BIO/TECHNOLOGY VOL.11 JUNE 1993
Is a Positive Western Blot Proof of HIV Infection?
Eleni Papadopulos-Eleopulos, Valendar F. Turner and John M.
Papadimitriou
It is currently accepted that a positive Western blot (WB) HIV antibody
test is synonymous with HIV infection and the attendant risk of developing
and dying from AIDS. In this communication we present a critical evaluation
of the presently available data on HIV isolation and antibody testing.
The available evidence indicates that: (I) the antibody tests are not standardised;
(II) the antibody tests are not reproducible; (III) the WB proteins (bands)
which are considered to be coded by the HIV genome and to be specific to
HIV may not be coded by the HIV genome and may in fact represent normal
cellular proteins; (IV) even if the proteins are specific to HIV, because
no gold standard has been used and may not even exist to determine specificity,
a positive WB may represent nothing more than cross-reactivity with the
many non-HIV antibodies present in AIDS patients and those at risk, and
thus be unrelated to the presence of HIV. We conclude that the use of the
HIV antibody tests as a diagnostic and epidemiological tool for HIV infection
needs to be reappraised.
"...we are not simply contending in order that my view
or that of yours may prevail, but I presume we ought both of us to be fighting
for the truth..."
from Philebus, the Dialogues of Plato
To date, the only routinely used methods for demonstrating the presence
of HIV in vivo are the ELISA and WB antibody tests. In the ELISA,
the "HIV proteins" are present as a mixture. For
the WB, the HIV proteins are dissociated and placed on a polyacrylamide
gel slab. After electrophoresis, which separates the proteins by molecular
weight and charge, the proteins are transferred to a nitrocellulose membrane
by electroblotting. In performing the antibody test, in both ELISA and
WB, the patient's serum is added to the antigen preparation. It is assumed
that if HIV antibodies are present, they will react with the HIV proteins
which, after washing, are visualised by an enzyme anti-human-immunoglobulin
chromogen reaction. In the ELISA the reaction is read optically. For the
WB, individual proteins are recognised and interpreted visually as coloured
bands, each of which is designated with a small "p" (for protein),
followed by a number, (which is the molecular weight in kilodaltons), for
example p41.
The WB is believed to be highly sensitive and specific, and a positive
result is regarded as synonymous with HIV infection. A positive HIV status
has such profound and far reaching implications that no one should be required
to bear this burden without solid guarantees of the verity of the test
and its interpretation. In this paper, the evolution of the antibody tests,
the basis of their specificity, and the validity of their interpretation
are evaluated. Acceptance of an antibody test for HIV as being scientifically
valid and reliable requires the following: (I) A source of HIV specific
antigens; (II) Standardisation; (III) Determination of the test's reproducibility.
Once these criteria have been met, and before the introduction of the antibody
tests into clinical medicine, the test's sensitivity, specificity and predictive
values must be determined by the use of a gold standard, HIV itself.
Proteins Considered to be HIV Antigens
The proteins considered to represent HIV antigens are obtained from
mitogenically stimulated cultures in which tissues from AIDS patients are
co-cultured with cells derived from non-AIDS patients-usually established
leukaemic cell lines. Following the detection of the enzyme reverse transcriptase
(RT) in the cultures, the supernatant, and more often the cell lysates,
are spun in density gradients. Material which bands at 1.16 gm/ml is considered
to represent "pure HIV" and consequently the proteins found at
that density are considered to be HIV antigens. The immunogenic HIV proteins
are thought to be coded by three genes, namely gag, pol and
env. The gag gene codes a precursor p53/55, which is then
cleaved to p24/25 and p17/18. The pol gene codes for p31/32, and
the env gene codes the precursor protein p160 which is cleaved to
p120 and p41/p45. (1)
The p120 protein.
