 ratio compared
with age and sex-matched controls" (Kessler et al., 1983);
4. In 1984, Tsoukas et al observed that amongst a group of 33 asymptomatic
haemophiliacs receiving factor VIII concentrates, 66% were immunodeficient
"but only half were seropositive for HTLV-III", while
"anti-HTLV-III antibodies were also found in the asymptomatic
subjects with normal immune function". They summarised their
findings as follows: "These data suggest that another factor
(or factors) instead of, or in addition to, exposure to HTLV-III
is required for the development of immune dysfunction in haemophiliacs"
(Tsoukas et al., 1984);
5. By 1986 researchers from the CDC concluded: "Haemophiliacs
with immune abnormalities may not necessarily be infected with HTLV-III/LAV,
since factor concentrate itself may be immunosuppressive even when
produced from a population of donors not at risk for AIDS"
(Jason et al., 1986);
6. In 1985 Montagnier (Montagnier, 1985) wrote: "This [clinical
AID] syndrome occurs in a minority of infected persons, who generally
have in common a past of antigenic stimulation and of immune depression
before LAV infection".
Thus, haemophiliacs, may develop immune deficiency before HIV infection,
that is, HIV is not necessary for the decrease in T4 cells observed
in haemophiliacs. Furthermore, to date, there is no evidence either
from the haemophilia studies or from studies in any other AIDS risk
group, that HIV can induce immune deficiency (Papadopulos-Eleopulos
et al., 1994). In other words, HIV is neither necessary nor sufficient
for the appearance of immune deficiency (decrease in T4 cells).
However, there is ample evidence which shows that:
1. Decrease in T4 cells in AIDS patients is not due to destruction
of T4 cells, but due a change to a T8 phenotype (Papadopulos-Eleopulos
et al., 1994);
2. There is no correlation between T4 cell numbers and the clinical
syndrome in any AIDS risk group (Allain et al., 1987; Papadopulos-Eleopulos
et al., 1994).
That is, decrease in T4 cell numbers is neither necessary nor sufficient
for the appearance of the clinical syndrome.
Clinical and classification considerations
In 1981, high frequencies of Kaposi's sarcoma (KS), Pneumocystis
carinii pneumonia (PCP) and a small number of other diseases induced
by other opportunistic infectious agents, that is by agents which
are ubiquitous but which usually produce clinical disease only when
the host defense mechanisms are depressed, were observed in gay
men in the United States.
Some of the gay men with KS or PCP were tested for immunological
abnormalities and a significant number were found to have low numbers
of T4 cells, that is, "cellular immune deficiency". Because
of this, the term gay related immune deficiency (GRID) was first
used to describe the disease in these patients but, not long after,
this was changed to AIDS.
In 1982, the CDC defined AIDS as "illnesses in a person who
1) has either biopsy-proven KS or biopsy- or culture-proven life-threatening
opportunistic infection, 2) is under 60, and 3) has no history of
either immunosuppressive underlying illness or immunosuppressive
therapy" (CDC, 1982). In addition to PCP, the "serious
OI" were "meningitis, or encephalitis due to one of more
of the following: aspergillosis, candidiasis, cryptococcosis, cytomegalovirus,
nocardiosis, strongyloidosisis, toxoplasmosis, zygomycosis, or atypical
mycobacteriosis (species other than tuberculosis or lepral); esophagitis
due to candidiasis, cytomegalovirus or herpes simplex virus; progressive
multifocal leukoencephalopathy; chronic enterocolitis (more than
4 weeks) due to cryptosporidiosis; or unusually extensive mucocutaneous
herpes simplex of more than five weeks duration".
It must be pointed out that no one of the diseases which constituted
AIDS, the AIDS indicator diseases, was new. What was new was the
high frequency of these diseases in gay men (CDC, 1982; CDC, 1982).
In the same year, Robert Gallo, Myron Essex and James Curran put
forward the hypothesis that the cause of AIDS is a virus, the retrovirus,
HTLV-I or a similar virus (Gallo, 1987). According to this theory,
the retrovirus induced immune deficiency by causing the destruction
of T4 cells, which in turn led to the appearance of KS, PCP and
other OI, that is, AIDS. In order to obtain evidence in support
of the above theory, that is, that AIDS was caused by an infectious
agent, the CDC formed a task-force, composed of 32 individuals,
mainly physicians, which "actively surveyed physicians in 18
major metropolitan areas in the United States by letter and telephone
to inquire about Kaposi's sarcoma in persons under 60 years of age
or opportunistic infections in patients without a known predisposing
factor since January 1979...a formal request was made to all state
health departments to notify the CDC of illnesses suspected of fitting
the [above] case definition" (CDC, 1982). Since HTLV-I was
claimed to be transmitted by blood and blood products, patients
with haemophilia became a specific target. In July 1982 the CDC
reported the first three cases of "Pneumocystis carinii pneumonia
among persons with Haemophilia A".
