IN SEARCH OF HIV
Analysis of the value of the tests used for 'HIV infection'
By Fabio Franchi
Leadership Medica Sept. 1998
"The task of researchers is to try to falsify as many theories as possible."
-- Karl Popper
"Is there a danger, in molecular biology, that the accumulation of data will
get so far ahead of its assimilation into a conceptual framework that the
data will eventually prove an encumbrance? Part of the trouble is that
excitement of the chase leaves little time for reflection. And there are
grants for producing data, but hardly any for standing back in contemplation".
-- John Maddox (1)
The diagnosis of HIV infection is made according to the results of
particular tests, of which the most important is the Western Blot, also
named “confirmatory test”. This one, on its turn, may or may not be corroborated
by other tests. But, a methodological and comparative analysis of anyone
of them let one conclude for a complete lack of specificity (and real validity).
Antibody tests, viral culture, direct detection of the virus, electronic
micrographs of HIV, HIV isolation allow one to find foundamental errors
in certain premises and in some essential logical constructions.
The argument that what is not proved by single tests can be proved
by them together is not valid from a scientific point of view. The paradoxical
result of this critic examination of the matter is that anyone of the phenomena
attributed to the virus is explainable without the need to postulate its
existence; Electronic micrographs of what has been considered “pure HIV”
- and only recently published - seem to confirm this thesis completely.
According to the AIDS infectious theory, the HIV infection is active
since from the first phases and during the years and it is very difficult
for the organism to get rid of it. Proper tests reveal its presence. Those
which at the moment are at our disposal are essential not only for the
diagnosis (2), but they are used as indicative elements with prognostic
and therapeutical purposes (3).
As science for definition is open to every control and all its demonstrations
must be proved on the basis of logical rules, this study has the object
of verifying if there is effective relation among the different “AIDS tests”
and the meaning attributed to them. We shall examine the most important:
the tests of the antibodies (the ELISA, the Western Blot and the p24),
the viral culture, the genetic identification with amplification methods,
the viral isolation, the micrographs of HIV. We shall analyse and criticize
the procedure followed at present, as presented in the updated specialized
texts including “AIDS, Biology, Diagnosis, Treatment and Prevention” (2)
published in 1997, edited by several top researchers including A. Fauci,
former manager of the National Institute of Health.
In other words we tried to answer to the crucial question as the great
clinician and science methodology scholar, prof. Augusto Murri would have
formulated it: “Why have I to believe that the AIDS test meaning is equivalent
to an infection with the HIV virus?” He thought it was essential, during
one's life, to be in the habit of criticizing before of accepting information.
According to the AIDS
infectious theory, the HIV infection is active since from the first phases
and during the years and it is very difficult for the organism to get rid
of it. Proper tests reveal its presence. Those which at the moment are
at our disposal are essential not only for the diagnosis (2), but they
are used as indicative elements with prognostic and therapeutical purposes (3).
As science for definition
is open to every control and all its demonstrations must be proved on the
basis of logical rules, this study has the object of verifying if there
is effective relation among the different “AIDS tests” and the meaning
attributed to them. We shall examine the most important: the tests of the
antibodies (the ELISA, the Western Blot and the p24), the viral culture,
the genetic identification with amplification methods, the viral isolation,
the micrographs of HIV. We shall analyse and criticize the procedure followed
at present, as presented in the updated specialized texts including “AIDS,
Biology, Diagnosis, Treatment and Prevention” (2) published in 1997, edited
by several top researchers including A. Fauci, former manager of the National
Institute of Health.
In other words we tried
to answer to the crucial question as the great clinician and science methodology
scholar, prof. Augusto Murri would have formulated it: “Why have I to believe
that the AIDS test meaning is equivalent to an infection with the HIV virus?”
He thought it was essential, during one's life, to be in the habit of criticizing
before of accepting information.
ANALYSIS OF THE ANTIBODY TESTS
The tests of the antibodies we shall examine are the Elisa, the Western
Blot, the p24 antigen capture assay.
Fig. 1 - ELISA test procedure scheme.
In Italy, the screening test
(ELISA), “provided with a good sensibility (near 99%) and a lower specificity,
valued about 90-95% in 1988”(4), is used at first to identify the HIV infection.
However these percentages must not deceive, in fact the test predictive
value changes depending on the infection prevalence in a certain population.