The generally accepted view is that p120 and p41 are cleavage products
of p160, which is found only in infected cells and not in the virus. However,
p120 is a component only of the knobs (spikes) on the surface of HIV particles;
The knobs are found only in the budding (immature) particles; and not in
cell free (mature) particles; immature particles are "very rarely
observed".(2)
Despite these findings, when "purified HIV" is tested against
AIDS sera, strong bands corresponding to p120 and p160 develop. The solution
to these contradictions was found when it was shown that p80 (vide infra)
and "the components visualized in the 120-160-kDa region do not correspond
to gp120 or its precursor but rather represent oligomers of gp41".(3)
The p41 protein.
p41 is one of the proteins detected by both Gallo's and Montagnier's
groups in the first HIV isolates. However, Montagnier and his colleagues
observed that AIDS sera reacted with a p41 protein both in HIV and HTLV-I
infected as well as non-infected cells, and concluded that the p41 band
"may be due to contamination of the virus by cellular actin which
was present in immunoprecipitates of all the cell extracts".(4) Although
Gallo's group did not find such reaction with p41 in non-infected cells,
they did find a p80 protein and concluded that the reaction was "non-specific"(5).
Actin is an ubiquitous protein which is found in all cells as well as
bacteria and several viruses. Well known retroviruses such as the mouse
mammary tumour virus and Rous sarcoma virus have also been shown to contain
actin of cellular origin and it has been postulated that this protein plays
a key role in both retroviral assembly and budding.(6,7) It is also known
that oxidation of cellular sulphydryl groups, as is the case in AIDS patients
(8), is correlated with assembly of polymerised actin (9), and that the
level of actin antibody binding to cells is determined by the physiological
state of the cells. For this reason actin antibody binding to cells has
been proposed "as a sensitive marker for activated lymphocytes"(10).
Platelets from healthy individuals also contain a p41/45 protein which
reacts with sera from homosexual men with AIDS and immune thrombocytopenic
purpura (ITP) and which "represents non-specific binding of IgG to
actin in the platelet preparation"(11).
The p32 protein.
In 1987 Henderson isolated the p30-32 and p34-36 of "HIV purified
by double banding" in sucrose density gradients. By comparing the
amino-acid sequences of these proteins with Class II histocompatability
DR proteins, they concluded that "the DR alpha and beta chains appeared
to be identical to the p34-36 and p30-32 proteins respectively"(12).
The p24/25 protein.
Detection of p24 is currently believed to be synonymous with HIV isolation
and viraemia. However, Apart from a joint publication with Montagnier where
they claim that the HIV p24 is unique, Gallo and his colleagues have repeatedly
stated that the p24s of HTLV-I and HIV immunologically cross-react (13);
Genesca et al.(14) conducted WB assays in 100 ELISA negative samples
of healthy blood donors; 20 were found to have HIV bands which did not
fulfil the then (1989) criteria used by the blood banks for a positive
WB. These were considered as indeterminate WB, (WBI), with p24 being the
predominant band, (70% of cases). Among the recipients of WBI blood, 36%
were WBI 6 months after transfusion, but so were 42% of individuals who
received WB-negative samples. Both donors and recipients of blood remained
healthy. They concluded that WBI patterns "are exceedingly common
in randomly selected donors and recipients and such patterns do not correlate
with the presence of HIV-1 or the transmission of HIV-1", "most
such reactions represent false-positive results";
Antibodies to p24 have been detected in 1 out of 150 healthy individuals,
13% of randomly selected otherwise healthy patients with generalised warts,
24% of patients with cutaneous T-cell lymphoma and prodrome and 41% of
patients with multiple sclerosis.(15)
Ninety-seven percent of sera from homosexuals with ITP and 94% of sera
from homosexuals with lymphandenopathy or AIDS contain an antibody that
reacts with a 25Kd membrane antigen found in platelets from healthy donors
and AIDS patients, as well as a 25 Kd antigen found in green-monkey kidney
cells, human skin fibroblasts, and herpes simplex cultured in monkey kidney
cells. This reaction was absent in sera obtained from non-homosexual patients
with ITP or non-immune thrombocytopenic purpura.(11)
Conversely, the p24 antigen is not found in all HIV positive or even
AIDS patients. In one study, the polymerase chain reaction (PCR) and p24
were used to detect HIV in patients at various CDC stages from asymptomatic
to AIDS. p24 was detected in 24% patients and HIV RNA in 50%.(16)
In another study, "In half of the cases in which a subject had
a positive p24 test, the subject later had a negative test without taking
any medications that would be expected to affect p24 antigen levels...the
test is clinically erratic and should be interpreted very cautiously".(17)
The p17/18 protein.