The first patient was a 62 year old individual with a one year
history of weight loss. The treatment and previous medical history
was not given. In December 1981, following the development of cough
and fever he was found to be "lymphopenic, and chest X-ray
revealed interstitial infiltrates compatible with viral pneumonia".
He was treated with corticosteroids resulting in an "overall
clinical improvement". In January 1982 he presented with "severe
respiratory distress" and PCP was proven by open lung biopsy.
The second patient, 59 years old, with a history of weight loss,
"apthous- like ulcers and anterior cervical adenopathy beginning
in October 1981" was diagnosed with oropharyngeal candidiasis
in February 1982. No previous medical history or treatment was given.
In May 1982 he was hospitalised "with symptoms including nausea,
vomiting, and recurrent fever. Pneumonia was diagnosed, and P. carinii
and cytomegalovirus (CMV) were repeatedly identified from lung tissue
or bronchial secretions". He also had decreased T4 cell number,
increased T8 cell numbers and a decreased T4/T8 ratio.
The third patient, 27 years old, with a history of fever and urinary
frequency and urgency (treatment not given), was diagnosed with
PCP in October 1981. In February 1982 he was treated with ketoconazole.
By April he developed fever, splenomegaly, anaemia, lymphopenia
and Mycobacterium avium was grown from a number of tissues. He also
had "a reduction in absolute numbers of circulating T-cells".
Subsequently, he was found to have decreased T4 cells, increased
T8 cells and a low T4/T8 ratio.
From these case histories it was concluded that "the clinical
and immunological features of these three patients are strikingly
similar to those recently observed among certain individuals from
the following groups: homosexual males, heterosexuals who abuse
IV drugs, and Haitians who recently entered the United States. Although
the cause of the severe immune dysfunction is unknown, the occurrence
among the three hemophiliac cases suggests the possible transmission
of an agent through blood products" (CDC, 1982).
As a consequence, the CDC "notified directors of hemophilia
centers about these cases and, with the National Hemophilia Foundation,
has initiated collaborative surveillance". In the same year,
Ragni and colleagues found two haemophiliacs with decreased T4 and
increased T8 cell numbers, elevated IgG and IgM levels and lymphadenopathy
and concluded that their findings were "consistent with the
diagnosis of AIDS" (Ragni et al., 1983).
By October 1983, the CDC had 23 reports of AIDS cases in haemophiliacs,
18 in the USA and 5 in other countries, none with KS. Two of the
above cases had other risk factors, one was an IV user, the other
gay (Jason et al., 1984). By the end of 1984, the number of haemophilia
AIDS cases increased to 67 (Levine, 1985). By this time, Gallo's
claim that AIDS in all risk groups, gay men, IV users, blood transfusion
recipients, and haemophiliacs is caused by a new retrovirus, HTLV-III,
later renamed HIV, became generally accepted. By the end of June
1985, 80 haemophiliacs in the USA and five in the United Kingdom
were reported with AIDS, none with KS (Jones et al., 1985). At about
the same time it became known that by 1982 the vast majority of
haemophiliacs tested positive for HIV. "Yet the attack rate
of AIDS in hemophiliacs is not steadily climbing per reported period,
[in gay men it was increasing exponentially]. In addition, the last
two reporting periods [last quarter of 1984, and first quarter of
1985 when HIV testing was introduced] contains a disproportionate
number of patients with mild and moderate disease" (Levine,
1985). Indeed, as has been seen, the only clinical symptoms in the
two patients reported by Ragini et al consistent with the "diagnosis
of AIDS", was lymphadenopathy.
Some published reports represent the gallant efforts made by some
researchers to prove that HIV infection in haemophiliacs, like HIV
infection in gay men, leads to neurological complications. Researchers
from the Royal Postgraduate Medical School in London reported two
fatal haemophilia cases. The first patient presented with "lethargy,
poor concentration, and difficulty with micturition. Examination
disclosed diminished cognitive function and brisk reflexes. Computed
tomography (CT) of the brain showed dilated lateral ventricles and
widened sulci consistent with cerebral atrophy". Four months
later "he was incontinent and had difficulty walking and showed
signs of a pyramidal tract lesion". One month later he was
"unable to walk, had paranoid delusions. Relentless neurological
deterioration followed with painful spastic quadriparesis and convulsions".