According to a theory expressed in 1988 by the AIDS National Commission
(4), in case of screening of the Italian prison population, in comparison
with 6,902 real positive individuals there should have been 1.751 false
positive; on the contrary, in case of blood donors, in comparison with
198 real positive, a good 999,990 should have been false positive (505
times more!). In the over mentioned text (2) the authors assert that the
progressive improvements took sensibility of the test to 95% and its specificity
to 99.8%, in high-risk population. Nevertheless they admit that “the positive
predictive value can greatly fluctuate depending on the population they
study”. Actually this happened. Depending on the information published
on the English authoritative medical magazine The Lancet (5), in 1990 on
20.2 million ELISA tests made in Russia, 20,000 were positive, but only
112 were confirmed by the WB; in 1991, on 30 million ELISA tests, a good
30,000 were positive, but of these only 66 were confirmed by the Western
Blot, that is a minimum percentage (0.002%).
“Then as the ELISA test
has been planned to optimize the sensibility to the detriment of the specificity,
it should not be used by itself for the HIV-1 infection diagnosis without
a confirmation test (2), although in some Countries is used alone.
Fig. 2 - Western Blot test procedure scheme.
The Western Blot is commonly
used as a confirmation test of HIV infection; the combination of the Elisa
and the WB should have a “positive predictive value higher than 99% for
low-risk and high-risk population” (2).
However even the WB does
not seem to be so specific as sustained if we analyze some data at our
A) Even official sources
acknowledge that “for reasonable disagreements it was very difficult to
code the definition on what a positive Western Blot should be considered”
(2) . Since 1993, in United States there were just 5 official criteria,
and only one (indicated in Du Pont's diagnostic kit) was approved by FDA
(Food and Drug Administration). This was the most restrictive one and was
used by a few laboratories. If only this criterion had been used , in United
States only the 50% seropositive individuals would have been confirmed
as really infected (6)! In Italy in a publication of the Health Ministry
directed towards G.P. (7), the authors denied there had ever been such
a problem. On this subject they affirmed: “in most cases, quite apart from
the criterion used, the test results correctly coded when the individual
The remedy to such a situation
was found simply standardizing the different criteria without reconsidering
the past and without explaining the scientific reasons for which they opted
in that way.
Afterwards the CDC identified
their criteria with the ASTPHLD's ones and in 1993 the United States Red
Cross conformed adopting the ASTPHLD/CDC's advice”(2). “According to the
American authority ASTPHLD, a positive result must present at least two
of the three main bands of diagnostic meaning (antigp-41, anti-gp120/160,
B) The reading of the WB
is visual and therefore is liable to interpretation from the operator,
a variability factor not negligible. “Its technique (note: the Western
Blot) was not standardized, the importance and the consequences occurred
in the laboratories have not been yet measured.
Their results must be interpreted;
the criteria for these interpretations vary not only from laboratory to
laboratory, but even from month to month..” (8). The Consortium for Retroviral
Serology Standardization (CRSS) made a quality control study*and sent 19
aliquots of the same serum to 19 different USA reference laboratories.
Results comparison has shown an incredible differences of the number of
bands and of their intensity (6).
Fig. 3 - WB results from the same serum and obtained
by 19 different laboratoires are shown.
C) The antigenic preparations are not purified.
“The presence of bands in
positions not corresponding to well-known virus antigens is a rather common
finding and we suppose it is the consequence of contaminants in the preparation...”
“The molecular weight of these aberrant bands may vary either from company
to company or even from lot to lot of the same producing company” (2).
Here is Zolla-Pazner's comment
in 1989: “A confusion on the identification of these bands (note: the results
of the Western Blot test ) resulted in incorrect conclusions in experimental
studies. [...] A re-interpretation of the already published results could
be necessary”(9). Re-interpretation that has never been made.
D) The bands considered
“specific” in many cases does not indicate an HIV infection. Some examples
- Even if the more restricting
criteria were employed, Lundberg noticed that 10% control sera, which included
blood samples from blood banks, had a positive WB (6).
- A study(10) shows
that from 2 to 49% tested patients can have a reaction to the WB (unspecified
WB), more frequently caused by cross-reacting antibodies.
- A simple vaccination against flu can give a positive result too (11).
- Transfusion of own
irradiated blood induces the formation of the same antibodies (12).
- Among the homosexuals
and bisexuals who resulted negative for ELISA and PCR, 20%-30% can
have either one or more bands present in the Western Blot.(2)
- There are even more amazing
data concerning dogs and published in 1990. Writing on Cancer Research,
Strandstrom at al. reported that 72/144 (50%) dog blood samples obtained
by the Veterinary Hospital of the University, California, and tested with
the Western Blot reacted with one or more HIV recombinant proteins
[gp120--21.5%, gp41--23%, p31--22%.p24--43%]”(13). As the Authors think
that dogs can not be infected with HIV, one must conclude that these data
are a further prove of the frequent antibody cross-reaction with many proteins
different from HIV ones”.