In addition to the p24 band, the p17/18 band is the most often detected
band in WB of healthy blood donors.(18)
Sera from AIDS patients bind to a p18 protein in mitogenically stimulated
HIV infected T-cells, but not to non-infected, unstimulated lymphocytes.
However, when the lymphocytes are mitogenically stimulated, but non-infected,
the AIDS sera bind to a p18 protein in these non-infected lymphocytes.(19)
A monoclonal antibody (MCA) to HIV p18, reacts with dendritic cells
in the lymphatic tissues of a variety of patients with a number of non-AIDS
related diseases;(20) and the "same pattern of reactivity was present
in normal tissue taken from uninfected individuals as in those taken from
HIV positive subjects".(21)
AIDS patients and those at risk have high levels of antibodies to the
ubiquitous protein-myosin,(22) which has two subunits of molecular weights
18,000 and 25,000. In view of all the above evidence it is difficult to
defend the view that the bands p41 (and thus p160 and p120), p32, p24 or
p18 represent specific HIV proteins. Even if it could be shown that all
these proteins are HIV specific, it cannot be automatically assumed that
antibodies that react with each of these proteins are specific to HIV infection.
Standarisation of HIV Antibody Tests
An antibody test becomes meaningful only when it is standardised, that
is, when a given test result has the same meaning in all patients, in all
laboratories, in all countries. From the first antigen-antibody reactions
performed by Montagnier's (4) and Gallo's (23) groups (fig.1, 2) it was
found that: not all of the "HIV proteins" react with all sera
from AIDS patients or even sera from the same patients obtained at different
times; and that sera from AIDS patients may react with proteins other than
those considered to be HIV antigens. Because of these variable reactions,
an essential requirement was to establish criteria as to what constitutes
a positive WB.
Initially, Montagnier's group considered p24 sufficient to define a
positive WB, whereas Gallo's group considered p41 sufficient. Most, if
not all other laboratories, used the criteria recommended by the CDC, namely
the presence of a band at either p24 or p41. By 1987 it became obvious
that those bands were not HIV specific. Furthermore, till 1987 "there
were as many WB procedures as there were laboratories doing the assay".(24)
Since then, all major laboratories have changed their criteria for WB interpretation
but in the United States there are still no nationally agreed criteria,
even among the major laboratories:
In 1987 the Food and Drug Administration (FDA) licensed a WB kit manufactured
by DuPont. The DuPont kit remains the only licensed WB kit and is used
by a minority of laboratories. It specifies "extremely stringent"
criteria for a positive result namely "specific bands representing
three different gene products: p24 (gag), p31 (pol), and
an env band, either gp41, gp120 or gp160" (24).
The American Red Cross defines a positive result as presence of antibodies
to at least one gene product from each of the gag, pol and
env genes, without specifying which bands.
The Association of State and Territorial Public Health Laboratory Directors/Department
of Defence/CDC consider a WB positive if two out of p24, gp41 and gp120/160
are reactive.
The Consortium for Retrovirus Serology Standardization (CRSS) defines
a positive WB as the presence of antibodies to at least p24 or p31/32,
and gp41 or gp120/160 (25).
All the other major USA laboratories for HIV testing have their own
criteria. For all laboratories, a negative result requires the absence
of any and all bands including bands which do not represent "HIV proteins".
All other patterns which do not satisfy a given laboratory's criteria for
a positive or negative test are regarded as WBI by that laboratory.
Thus, in the scientific literature, no strips have been published of
a standard positive WB. Fig.0 is reproduced from the instruction manual
of a WB kit manufacturer, Bio-Rad. Although given as "Examples of
a typical reactive patient serum sample and reaction with a strong, weak
and non-reactive control" it is also stated, "This example shows
typical reactive patterns only, and is not to be used as a reference for
comparisons with results from unknown serum samples...Patient samples may
show varying degrees of reactivity with different proteins, thus showing
different band development patterns...Each laboratory performing Western
Blot testing should develop its own criteria for band interpretation. Alternatively,
band interpretation may be left to the clinician".