The second patient presented with "weight loss, confusion,
unilateral cerebellar dysfunction, and diplopia which was diagnosed
clinically as an internuclear opthalmoplegia. A cerebral CT scan
showed low attenuation areas in the white matter of the frontal
lobes and also in the right parietal lobe". The above clinical
signs were followed by coma. Although no general or neuropathological
examination was conducted in either of these patients (permission
for autopsy was refused), both these cases of "subacute encephalopathy"
were attributed to HIV because the patients were HIV positive and
had a low T4/T8 ratio (Rahemtulla et al., 1986). Similarly, cerebral
toxoplasmosis attributed to HIV, also without neuropathological
examination, has also been reported in haemophiliacs (Esiri et al.,
The introduction of "mild and moderate disease" as indicating
AIDS, which commenced in the last quarter of 1984, co-incided with
the acceptance of HIV as the cause of AIDS in all risk groups and
the redefinition of AIDS by the CDC. Before this date practically
all AIDS was KS and PCP. According to the 1985 CDC definition, "a
case of acquired immunodeficiency syndrome (AIDS) is an illness
I. one or more of the opportunistic diseases listed below (diagnosed
by methods considered reliable) that are at least moderately indicative
of underlying cellular immunodeficiency; and
II. absence of all known underlying causes of cellular immunodeficiency
(other than LAV/HTLV-III infection) and absence of all other causes
of reduced resistance reported to be associated with at least one
of those opportunistic diseases.
Despite having the above, patients are excluded as AIDS cases if
they have negative result(s) on testing for serum antibody to LAV/HTLV-
III, do not have a positive culture for LAV/HTLV-III, and have both
a normal or high number of T-helper (OKT4 or LEU3) lymphocytes and
a normal or high ratio of T-helper to T-suppressor (OKT8 or LEU2)
lymphocytes. In the absence of test results, patients satisfying
all other criteria in this definition are included as cases"
A number of additional AIDS indicator diseases were added to the
1982 definition. These included: lymphoma limited to the brain,
disseminated histoplasmosis, isosporiasis and non-Hodgkin's lymphoma.
Although HIV was accepted as being the cause of AIDS in all AIDS
risk groups, there were significant differences between the groups.
1. If HIV is the cause of AIDS in all the above risk groups, one
would expect the rate of conversion to clinical AIDS in all HIV
positive individuals to be the same. This is not the case. In a
cohort of gay men in the USA, the three year actuarial progression
rate was 22% (Moss et al., 1988). In a cohort of haemophiliacs,
the annual incidence of AIDS ranged from zero during the first year
after seroconversion to 7% during the eight year followup with an
8 year cummulative rate of 13.3% (Goedert et al., 1989). In the
United Kingdom three percent of haemophilia patients developed AIDS
by 3 years after seroconversion and 7% by 5 years post seroconversion
(Darby et al., 1989);
2. The clinical syndrome in haemophiliacs is different from that
in gay men. KS, one of the two most significant and frequent diseases
in gay men, for whose explanation the HIV hypothesis was put forward,
is practically non- existent in haemophiliacs. Such is also the
case with oral hairy leukoplakia (Greenspan & Greenspan, 1989).
To determine the forms of neuropathological and systemic pathology
in HIV positive haemophiliacs as compared to other HIV positive
subjects, Esiri et al examined the brains of 42 HIV seropositive
individuals. Amongst these were 11 haemophiliacs and 29 gay men.
Four of the haemophiliacs were classified as AIDS as were the majority
of the gay men. "The prevalences of opportunistic infections
of the central nervous system were significantly higher in the non-haemophiliacs
(cerebral toxoplasmosis 23% (7), progressive multifocal leucoencephalopathy
10% (3), and cerebral cytomegalovirus infection 19% (6) in the non-haemophiliacs
v no cases in the haemophiliacs). The prevalences of fresh and old
intracranial haemorrhages and cirrhosis of the liver were significantly
higher in the haemophiliacs (fresh intracranial haemorrhage 45%
(5), old intracranial haemorrhage, 36% (4) and cirrhosis of the
liver 27% (3) in the haemophiliacs v no cases in the non- haemophiliacs)".