Remarks: The fact that the
bands are considered unspecific if they are found separately, but virus-specific
if they are present in a number of two or more, seems to refer not to considerations
of a biological kind, but to a convention, a legal agreement changeable
in time and space (in fact it is different from nation to nation). According
to a convention accepted since a few time ago by many authors, three bands
in a clinically healthy blood donor's WB were considered false positive
reactions. According to the same convention, three bands of the same blood
donor, this time presented to an Insurance Company for a life policy, should
be considered true positive.
The researchers group from
Perth who analyzed in depth the WB meaning, concluded that the proteins
considered viral proteins are not peculiar to HIV, but one cannot distinguish
them form proteins of a cellular source. In particular, according to the
review by Eleopulos et al.(14) which has not yet been refuted up to now,
the gp41 should correspond with actine; the p18 and p24/25 with the two
myosin sub-units (p24 is the unspecific band found more frequently in “uninfected”
individuals); the p32 should be identical to the beta chain of the antigen
DR of the histocompatibility class II; the gp120-160 should not be other
than gp41 oligomers.
The p24 antigen
In U.S.A. the evaluation
of the p24 antigen capture test was included among the assays which are
carried out to each blood unit (and it is considered as an “HIV direct
evidence”). Fauci et al.(2) think that: a) the p24 corresponds to virus
capsid antigen (core) which is highlighted by a serum containing the related
monoclonal antibodies (anti-p24) associated to a detector system; b) however
the test is not useful like a primary screening mean in establishing a
precocious HIV infection diagnosis; c) the p24 level increasing corresponds
presumably to the blast in virus replication, which can be observed by
other methods like plasma viraemia; d) the p24 level usually falls under
the determination threshold with conventional methods and like the antibody
anti-p24 starts developing during seroconvertion and can remain in that
state during the following asymptomatic infection years. Only 20-30% of
the individuals with asymptomatic infection has detectable levels of p24
However there are many perplexities
on its real meaning. Quantity data indicate that 50 pg and sometimes more
of core protein (p24) may be found in seropositive patients. If a retrovirus
weight is of 10-3 pg and half of its weight is constituted by p24
(15), then this quantity corresponds to about 100,000 viral particles,
according to other authors and on other premises even to 500,000 particles
(16)! Then if all this protein came from virions, these patients should
be highly viraemic (17, 18). In other words there should be a direct correspondence
among the p24 levels, “virus isolation”, “viral RNA” levels (said also
“viral load”). On the contrary it is not so. Results are irremediably different
from the ones one should expect. For example a paper reports the presence
of antigenaemia, but that no virus could be isolated by 31 antigenaemic
patients, even after an extended culture (19).
Other studies report similar
remarks. We shall mention some of them:
1) In half of the cases
when a person had a positive test, afterwards he had a negative one without
making any treatment which could alter the p24 levels... the test is clinically
erroneous and should be interpreted with much caution”.(20)
2) The p24 antigen is found
neither in all seropositive subjects nor in individuals with clear AIDS.
In a study , the PCR (Polymerase Chain reaction) and p24 were used to detect
HIV in patients in in various stages, from asynptomatical to AIDS individuals.
They found the p24 in 24% and HIV-RNA in 50% of patients (21).
3) More than 29% of the
patients who receive a transfusion from seronegative individuals, become
positive for the anti-p24 antibody (22), even “on infection absence”.
4) The unspecificity of
the p24 antigen assay is so obvious to be accepted by an authority recognized
in the field, Philip Mortimer.et al- from Public Health Laboratory Service
in England.” “Experience proved that neither virus culture, nor p24 tests
are of great value in diagnostic tests. They can be either insensible or
The antibody tests (the
ELISA, Western Blot and p24 antigen capture assay) for the many reasons
over explained, are non able to indicate with reasonable assurance they
can face an HIV infection and so the assertion that WB positive predictive
value (24) can be considered higher than 99% does not correspond with reality.