In addition to the obvious problems associated with the lack of standardisation,
all of the above interpretations possess major problems:
When the FDA criteria are used to interpret the WB, only a minimal number
(less than 50%) of AIDS patients have a positive WB, that is, are infected
with HIV. If the criteria of the CRSS are used, the percentage of AIDS
patients testing positive increases to 79%.
More importantly, even when the most stringent criteria are used, 10%
of control samples, which include "specimens from blood donor centers",
have a positive WB (25).
As already mentioned, Henderson and his colleagues have shown that p31/32
is a non-HIV protein. Pinter and his colleagues have shown that p160 and
p120 are oligomers of gp41. They have also shown that the WB pattern obtained
is dependent on many factors including temperature and the concentration
of sodium dodecyl sulphate used to disrupt the "pure virus",
and concluded:
"Confusion over the identification of these bands has resulted
in incorrect conclusions in experimental studies. Similarly, some clinical
specimens may have been identified erroneously as seropositive, on the
assumption that these bands reflected specific reactivity against two distinct
viral components and fulfilled a criterion for true or probable positivity.
The correct identification of these bands will affect the standards to
be established for Western Blot positivity: it may necessitate the reinterpretation
of published results"(26).
The finding that the p31/32 band represents a cellular protein, and
that p120 and p160 are oligomers of p41, reduces the criteria of the CRSS
and that of the American Red Cross to two bands, p24 and p41, which according
to Colonel Donald Burke are "less than perfectly specific",(27).
The above findings reduce the criteria of the Association of State and
Territory Public Health Laboratory Directors/Department of Defence/CDC
to p24 or p41, generally accepted as being non-specific.
Despite the above evidence, even at present, the p160, p120 and the
p41 bands are considered to represent distinct viral envelope glycoproteins.
In fact, the current WHO guidelines consider a serum positive for HIV-1
antibodies if "two envelope glycoprotein bands (with or without) other
viral specific bands are present on the strip"(28).
To date, AIDS in Africa is defined on clinical grounds. Recently, the
CDC recommended the future inclusion of serological evidence for HIV infection
in the African definition of AIDS. The test recommended is ELISA, (29)
which cannot be considered specific.
In Russia, in 1990, out of 20,000 positive screening tests "only
112 were confirmed" using the WB as a gold standard. In 1991, of approximately
30,000 positive screening tests, only 66 were confirmed (30).
In the Latin American and Caribbean AIDS definitions the "clinical
findings of HIV infection" are confirmed "by antibody testing
using ELISA, immunofluoresence or Western blot methods". No criteria
are given for WB interpretation (31).
Reproducibility
The problems associated with reproducibility may be best illustrated
by two examples. Fig.3 represents WB strips of a serum specimen from a
patient with AIDS, tested by 19 laboratories that participated in the second
CRSS conference on WB test standardisation (25). As can be seen, the band
pattern obtained with one and the same serum, varies from laboratory to
laboratory, although all laboratories reported this specimen as positive.
The Transfusion Safety Study (TSS) Group in the USA submitted approximately
100 patient samples weekly for WB testing to three reference laboratories
over three separate periods of several months. With the 100 patient samples,
they submitted aliquots from four quality control (QC) plasmas, two positive
and two negative. HIV positivity or negativity "was based on the collective
experience with each plasma using: (a) licensed EIA systems of five manufacturers,
(b) an immunofluoresence assay, (c) IB in four reference laboratories,
and (d) a radioimmunopreciptation assay in an additional laboratory".
(EIA=ELISA;IB=WB).
The samples were then sent to reference laboratories which were aware
of quality control testing, but "the labels and codes did not permit
identification of the QC specimens as such or linkage to previous QC specimens".
QC1#(+) was submitted 40 times to laboratory A, 5 times to laboratory
B and 45 times to laboratory C. A reported the following band patterns:
p24, p32 and gp41/120, 7 times; p24, gp41/120, 28 times; p24 only, 5 times.
B reported: p24, p32, gp41/120, 4 times; p32, gp41/120, on one occasion.