Discussing their results Esiri and colleagues wrote: "The rarity
of opportunistic infections in the central nervous system and elsewhere
in haemophiliacs is in keeping with many of them dying at an earlier
(pre-AIDS) stage in the development of HIV associated immunodeficiency
than do most subjects with HIV infection. [To the contrary, as has
been stated above, HIV seropositive haemophiliacs live longer than
HIV seropositive gay men]. Consistent with this suggestion is Hilgartner's
view that the pattern of disease due to HIV infection in haemophiliacs
differs from that in other groups at high risk, and the observation
of Darby et al that a substantial burden of fatal disease occurs
among haemophiliacs who are positive for HIV and not formally diagnosed
as having AIDS. If our cases of haemophilia are representative of
others much of this fatal disease would seem to be accounted for
by cerebrovascular and liver disease" (Hilgartner, 1987; Esiri
et al., 1989; Darby et al., 1990).
Once again, it is of pivotal significance to note that even in
the early years of the recognition of AIDS, it was agreed that in
haemophiliacs, there was "an immunodeficiency independent of
HTLV-III infection" (Hollan et al., 1985; Madhok et al., 1986).
That is, haemophiliacs have "known underlying causes of cellular
immunodeficiency (other than LAV/HTLV-III)", HIV). Thus, according
to the 1985 AIDS definition, haemophiliacs cannot be AIDS cases.
Furthermore, although a prerequisite of the diagnosis of AIDS in
the 1985 definition was a positive test for HIV, of all AIDS cases
reported in the two year period 1985-1987 in New York City and San
Francisco, which constituted approximately one third of all AIDS
cases in the USA, "less than 7% have been reported with HIV-antibody
test results" (CDC, 1987).
Like the 1982 definition, the 1985 required the diseases which
constituted AIDS, the AIDS "indicator" diseases, to be
definitely diagnosed. However, the New York State Health Department
found that although 13% of the 1329 AIDS cases reported by the beginning
of 1987 did have a positive HIV antibody test, clinically these
individuals' symptoms were suggestive of AIDS but were not definitely
diagnosed. In a similar study researchers from the CDC and the Department
of Health in New Jersey, Puerto Rico, Boston, Washington, D.C. and
Connecticut found that approximately 11% of cases had a presumptive
diagnosis because, according to one AIDS epidemiologist "Many
physicians are familiar enough with AIDS now that when they see
a young man with pneumonia, they can make a reasonable presumptive
diagnosis [of PCP] without resorting to biopsy," (Anonymous,
1987). Thus a significant number of reported AIDS cases did not
meet either the 1982 or the 1985 AIDS definition.
To accommodate the non-compliance with the 1985 AIDS definition,
the CDC claimed that their 1985 definition made it "unnecessarily
difficult to diagnose" AIDS, and thus it underestimated the
number of AIDS cases. In 1987, the CDC yet again redefined AIDS.
The 1987 definition permitted reporting of Acquired Immunodeficiency
(AIDS) cases even if there was no evidence of immune deficiency,
or of a definite diagnosis of at least some of the AIDS indicator
diseases. More importantly, although the definition considered HIV
to be the sole cause of AIDS, individuals could be reported as AIDS
cases even when there was evidence against HIV infection. The major
features of the 1987 definition are:
I. Without laboratory evidence of HIV infection (patients not tested
for HIV or if tested the results were inconclusive), the 1985 indicator
diseases "if reliably diagnosed and other causes of immune
deficiency are ruled out", (that is, immunosuppressive therapy
=F3 3 months before the onset of the indicator disease, a small
number of neoplastic diseases diagnosed =F3 3 months after diagnosis
of the indicator disease, an even a smaller number of congenital
immunodeficieny diseases), "are still accepted as a diagnosis
II. "Regardless of the presence of other causes of immunodeficiency
in the presence of laboratory evidence of HIV infection":
A: Twelve new AIDS indicator diseases, when definitely diagnosed,
indicate AIDS. These include:
(i) extrapulmonary tuberculosis;
(ii) wasting syndrome, that is, involuntary weight loss of >10%
of body weight and either chronic diarrhoea (at least 2 loose stools
per day for =F2 30 days) or chronic weakness and documented fever
(for =F2 30 days, intermittent or constant);
(iii) HIV encephalopathy (shizoid behaviour, general fatigue, malaise,
diminished cognitive function (Gomperts, 1990);
(iv) bacterial infections, multiple or recurrent (any combination
of at least two within a 2-year period) of the following types affecting
a child less than 13 years of age: septicaemia, pneumonia, meningitis,
bone of joint infection, or abscess of an internal organ or body
cavity (excluding otitis media or superficial skin or mucosal abscesses)
caused by Haemophilus, Streptococcus, (including Pneumococcus),
or other pyogenic bacteria.