These tests show reactions of a variable or rather undetermined specificity,
so that they cannot allow a distinction between cross-reaction and “HIV
THE DIRECT IDENTIFICATION OF THE VIRUS
It is a common conviction
that the antibody tests should be ratified and in a certain way verified
from other ones which are able to highlight the virus itself, tests able
to identify it directly. However, this direct determination is such only
apparently. It can only record some phenomena which are considered associated
to the HIV in an unequivocal way. Virus direct identification is divided
into 2 main categories (2): the virus culture and the direct identification
of the viral nucleic acids (Southern Blot, amplification techniques like
the PCR,TR PCR, bDNA, NASBA).
The viral culture
Usually virus isolation involves
a patient's cell co-culture with a donor's uninfected cells which have
been stimulated with phitoemoglutinine (PHA) for 3 days. These co-cultures
are controlled about every 3 days for 28 days or more to control either
the syncitia formation and p24 presence or the reverse transcriptase (RT)
in culture supernatant (2). The presence either of syncitia (a) or the
p24 antigen (b) or the RT (c) is considered a virus replication evidence
(2). According to Gallo et other authors even the finding of virus-like
particles in culture must be considered a sufficient criterion.
a) The appearance of sincytia.
Sincytia appearance is observed
only in considered infected cultures, but these are present even in “uninfected”
cell lines and are similar to the ones used for HIV (25). Then such morphological
feature could be a characteristic either of the same cell lines, or of
the result of the culture conditions or both these factors (14).
b) The p24 antigen.
Its specificity lack has
already been mentioned. The objections to use it like a test on the patient's
serum are efficacious even for its use in cultures. In a study where this
“virus isolation” method was employed ( the determination of the p24 in
cultures with unfractioned entire blood), positive results were obtained
in 49 of 60 “presumably uninfected but serologically undetermined individuals”(82%)
and in 5 of 5 seronegative blood donors (26).
c) The reverse transcriptase (RT).
In all the HIV research,
the ability of copying template-primer A(n)dT15 - when it is incubated
with the supernatant or material coming from infected material co-cultures
with the AIDS virus which separates at a 1.16 gm/ml level (sucrose density
gradient) - is considered the proof of a reverse-transcriptase activity
of HIV. In many cases this activity is considered synonymous of “HIV isolation”
. However: also the template-primer itself is copied when it is incubated
with material which separates in 1.16 band from leukemic cell cultures
(27) and uninfected spermatozoa(29); b) the template-primer A(n)dT15 can
be transcripted not only by RT, but also by other DNA cell polymerases.
All the DNA cell polymerases, alfa, beta and gamma are able of copying
the A(n)dT15 (29). In fact. in 1975, an International Conference on DNA
which included Baltimore and Gallo (30) on eukariotic polymerases defined
the DNA polymerase gamma, “a normal cell component” (31) which activity
can be increased by many factors including the PHA stimulation (32), an
enzyme which “copies the A(n)dT15 with high performance, but does not copy
well the DNA” (33). From what over explained we may deduce that it is wrong
to affirm that such an enzyme activity is peculiar of retroviruses, and
less that this is an unequivocal index of the presence of HIV. Particles
and proteins which separate at 1.16 mg/ml band level could reflect fully
unviral material, most of the same material is certainly cellular and coming
from cultures stimulated with PHA (as we shall see better later), since
transcriptase activity could derive from it.
d) Objects with a retrovirus appearance.
Gallo considered the finding
of retrovirus-like particles in culture like the proof of the HIV isolation
(at that time HTLV-III) (34). However Gallo used H9 (HUT78) cell line to
isolate HTLV-IIIB, and this cell line releases retrovirus-like particles
even when it is not infected with HIV” (35). Weiss obtained his “isolate
CBL-1 from the leukaemic cell line CEM, a cell line which hosted retroviruses
even when they were not infected with “HIV”(36). In 1970, these particles
were frequently observed in leukaemic tissues (37), in embryonic tissue
cultures (38, 39) and in most if not in all human placenta (40). Mature
and *extrobended* particles of the C pattern appear in linphoma cells which
are metabolically damaged but are not infected with HIV (41). “Retrovirus
particles” antigenically similar to HIV were found in extracts from Sjorgen
syndrome patient's salivary glands(42). And more important thing, in the
only study of electron microscopy(43), both in vivo and in vitro where
suitable controls were used and where an extensive assay in cieco of controls
and test material was made, “virus particles indistinguishable from HIV
were found” in a variety of reactive linphoadenopathies which are not HIV-associated,
so that the authors concluded: “The presence of such particles does not
indicate, for itself, an HIV infection”.
Fig. 4 - Can these animals be considered quasi species of human being?