C reported: p24, p32, gp41/120, 26 times; p24, gp41/120, 10 times; p24,
p32, twice; p24 only, 5 times; "others", once; no bands, once.
QC#2(+) was sent a total of 89 times to the three laboratories and was
reported: p24, p32, gp41/120, 64 times; p24, gp41/120, 19 times; p24, p32,
once; p32, p41/gp120, 4 times; no bands, once.
A total of 101 aliquots of the two quality control negative samples
QC#3(-) and QC#4(-) were sent to the three laboratories. These were reported:
no bands, 67 times; "other" bands, 13 times; gp41 only, once;
p24 only, 18 times; p24,p32,gp41/120, twice.
A special panel of QC samples was sent to laboratories B, C and an additional
laboratory D. The panel consisted of three aliquots of each of eight samples,
including batches QC#1(+), QC#2(+), QC#3(-) and QC#4(-).
Discussing the latter results the authors state: "Only Laboratory
C's reports with the panel were consistent with the data accrued from all
other evaluation of reactivity... Laboratory B reported the three aliquots
of QC#1 (+) as respectively positive on the basis of three bands (gp41,
p55 and p65),indeterminate on the basis of a single band (gp41), and negative
(no bands observed). In addition, all three aliquots of QC#6(-)were considered
indeterminate because only a single band (gp41) was seen. Laboratory D
reported one aliquot of QC#6(-) as positive (p15, p24, p32, gp41, p65)
and the other two aliquots as negative (no bands observed). It also reported
a band at p55 for all three aliquots of QC#3(-)".(32)
In considering the results detailed above, one must bear in mind that
they occurred in Reference Laboratories, that is, first class laboratories
which constitute only a small number of the total number of laboratories
which perform WB testing in the USA.
In addition, many laboratories continue to use unlicensed WB kits because
of cost and the "stringent criteria required for interpreting the
licensed test".(33)
Specificity of the HIV Antibody Tests
The task of authenticating a new diagnostic test in clinical medicine
requires an alternative independent method of establishing the presence
of the condition for which the test is to be employed. This method, often
referred to as the gold standard, is a crucial sine qua non, and represents
the tenet upon which rests the scientific proof of validity.
The only possible gold standard for the HIV antibody tests is the Human
Immunodeficiency Virus itself. Obviously, the clinical syndrome and the
decrease in T4 cells cannot be considered a gold standard. Although HIV
has never been used as a gold standard there is general consensus that
proof of the specificity of the HIV antibody tests is firmly established.
For the ELISA, Gallo's best figures, obtained from AIDS patients and 297
healthy blood donors, were 97.7% sensitivity and 92.6% specificity assuming
borderline tests as positive, and using the clinical syndrome as gold standard.(34)
Colonel Donald Burke and his colleagues from the Walter Reed Army Institute
in the USA are credited as having most thoroughly researched the problem
of defining HIV antibody specificity in a large population and his data
is widely believed to represent the current state of the art.(35)
Burke et al (36) tested a highly selected healthy subpopulation of 135,187
individuals chosen for a very low prevalence of HIV infection--1/10th
that of a much larger pool of applicants (1.2 million), for US military
service.
All applicants were screened with an initial ELISA. All reactive ELISA
tests were repeated in duplicate. Then an initial WB was performed and,
if diagnostic or reactive, a second WB was performed on another fresh blood
specimen. Initially the criteria for a positive and diagnostic WB were
the "presence of a band at 41kd, a combination of the bands 24 and
55kd, or both.
Beginning in May 1987, the method of preparing blot strips was modified
so that antibodies to gp120 and gp160 could be detected reproducibly, and
criteria for a reactive and diagnostic blot pattern were changed to those
of the Association of State and Territorial Public Health Laboratory Directors".
A positive WB was diagnosed if and only if the first and second serum
samples were diagnostic on WB. All of the diagnostic WB samples were then
assayed with four other antibody tests. A WB was considered "true
positive if all four assays on all available serum samples from an applicant
were reactive and diagnostic", but was considered "false positive
if all four assays on all available serum samples from an applicant were
non-reactive, non-diagnostic or both".