B. The diseases listed below, even if presumptively diagnosed,
"1. Candidiasis of the oesophagus.
2. Cytomegalovirus retinitis with loss of vision.
3. Kaposi's sarcoma.
4. Lymphoid interstitial pneumonia and/or pulmonary lymphoid hyperplasia
(LIP/LPH complex) affecting a child <13 years of age.
5. Mycobacterial disease (acid-fast bacilli with species not identified
by culture), disseminated (involving at least 1 site other than
or in addition to lungs, skin, or cervical or hilar lymph nodes).
6. Pneumocystis carinii pneumonia.
7. Toxoplasmosis of the brain affecting a patient >1 month of
age."For example: (i) presumptive diagnosis of candidiasis
of the oesophagus is:
"a.Recent onset of retrosternal pain on swallowing; AND b.
oral candidiasis diagnosed by the gross appearance of white patches
or plaques on an erythematous base or by the microscopic appearance
of fungal mycelial filaments in an uncultured specimen scraped from
the oral mucosa".
(ii) presumptive diagnosis of KS is:
"A characteristic gross appearance of an erythematous or violaceous
plaque-like lesion on skin or mucous membrane" (WHO, 1988).
III. If there is "laboratory evidence against HIV infection",
that is, the laboratory tests for HIV infection are negative, but
the patient has all above mentioned causes of immunodeficiency excluded
(a) Pneumocystis carinii pneumonia diagnosed by a definite method
(b) any of the 1985 AIDS indicator diseases diagnosed by a definite
method AND; a T4 cell count <400/mm3.
the patient has AIDS.
Thus the 1987 AIDS definition legitimised the reporting of a person
as suffering from Acquired Immune Deficiency Syndrome, accepted
to be caused by HIV when;
1. Evidence of HIV infection was "not performed or gave inconclusive
results", or even when all the tests were negative that is,
when there was definite evidence that the patient was not infected
2. The absence of any evidence of immune deficiency and even when
the cause of immune deficiency could have been other than HIV;
3. Both in the absence of HIV infection and immune deficiency.
In 1987, it was known that many, including KS and PCP, indicator
diseases of the 1985 definition were difficult to diagnose both
clinically and histopathologically (CDC, 1981; Follansbee et al.,
1982; Hughes, 1987; Beral et al., 1990). Yet, the definition permitted
a person to be reported as suffering from AIDS, even when the indicator
diseases were presumptively diagnosed that is, on the basis of non-specific
findings. In fact, the 1987 definition allowed so many degrees of
freedom that nearly anybody, especially those belonging to a "risk
group", could be reported as an AIDS case. This can best be
illustrated by the following example:
In 1992, "The AIDS Reporting System was searched for all persons
who had been given a diagnosis of AIDS" by investigators from
the CDC (Smith et al., 1993). In seven reported haemophilia "AIDS"
(a) the HIV tests were all negative;
(b) not one of the patients had an AIDS indicator disease: 4 had
common diseases especially frequent in haemophiliacs, haematomas,
hepatitis C infection, thrombocytopenia and oral herpes, and two
patients were asymptomatic;
(c ) all patients had a low (<300/mm3) T4 cell count.
Interestingly, in 1987, merely by redefining AIDS, there was a
sharp increase in AIDS cases in all AIDS risk groups including haemophiliacs.
In 1988, there were 552 cases in haemophiliacs of which 31 were
known to be gay and 12 drug users (Koerper, 1989). Not only did
the 1987 AIDS definition fail to solve the major problems arising
from the 1982 definition, but by then other problems associated
with the HIV hypothesis of AIDS in haemophiliacs became apparent:
1. It is accepted by all HIV researchers that heating factor VIII
preparations destroys HIV. Yet AIDS has been diagnosed in haemophiliacs
who exclusively received heat treated factor VIII (CDC, 1987);
2. Unlike other AIDS risk groups, in haemophilia, thrombocytopenia
and older age are risk factors for the development of AIDS. (Eyster
et al., 1987).
(a) it is well known that older age is associated with both immune
deficiency and increased incidence of OI and malignancies. Indeed,
of the first 3 cases reported to the CDC with PCP in haemophiliacs
in 1982, one of these was not considered to be an AIDS case because
he was 62 years of age;
(b) before the AIDS era the rate of thrombocytopenia in haemophiliacs
was significantly different (p<0.0003) than the normal population.