Their genetic difference is comparable to that presnt among various "HIV" genotypes
It is inevitable
to agree that neither syncitia presence, nor p24 finding, nor reverse transcriptase
activity, nor virus-like particle presence (well, each element which settles
that an HIV culture is positive) are “a virus replication evidence, though
just this is the significance given to it.
Direct identification of the nucleic acids
For direct identification
of the nucleic acids one intends the Southern Blot, PCR techniques, the
quantitative amplification techniques (RT PCR, bDNA, NASBA). The identification
is based on the identification and amplification of little nucleotide sequences
attributed to HIV, not of the entire viral genome. This research is made
not on cultures, but direclty on patient's tissues (including blood).
a) Southern blot technique
(technique which allows to separate and highlight nucleic acids of the
In 1984 Gallo carried out
the first study. Using the Southern blot hybridization technique (capable
theoretically of identifying only one infected on ten cells), AIDS patient's
tissues were examined, but weak bands were found only in a minority of
b) The PCR (polymerase chain reaction).
The PCR sensibility is valued
92-100% (45) and is able to detect one “infected” cell on 100.000 (2).
However, “for its high sensibility, it is greatly liable to false positivity
either for even minimum contaminations or other common laboratory mistakes”
(2). Retrovirus-like sequences can be found in human genome in every cell
(46). Already in 1991, Imagawa and Detels pointed out a potential
fallacy of the PCR. “The determination of a part of the genome- they affirmed-
does not mean to detect an entire virus” (47).
The problem of the false positives.
Many researchers challenge
the PCR reability for the great number of false positive that this test
should produce(48). The PCR did not prove reproducible and false positives
and false negatives were observed in all the 7 reference laboratories involved
in an important French study (the concordance with serology varied from
40% to 100%). Moreover, the number of positive results was not significally
different among high and low risk seronegatives (49). There are many other
confirmations to these remarks:
1) A validity study in evaluating
the HIV PCR performance and in identifying free-cell viral
DNA showed “a disturbing high rate of unspecific positivity“ employing
commonly used primers (SK38, both for the p24 and the gag gene). Really,
similar positivity rates were found either for antibody negative samples
than for antibody negative samples(18% against 26%) (50).
2) The PCR (qualitative
DNA-PCR) made on uninfected infants appeared positive on many occasions
3) In a PCR validity study
coordinated by the OMS Work Group on the PCR, 54% of involved laboratories
had problems with false positive results; 9.3% of uninfected samples were
reported as positive(52).
4) The authors of a multicenter
study of quality evaluation stated “this study showed that false positive
results, in spite of rigorous procedure algorithms, happen among uninfected
individuals with such a frequency to remain a serious problem”(53). The
discover of a considerable number of “false positives” gives evidence of
a remarkable PCR specificity lack.
c) Quantitative techniques.
The “quantitative” techniques”
should be able to calculate, in an approximate way, the number of virions
per milliliter of plasma using opportune modifications of some amplification
techniques (either substrate amplification, like RT-PCR and NASBA, or signal
amplification, like bDNA.The substrate consists in “preserved regions”
of a virus-presumed gene. The calculated sensibility is: for the RNA RT-PCR
of 100 HIV RNA copies per milliliter of plasma(2); for NASBA of 1,000 molecules
per milliliter (54); for bDNA (branched DNA) of 10,000 molecules per milliliter
(2). Up to 1994 HIV was considered to be present in small quantities in
asymptomatic and AIDS subject's tissues (1 lymphocyte on 1,000-100,000).
In 1995 two studies (55, 56) seemed, on the contrary, to show that HIV
replicated with much activity since from the first phases of the infection.
The over-mentioned techniques in appearance allowed huge virus quantities,
which in a first time had escaped from observation, to be detected.
However if a massive infection
were present in infected individuals, as some of the most known experts
claim, the Southern blot hybridization would have been more than sufficient
to detect it. Don't forget that “either virus DNA scarcity or lack in a
considerable patient's part was a surprising result”(57). A John Maddox'
significant Freudian slip confirms suspects: according to Maddox “both
Ho than Wei and their colleagues were able to come to their alarming conclusions
only after a ten years research of the HIV as they teamed up with mathematicians
and because they were able to use new techniques to detect the low levels
of virus involved “(58)!
Logical incompatibilities of results are macroscopic and evident.
A) In fact there is a lack
of correlation between p24 and gene amplification techiniques. (We have
already mentioned the fact that the p24 is considered to be “ the direct
proof of HIV identification”).