From the 135,187 applicants, there were 16 positive tests. In one of
these, the serum was unavailable for further testing and one applicant
declined to provide a second sample. Serum from 27 of the 29 samples from
the 15 applicants found positive were tested by the four other antibody
tests. Fourteen samples were found positive by all four assays and all
four were negative for one applicant.
From this Burke and his associates calculated the false positive rate
as 1 in 135,187 or 0.0007%. They also speculated on the implications that
this data might hold for their entire population of 1.2 million applicants.
They calculated the overall prevalence of 1.48 per 1000 in the entire pool
as equivalent to 200 per 135,187. Assuming that the false positive rate
is the same for the whole population they estimated that since there will
be 200 true positive tests per 135,187 persons of which only one will be
a false positive then the "predictive value of a positive diagnosis
in the program is 99.5%, and a specificity of 99.9%".(35,36)
Much of Burke's and his colleagues' reasoning is open to criticism:
(I) There is no gold standard for defining HIV infection. Testing the
positive WB in the 15 remaining applicants against four other antibody
tests does not enable an independent establishment of "true"
HIV infection as they are the same test;
(II) They define: (a) the true positive tests as samples which repeatedly
test positive in four similar tests. (b) the false positive tests as samples
which repeatedly test negative in four similar tests. The number of samples
tested and the repeats is arbitrarily defined. It would be impossible to
say what the outcome would be if for example the ELISA tests were repeated
three instead of two times or if the samples which tested negative in the
first ELISA were tested again with another ELISA or WB. There are well
documented reports in which the ELISA is negative and the WB positive.(37)
(c ) the false positive rate as the number of false positive results divided
by the number of samples tested. These definitions bear no resemblance
whatsoever to those described in standard texts.(38) The correct definitions
are:- (i) A true positive is a positive test occurring in an individual
who is HIV infected as defined by an independent gold standard; (ii) A
false positive is a positive test which occurs in an individual who, by
application of the gold standard, does not have HIV infection, (but is
not necessarily healthy); (iii) The false positive rate is the number of
false positive tests as a fraction of all positive tests, both true and
false positive.
(III) The Burke et al premises are quite the opposite to those of Gallo
et al where all positive test results in healthy individuals are regarded
as false positive. Based on Gallo and his associates' premises we must
regard all sixteen cases as false positives as there is no compelling reason
for regarding healthy military applicants as significantly different from
healthy blood donors.
(IV) Burke's extrapolation to the entire 1.2 million applicants is invalid.
This extrapolation can only be done if the 135,187 applicants were randomly
selected from the entire pool, which they were not. In the rest of the
population the false positive rate may have been much higher for example
as a result of higher concentrations of globulins in general or of autoantibodies
in particular.
Their stated figure of 99.5% positive predictor value is impossible
to arrive at without knowledge of the sensitivity of the WB test and the
prevalence of true HIV infection, (38) even if the specificity and the
extrapolation were correct.
(V) It is impossible to define specificity, sensitivity and predictive
value with the algorithm used by Burke and his associates. The best they
can do with their algorithm is to determine the reproducibility of ELISA
and WB. In this regard, in Burke's larger study of 1.2 million healthy
military applicants, approximately 1% of all initial, 50% of all repeat
ELISAs were positive; and 30-40% of first WB were positive and 96% of second
WB were positive. In other words Burke's larger study reveals: (a) 6,000
individuals with an initially positive but subsequently negative ELISA.
(b) 4,000 individuals with two positive ELISA's followed by a negative
WB. (c ) 80 individuals with two positive ELISA's, an initially positive
WB and a negative repeat WB.
This cannot be regarded as a trivial problem since: (I) both ELISA and
WB are regarded as highly sensitive and specific.(24) (II) Several thousand
healthy individuals have antibodies that react with "HIV proteins"
but who are ultimately deemed not to be HIV infected; (III) Even in the
best laboratories, 80 of Burke's healthy applicants would be diagnosed
as HIV infected since, unlike Burke, only one WB is performed.
The problem becomes even more serious when one realises that by September
1987 by which time, based on the antibody tests, a causal relationship
between HIV and AIDS was generally accepted, a single positive ELISA or
a positive WB, one band (either p24 or p41) was sufficient to confirm HIV
infection.