Eyster et al examined data collected by the Hemophilia Study Group
from 1975 to 1979 on 1551 patients, "To determine whether there
was clinical or laboratory evidence to suggest an abnormality of
immunoregulation in persons with haemophilia before the recognition
of AIDS". Twenty six of 518 (5%) patients whose platelet number
were determined were found to be thrombocytopenic (4 developed idiopathic
thrombocytopenic purpura [ITP]) and 9.3% (94/1013) had lymphocytopenia
(Eyster et al., 1985);
(c ) to elucidate "The attention given to infectious diseases
in haemophiliacs", which has "given rise to the concept
of a novel form of 'immunosuppression' in this population group",
Aronson obtained data from the National Center for Health Statistics
USA regarding primary and associated causes of death in haemophiliacs.
For the years 1968-79, 949 deaths were recorded, "2 patients
had candidiasis listed as the primary cause of death. 66 deaths
were related to pneumonia (10 primary, 56 associated) usually from
unidentified organisms. Many of these pneumonia-associated deaths
occurred in the younger age groups, (25/66 [38%] were in patients
below the age of 45 while only 8% of pneumonia deaths in the normal
male population are below the age of 45)". Aronsen concluded
"it seems possible that many of the unspecified pneumonias
in haemophiliacs in the past would be classified today as AIDS"
(Aronson, 1983). "Death reports from the United States Vital
Statistics System and from the hemophilia center survey" for
the above period 1968-1979 were also analysed by workers from the
CDC and the National Hemophilia Foundation. They found that "an
average of six deaths were reported to the National Center for Health
Statistics annually in 1968-1979 for conditions which could possibly
be related to AIDS" (Johnson et al., 1985). Of 89 haemophilia
deaths in the UK between 1976 and 1980, 11 (12%) died of unknown
causes, 4 (4.5%) of unspecified pneumonias, 7 (8%) of neoplasms
(Rizza & Spooner, 1983).
Thus, AIDS-like diseases in an appreciable number of haemophiliacs
were reported before the AIDS era, 1980. A high frequency of reporting,
or even true incidence of these diseases in this group since 1980
may be due to a number of factors other than HIV:
1. Underreporting of specific causes of death in haemophilia patients
before 1980. In the above mentioned survey by workers from the CDC
and the National Hemophilia Foundation it was found that "The
number of deaths reported among factor VIII-deficient patients in
the hemophilia treatment center survey decreased from 26 deaths
and 24 deaths in 1978 and 1979, respectively, to 18 and 19 deaths
in 1980 and 1981, respectively. The number of deaths then more than
doubled, with 53 deaths reported for 1982. The two-to three-fold
increase in deaths in 1982 include the first five reported of immunodeficiency,
an increase in deaths assigned to haemorrhage unrelated to trauma,
and an increase in deaths unrelated to AIDS or hemophilia. The sharp
increase in deaths across all categories is most likely due to underreporting
of deaths, as a result of hemophilia treatment centers inability
to identify deaths in previous years" (Johnson et al., 1985);
2. The increased reporting of PCP in haemophiliacs may be due to
a true increase of the incidence of PCP or due to:
(a) under diagnosis of PCP in this population before 1980 as a
(i) lack of awareness, one searched for PCP only in immunosuppressed
patients, but before 1980 no one was aware that haemophiliacs were
immunosuppressed. (No immunological tests were carried out in this
(ii) the inadvisability, in haemophiliacs, of performing invasive
procedures which were required for definitive diagnosis;
(b) in the AIDS era, overdiagnosis of PCP after 1980, that is,
pneumonias of unknown aetiology are presumed to be PCP. Even when
pneumonias are "definitely diagnosed" as PCP, this may
not be the case: "one might expect to find P. carinii in the
fluids from bronchoalveolar lavage of about 40 percent of patients
with AIDS who present with symptomatic pneumonia caused by other
organisms" (Hughes, 1987). However, regarding the method for
definite diagnosis of PCP, the CDC definition states: "Pneumocystis
carinii pneumonia (on histology, or microscopy of a 'touch' preparation,
bronchial washings or sputum)" (WHO, 1986);
3. Over diagnosis of AIDS cases. For example, between July 1986
and June 1987, the CDC had 3001 death certificates "that indicated
HIV infection/AIDS", but only 85% met the CDC AIDS definition
4. Increased life span of patients with haemophilia. PCP "in
infants and children with congenital immunodeficiency did not evolve
until after the development of antibiotic therapy which allowed
these children to live long enough to develop a nonbacterial infection"
(Burke & Good, 1973). That this may also be the case in haemophiliacs
is suggested by the following:
(a) in haemophiliacs the risk of AIDS is directly related to age.