A) No relation was found
between the p24 level and the RT-PCR (59). The determination of the viral
charge was highly sensible when it was compared with the p24 measurement
(more than 95% of patients had detectable virus charge while only 40% had
detectable p24), but in positive samples antigen and viral charge level
had only a borderline significance (60).
B) No relation was observed
between viral charge quantified either with the NASBA or the Amplicor and
the concentration with the p24 antigen. No relation was observed between
the infectious dose, the viral virl load found either with the NASBA or
the Amplicor(RT-PCR) (61).
C) There is a relation lack
between RT-PCR and other assays. In a study on vaccinated subjects, a group
of four serum samples resulted positive with RT PCR during a 11 months
period, including a result of 104-105 copies/mL, and an year later a fifth
positive result in a vaccinated subject, but not infected” (62). For this
reason, the authors advanced “doubts concerning specificity of HIV RNA
measurement as a golden standard for the screening and the infection confirmation”.
D) There is finally a lack of correlation between “virus” and “viral load”.
1) According to Piatak,
the high “virus” level in plasma was 999 per 1,000 “unculturable” (63).
2) Ho et al. showed that
10,000 plasma “virions” counted with the branched PCR correspond with less
than one “infectious” virus per ml (64).
3) In a more recent study
by Ho (65) one may notice that 0 (zero!) “cultured viruses” corresponded
to a load of 500,000 units! According to the reported data, to patient
105 before the treatment (with Ritonavir) 643,000 “virions”/ml were detected,
where the number was calculated with the branched DNA assay, more than
1,000 TCID-50/ml corresponded to it (50% tissue culture infectious doses).
After a two day therapy and in following days the “cultivable virus” was
0(zero). In the meantime, at 2, 3 and 4 days “free virions” (that is the
so-called “viral charge”) remained over 500,000. Till 7 day “virions” were
again over 100,000. A virion is defined as a complete viral particle constituted
with the viral nucleic acid, capsid and pericapsid (when present). The
possible objection for which a defective virion is “seen” by branched DNA
assay, but is not detected by culture samples, does not resolve the problem
of non-correspondence with the other tests and the impossibility of seeing
these particles at the microscope directly from patient's tissues, without
culture transfers. If it were so, Gallo would not need for applying to
frauds (67, 68), he would not call for three year tireless researches in
order to find something which had the appearance of a virus. He would have
come to it immediately with other researchers.
Mark Craddock, professor
at the School of Mathematics and Statistics, The University of Sidney,
Australia commented: “What is this viraemia of billions of RNA particles
that can only be seen with an undocumented branch-PCR or PCR but not with
a functional infectivity test?”. (68)
As already stated, genomic
amplification techniques concern small segments, not surely the complete
“viral” genome and the results which are obtained do not give the assurance
to have the true “HIV”, besides they are frankly discordant. At this point
it's legitimate to ask what is really and how it's made the original viral
genome. The reserch group from Perth (69) notes that if an unique AIDS
retrovirus existed, then less than 1% genomic differences should be the
rule, not the exception. For instance, the type 3 Sabin poliovirus vaccine
differed from its neurovirulent progenitor at only 10 nucleotide positions
after 53 in vitro and 21 in vivo passages in monkey tissues. In 1977, H1N1
influenza A virus reappeared in the human population after 27 years of
dormancy with sequences mainly identical to those of the 1950s virus”.
Although Eigen's quasispecies model has been used to describe the genome
of RNA viruses, even 1% sequence differences in these genomes are considered
to represent “extreme variability”. “Many selective forces may stabilize
virus populations. These stabilizing factors may include the need for conservation
of protein structure and function, RNA secondary structure, glycosylation
sites, and phosphorylation sites”. Let us see what is the picture offered
by HIV research. Not long afterwards it was discovered that “If you were
to test two HIVpositive people at random and analyse the genetic material
of their strains, they would differ, on average, by about 13 percent” (70).
Besides what is considered to be the genotype of HIV comes from sequence
analysis of subgenomes that HIV genotype consignments are derived
from sequence analysis of subgenomes measuring 2% to 30% of the total (71).
The data is that such “genomes” vary between 3-40% (72,73). “If 30% of
the HIV genome varies as much as 40%, how much does 100% of the HIV genome
vary? This is the legitimate question of Eleopulos and colleagues (69).