At present, the general opinion is that the ELISA tests have a "sensitivity
and specificity of over 98%, many approaching 100%",(24) and the CDC
AIDS definition "accepts a reactive screening test for HIV antibody
without a confirmation by a supplemental test because a repeatedly reactive
screening test result, in combination with an indicator disease, is highly
indicative of true HIV disease".(39) (screening test=ELISA).
Burke et al, like Gallo et al, determined specificity without reference
to sick individuals. The definition of specificity requires that the test
is evaluated in persons who do not have the disease which is under scrutiny,
including sick individuals who have other diseases where antibodies, some
of which may interact with HIV antigens, may be produced for other reasons.
The specificity of the HIV antibody tests must be determined by testing
individuals who are immunosuppressed and/or who have symptoms and clinical
signs similar to AIDS, but who are not considered to have AIDS or HIV infection.
This point is well illustrated by the serological tests for syphilis. A
healthy person who is not infected with Treponema pallidum would very seldom
test positive (false positive).
However several authors attest to the presence in various unrelated
disorders of biological false positive tests to syphilis (BFPS), which
may occur in patients with auto-immune haemolytic anaemia, systemic lupus
erythematosus (SLE), idiopathic thrombocytopenic purpura, leprosy and in
drug addicts. More than 20% of drug addicts test positive and have the
highest incidence of BFPS's.(40)
Persons with BFPS were also found "to have a high frequency of
other serological abnormalities including anti-nuclear factors, autoantibodies,
and alterations of gamma globulin". This led researchers to conclude
that "a BFP reaction often is a marker for an unidentified disorder
of the immune system that predisposes to autoimmune diseases".(40)
It is of significance that a high proportion (14%) of AIDS patients were
also found to have false positive syphilis serology.(41)
At least two groups of researchers raised the possibility that the HIV
antibody test in Africans and IV users may also be a BFP reaction. Jaffe
et al (42) tested 1129 serum samples from IV drug users and 89 controls
from non-users. All samples were collected during 1971-1972 and tested
by two commercial ELISAs and WB. Seventeen of the samples from the IV drug
users, but not one of the controls was found positive.
They concluded: "On the basis of our positive Western Blot data,
it appears that parenteral drug users may have been exposed to HTLV-III
or a related virus as early as 1971. An alternative but equally viable
explanation is that the HTLV-III seropositivity detected in these specimens
represents false positive or non-specific reactions".
Biggar and his colleagues (43) found that in healthy Africans the probability
of finding a positive HIV antibody test increased significantly with increasing
immune-complex levels. They concluded "reactivity in both ELISA and
Western Blot analysis may be non-specific in Africans....the cause of the
non-specificity needs to be clarified in order to determine how they might
affect the seroepidemiology of retroviruses in areas other than Africa,
such as the Caribbean and Japan".
That a positive WB in all individuals may represent a BFP reaction is
suggested by evidence from both retrovirology in general and HIV antibody
testing in particular.
It is known that all antibodies including MCA are polyspecific and are
capable of reacting with immunising antigens as well as other self and
non-self components.(44,45) In relation to retroviruses, the scientific
literature abounds with data which convincingly show the widespread presence
of non-specific interaction between retroviral antigens and unrelated antibodies.
Much of this work has appeared as a result of the search for a viral origin
for animal and human neoplasms.(46-50)
In 1975 Gallo discovered that patients with leukaemia have widespread
infection (antibodies) to a retrovirus which Gallo claimed to have isolated
from cultures and fresh tissues of these patients and which he named HL23V.
Gallo suggested that this virus was aetiologically associated with the
disease but HL23V was later shown to be a "cocktail" of two monkey
viruses.
In 1980 Gallo discovered HTLV-I which he and his associates claim causes
adult T-cell leukaemia. Up to 25% of AIDS patients have antibodies to this
virus, (51) however AIDS patients do not develop leukaemia any more often
than the general population. This can only be interpreted as either HTLV-I
does not cause adult T-cell leukaemia or some retroviral antibodies detected
in AIDS patients are non-specific.