In one USA study, eight-year cumulative incidences of AIDS in HIV
positive haemophiliacs have been found to be as follows: 1-11 years
old, 3%; 12-17 years old, 9.2%; 18-25 years old, 14.9%; 26-34 years
old, 19% and 35-70 years old, 29.7% (Goedert et al., 1989). The
CDC also reported that the AIDS cases are "older than the other
hemophilia treatment center patients (p<0.005), with a median
age of 34 years" (Johnson et al., 1985);
(b) before the AIDS era, the life span of patients with haemophilia
was much lower than that of the rest of the population, in 1972
the median age was 11.5 years. This compared with a median age of
26.8 years for the USA male population in 1970. As a result of treatment
with factor VIII the median age increased to 20 years in 1982, and
was 25 years in 1988 (Johnson et al., 1985; Koerper, 1989);
5. "Because of advances in medical practice", in the
past few decades, there has been an increase in the incidence of
immunosuppression and OIs. This may especially be expected in haemophiliacs
since steroids have been used for the treatment of joint problems
(Muller, 1960), for factor VIII inhibitors (Lian et al., 1989) and
for ITP (Eyster et al., 1985), all of which are present in haemophilia
patients more often than in the general population. Joint problems
have also been treated with other immunosuppressive agents such
as radioactive gold or technetium (Fernandez-Palazzi et al., 1984).
In fact, before the AIDS era, "Pretreatment with antihistamines,
corticosteroids and adrenergic agents was recommended for all patients
with hemophilia being treated at home" with factor VIII (Helmer
et al., 1980);
6. HIV positive individuals including haemophiliacs are treated
with AZT. The toxic effects of AZT have repeatedly been stressed
by Lauritsen (Lauritsen, 1990) and Duesberg (Duesberg, 1992). Here,
only some of these properties, especially those of significance
to haemophiliacs, will be mentioned:
(a) bone marrow failure including anaemia, neutropenia and thrombocytopenia
(Callaham, 1991). Many patients require blood transfusion within
weeks of commencing AZT. It is important to note that "the
frequency of lymphocytopenia and thrombocytopenia was increased
in multitransfused factor VIII-deficient hemophiliacs before the
advent of AIDS" and that the latter is a contributing factor
in the development of AIDS in haemophilia (Eyster et al., 1985).
Furthermore, haemophiliacs with thrombocytopenia "usually need
treatment with drugs as zidovudine, corticosteroids or immunoglobulins,
which interfere with the immune system" (Mannucci et al., 1992).
(b) peripheral neuropathy;
(c ) myopathy, "up to one-third of patients taking the drug
for more than a year, at a dose of around 1g daily, develop myopathy".
It is manifest clinically as symmetrical proximal weakness, usually
preceded by and associated with myalgia, together with muscle wasting.
This leads to difficulty in walking and patients may become wheel-chair
or bed bound. (Lane et al., 1993). The toxic effects on muscle eventually
lead to heart and other cardio-vascular and pulmonary problems.
Since the major long term disabilities in haemophiliacs, irrespective
of AIDS, are musculoskeletal disease (Levine, 1985), the above toxic
effects of AZT are of particular interest in this group of individuals.
(d) In the 1960s, before the AIDS era, AZT was developed as an
agent to treat neoplasms. All drugs presently used to treat cancer
are known to be immunosuppressive and to lead to the appearance
of OI. They are also known to be carcinogenic (Papadopulos-Eleopulos,
1982). Before the AIDS era animal evidence showed that AZT is no
exception (Callaham, 1991) and the widespread use of AZT in HIV
positive individuals in the AIDS era has shown that this is also
the case in humans. Thus lymphomas develop in 9% "of AZT-treated
AIDS patients, with Kaposi's sarcoma, pneumonia and wasting disease"
within one year of commencing therapy and it has been calculated
that the "annual lymphoma risk of AZT recipients is about 30
times higher than that of untreated HIV-positive counterparts"
(e) AZT induces liver damage and may cause hepatic failure and
death (Touchette, 1993). This is of particular interest in haemophiliacs
who, regardless of their HIV status, can suffer from chronic liver
disease, which may also "contribute to AIDS-related diseases",
and since the introduction of factor VIII for the treatment of bleeding,
has become the leading cause of death in haemophiliacs (Eyster et
al., 1985; Eyster et al., 1987).