Among researchers there
is disagreement also about the number of HIV genes. In 1988 they said HIV
genome had eight genes (74), in 1990 ten (75). In 1996 Montagnier referred
that HIV had eight genes (76), and according to Barré-Sinoussi,
HIV has nine genes (77) Not even the number of nucleotides of HIV genome
seems to be constant (69)
Because great genomic differences
were found - even the same person can harbor more than 106-108 genetically
distinct variants (78) - to try to find a justification for this phenomenon,
the concept of quasi-species has been introduced. Prior to the 1990s, the
HIV sequences were classified as African and USA/European with sequence
differences of 20-30 percent between these two groups. (79). In the 1990s,
HIV researchers started to divide the “HIV genome” into subtypes A, B,
C, D, E, etc. The basis for this classification system is: “(a) subtypes
are approximately equidistant from one another in env; (b) the env phylogenetic
tree is for the most part congruent with gag phylogenetic trees; (c) two
or more samples are required to define a sequence subtype”. However, “Subtype
naming problems have arisen. A small but not insignificant number of viral
sequences are hybrid, clustering with one sequence subtype in gag and another
sequence subtype in env, for example;... Naming becomes problematic when
highly divergent forms of a given subtype arises: such forms are sometimes
designated A', B', F', etc”. It is increasingly necessary to have sequence
data from both gag and env coding sequences when a new form or subtype
is being claimed” (80). By the middle of 1996 “at least ten” (A-J) prevalent
major (M) and a low prevalence, O, HIV-1 genotypes were described and new
genotypes are still reported (81,82).
Is it possible then to describe
the “HIV DNA” even if it has variation of 10% , not to mention 20 or 30
or 40% as is the case, as a ...”population of closely related genomes,
referred to as a quasispecies” (69)? The extreme variability of HIV genome
make us ask what is the sense to refer to HIV as a clear distinct entity.
The genomic difference among human being of different race is about 1 per
1.000, between man and orang-utan about 2%, but to find a 30% difference
is necessary to compare non the less man and mouse, man and ass, man and
elephant! Can one consider mouse, ass and elephant as quasi-species of
Fig. 5 - Electronic microscopy of a cell surrounded by a small particles.
Without any proof these are considered viral particles (HIV), while
they can simply be artefacts of staining or cellular particles.
Particle detection Retrovirus
are defined as particles of about 100-120nM of diameter with a core which
includes a proteic coat and a ribonucleoprotein complex. Retroviruses are
classified in three subfamilies: Spumavirinae, Lentivirinae and Oncovirinae.
Retroviruses belonging to the last subfamily are divided in type A, B,
C, and D particles. To which of these should belong HIV, is not very clear.
According Gallo in 1984, it was a type C particle, but in 1985 he admitted
it could be also a type D (83). Montagnier and coll. initially reported
it was a type C particle (84), then a type D (85), and then a lentivirus
(86), that is to say a different subfamily. According to Munn and others
AIDS virus shows characteristics of type C, of type D and of lentivirus
(87). Fauci and others described HIV in monocyte/macrophages cultures as
retroviral particles with characteristics of type A (88). HIV identity
seems to be so vague, so “shot”, that it adds further reasons of perplexity.
Photographs Several researchers in the AIDS field have shown HIV photographs,
but those that can be seen are only virus-like viral particles indistinguishable
from normal cellular microvescicles. These particles, in contrast with
viruses, are very unstable when removed from their contest, and it's difficult
to isolate them and photograph them in a condition of a true isolation.
On the contrary, viruses are stable because they have to leave again the
cells and host organisms. The use of centrifugation techniques doesn't
constitute a problem to separate viruses from various contaminants and
so get their isolation - then photograph them, to put in evidence their
proteins and their genetic constituents in a direct way. True viruses are
so stable that it's easy to photograph directly them as tridimensional
particles by an electronic scanning microscope, without the need to first
fix them chemically. On the contrary, the microvescicles are so unstable
that can be photographed only by a transmission electronic microscope which
requires that they be previously fixed chemically in ultra-thin sections.
What has been shown to the world as HIV micro graphs are ultra thin sections
which include particles indistinguishable from the cellular ones (89) (pict
The evidence of HIV (non-)existence
A noteworthy confirmation to the argued critics of the researchers from
Perth came in march 1997: two groups, one French/German (90) and one from
the American NCI (91), have published the photographs of density gradients
in which pure HIV virus had to be present (pict. 9). These data should
have been available by the first isolation of the virus (1983-84), but
nobody showed them before. The most likely reason is the lack of correspondence
between what they found and what they expected.
In the Franco/German study
the pictures are from the 1.16 gm/ml band. It is impossible to tell
from which density the pictures in the American study are taken but presumably
it's the same 1.16 density for retroviral particles. Eleopulos and colleagues
note that “. The authors of both papers concede that the particles which
are present in the banded material and which are said to be HIV represent
only a very small fraction of the total material. Gelderblom et al.
state that the material contains “an excess of [cellular] vesicles with
a size range 50-500nm, as opposed to a minor population of virus particles...
cellular vesicles appear... to be a major contaminant of HIV preparations
enriched by sucrose gradient centrifugation”. “For the small number of
particles deemed to be “HIV” no evidence is given that they are even a
retrovirus-like particle. Indeed, to the contrary: (a) the particles do
not appear to have surface spikes (knobs)... In other papers published
by many researchers including Gelderblom and his associates such projections
are noted to be absent (93, 94); (b) the particles referred to as “HIV”
are not spherical and have diameters exceeding 100-120 nM. In the EM in
Gluschankof et al. (90) there are arrows pointing to five “HIV” particles
devoid of surface projections whose dimensions are 121 X 145; 121 X 169;
121 X 145, 121 X 145 and 133 X 145 nM respectively.
In Bess et al. (91) there
are a total of six “HIV particles” also devoid of surface projections whose
dimensions are 160 X 240; 200 X 240; 280 X 280; 208 X 250; 167 X 250 and
250 X 292 and nM respectively. “Actually, the authors maintain these are
HIV particles, but without any proof. In the material of that density band
there ought to be many viral particles, on the contrary they point only
some, among many impurities.
They bear only the vaguest
resemblance to retroviral particles”. “The point is that any genuine retroviral
particle contains a fixed amount of RNA and protein. No more and no less.
This means that if we consider diameters of particles pointed in the two
studies, they are 1.14 times to 1.96 times larger than the estimated
maximum for a normal HIV particle. Translating this in volumes, and comparing
them to a particle with a diameter of 120nM, the Franco/German particles
have 50% more volume than a retroviral particle and the US particles have
750% more volume”, that is to say incompatible with the same definition
of a retrovirus! Even if the authors do not admit it, the data of the two
cited papers contribute to show that where should be present pure HIV,
of HIV there is no trace.
None of the tests examined
seems to be sufficiently sure to detect an HIV infection. Paradoxically
not even the “direct virus detection” is able to do answer to the same
question. As a matter of facts there is an incompatible discordance among
the results obtained with different methods. These discordances are by
themselves logical incompatibilities that invalidate them. Besides, the
existence of an entity that one can reasonably name “HIV virus” is not
proved, as far as its proteins, its genome, its physical particle are concerned.
The true meaning of the tests is better understood if its origin is examined.
To show the existence of a new virus, both Montagnier group in 1983 and
the group of Gallo in 1984 separated supernatant of cultures they considered
infected in density gradients. They erroneously considered that the material
that banded at 1,16 mg/ml was constituted by “pure HIV particles”. The
proteins which were present in this band and were reactive with AIDS
patients sera (but not only with their sera) were considered HIV proteins.
In the same manner, a particular fraction of RNA which they found in the
same band was considered the genome of the new virus. By then, some of
these proteins are used to get the reagent for some kind of tests
or as immunogens to produce in laboratory animals the antibodies used for
other kind of tests ( similar procedure was followed to obtain RNA
At this point the explanation
of Gallo experiments in 1984 appears clearer: he used a mixture of proteins
(believed to be viral but in fact cellular) to immunize the rabbits and
obtain a serum which obviously reacted with the same proteins that were
in the culture from which the initial mixture came (95).
In other words Gallo believed
to show an hypothesis (the existence of the virus) with other hypothesis
shown to be false (that is to say that inverse transcription activity,
the presence of virus-like particles, the antibodies obtained in the way
just mentioned were all virus specific).
In this way were born the
tests which to-day are still used and perfectioned to put so engaging a
diagnosis. An important prove of the correctness of this interpretation
of the facts stays in the results that they obtained on mice and monkeys
in 1991 (96).
In those studies, with the
scope to evaluate a candidate vaccine, some laboratory animals were injected
with cellular components from cultures which were “not infected by HIV”:
the results were similar to those obtained with “the real vaccine” (i.e.
with virus from “infected cultures”).
No significant difference
was found, so that the comment - perfectly suited and still up to
date of John Maddox - was: “AIDS research turned upside down”.
That is to say that it wasn't
necessary at all to hypothesize the presence of a virus to obtain those
Dr. Fabio Franchi MD, Specialista in Igiene, Medicina Preventiva e in Malattie Infettive, Trieste
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