7. Factor VIII. As Levine has pointed out: "To understand
the occurrence of AIDS in haemophilia, it is important to recognize
that each vial of factor VIII concentrate will contain, depending
on manufacturer and lot number, a distillate of clotting factors,
alloantigenic proteins, and infectious agents obtained from between
2500 and 25,000 blood or plasma donors. Until recently, of all the
protein injected in "factor VIII preparations", factor
VIII accounted for only about 0.03-0.05% of the total. The rest
included: albumin, fibrin(ogen), immunoglobulins and immune complexes
(Eyster & Nau, 1978; Mannucci et al., 1992). Even the recent
"high-purity" factor VIII contains "potentially harming
proteins" such as isoagglutinins, fibrin(ogen), split products,
immunoglobulins and, when monoclonal antibodies are used for factor
VIII preparation, murine proteins in addition to albumin (Beeser,
Factor VIII was first introduced in the late 1960s. "In 1975,
the average patient received an estimated 40 000 units of factor
VIII per year (a unit being the equivalent of 1mL of fresh frozen
plasma as to factor VIII content). By 1981, the average patient
was consuming 60 000 to 80 000 units per year" (Levine, 1985).
The introduction of factor VIII led to a dramatic decrease in haemophilia
deaths from bleeding but it also had some harmful effects including
myocardial ischaemia, visual disturbances, headache, dyspnoea, bronchospasm,
hypotension and anemia (Eyster & Nau, 1978; Kopitsky & Geltman,
1986; Beeser, 1991). As previously stated, factor VIII preparations
contain immunoglobulin which may produce systemic reactions such
as pruritus, chills, fever, tremor, flushing, malaise, nausea, vomiting,
back pain and joint pain (van Aken, 1991). Before the AIDS era,
no immunological studies were carried out in haemophiliacs but subsequently,
as has been mentioned, in 1985, Eyster et al showed that the frequency
of lymphocytopenia and thrombocytopenia was increased in haemophiliacs
prior to the AIDS era (Eyster et al., 1985). More recently performed
immunological studies including determination of T4 cell numbers,
led to the generally accepted view that factor VIII itself is immunosuppressive.
Recently, researchers from the UK showed that progression to AIDS
in HIV seropositive haemophiliacs is determined by abnormalities
induced by factors other than HIV all of which existed before seroconversion
(Simmonds et al., 1991). In other words, HIV is not sufficient for
the development of AIDS in patients with haemophilia.
In conclusion, HIV is not necessary for the development of AIDS
in patients with haemophilia. Nonetheless, since:
1. According to the new 1993 CDC AIDS definition, any individual
who is HIV seropositive and who has one ("the lowest accurate,
but not necessarily the most recent") T4 cell count less than
200 cells/uL, irrespective of the clinical situation even if asymptomatic,
has AIDS (CDC, 1992) and,
2. (a) most haemophiliacs test positive for HIV (but AIDS experts
accept that in haemophiliacs a positive antibody test does not prove
HIV infection); (b) most haemophiliacs have a low numbers of T4
cells (but AIDS experts accept that in haemophiliacs the immune
deficiency may be caused by factors other than HIV);
in the future, by definition, virtually all haemophiliacs will
die from no other disease but AIDS caused by HIV.
Note added in proof
After this paper was accepted for publication the
CDC forwarded the authors a copy of its fact sheet (CDC 1994) on
HIV transmission. Given the perilous future for haemophilia patients
enshrined in the CDC's 1993 AIDS definition and cognisant of the
fact that factor VIII has long been supplied as a freeze-dried powder
which may spend many weeks or months waiting use, it is incomprehensible
that the CDC would also, in 1994, communicate the following experimental
data and conclusion: "In order to obtain data on the survival
of HIV, laboratory studies have required the use of artificially
high concentrations of laboratory grown virus...the amount of virus
studied is not found in human specimens or anyplace else in nature,...it
does not spread or maintain infectiousness outside its host. Although
these unnatural concentrations of HIV can be kept alive under precisely
controlled and limited laboratory conditions, CDC studies have shown
that drying of even these high concentrations of HIV reduces the
number of infectious viruses by 90 to 99 percent within several
hours. Since the HIV concentrations used in laboratory studies are
much higher than those actually found in blood or other body specimens,
drying of HIV-infected human blood or other body fluids reduces
the theoretical risk of environmental transmission to that which
has been observed-essentially zero". It is thus inexplicable,
given their own data, that the CDC continues to regard patients
with haemophilia at risk for HIV infection via contaminated factor
VIII concentrates and enigmatic that another explanation for "HIV"
and AIDS in haemophiliacs has not been sought. *