In 1986 Essex obtained serological evidence for, and isolated, another
"human retrovirus", HTLV-IV. Essex's HTLV-IV was later shown
to be a monkey virus, now called Simian Immunodeficiency Virus.
That a positive WB may not represent proof of HIV infection but is only
a non-specific marker for AIDS, is suggested by the following data:
In drug addicts there is a strong association between high serum globulin
levels and a positive HIV antibody test and this was the "only variable
which remained significant in a logistic regression model"; (52) In
children, using WB as a gold standard, hyperglobulinaemia identified HIV
infected children with a specificity of 97%.53 Sixty three sera obtained
from 23 patients before and immediately after immunoglobulin infusion were
tested for HIV antibodies using WB. Of the 63 sera, 52 (83%) were found
positive. "Several samples tested in an HTLV-III p24 radioimmunoassay
were also positive. The amount of antibody detected was greatest immediately
after infusion and decreased between infusions".(54)
An individual was given six 5ml injections of donated Rh+ serum, administered
at 4 day intervals. "The donor serum was shown to be negative on HIV
antibody and antigen ELISA, so was blood taken from his wife and child".
"Blood taken after the first immunization was shown to be negative
on HIV antibody ELISA and immunoblot assay. After the second immunization
a weak signal on ELISA, slightly above the cut-off level, was monitored.
After the third immunization the signal was strong and immunoblot revealed
distinct interaction with p17 and p55 proteins. An even stronger signal
was monitored after the fifth immunization. Interaction with p17, p31,
gp41, p55 and some other proteins was evident".(55)
Since individuals from the main AIDS risk groups, that is, gay men,
drug users and haemophiliacs are exposed to many foreign substances such
as semen, drugs, factor VIII, blood and blood components; and individuals
belonging to the above groups commonly develop infections unrelated to
HIV; one would expect these individuals to have high levels of antibodies
directed against antigens other than HIV. In fact at present, evidence
exists that individuals with AIDS, AIDS-related complex (ARC) and those
at risk, have circulating immune complexes, rheumatoid factor, anti-cardiolipin,
anti-nuclear factor, anti-cellular, anti-platelet, anti-red cell, anti-actin,
anti-DNA, anti-tubulin, anti-thyroglobulin, anti-albumin, anti-myosin,
anti-trinitrophenyl and anti-thymosin antibodies.(22,56)
Anti-lymphocyte auto-antibodies have been found in 87% of HIV+ patients,
and their levels correlate with clinical status.(57,58) Unlike normal sera,
37% of HIV+ sera were found positive for Type-D retroviruses, (59) whereas
HIV is thought to be a Lentivirus.
It is also known that serum IgG levels are higher in Black blood donors
than in Caucasians; (60) that some risk groups, drug users and gay men
are exposed to high levels of mitogenic agents, semen and nitrites, (61,62)
and that animals treated with such agents develop antibodies which react
with retroviral antigens.(63)
That the positive HIV antibody test may be the result of antigenic stimulation,
other than HIV, is further supported by the following data:
(I). HIV is thought to be transmitted by infected needles, yet a higher
percentage of prostitutes who use oral drugs (84%), than IV (46%), test
positive; (64)
(II) "Mice of the autoimmune strains MRL-lpr/lpr and MRL-+/+ made
antibodies against gp120". Mice that have been exposed to T-lymphocytes
from another murine strain were shown to make antibodies against gp120
and p24 of HIV.(65)
(III) Recipients of negative blood seroconvert and develop AIDS while
the donors remain healthy and seronegative.(66)
(IV) In healthy individuals, partners of HIV positive individuals, organ
transplant recipients and patients with SLE, a positive WB may revert to
negative when exposure to semen, immunosuppressive therapy or clinical
improvement occurs; (67,68,69)
(V) While the frequency of positive HIV antibody tests in healthy blood
donors and military applicants is low, patients with tuberculosis (TB),
including those with TB localised to the lungs, both in the USA70 and Africa,
(71) have high frequency, up to 50%, of positive WBs. In the USA72 (26
hospitals studied), patients who are not at risk of developing AIDS, and
who do not have any infectious diseases, have a high rate of positive WB,
(1.3% to 7.8%).
The above data may be interpreted either as proof that HIV is spreading
to the heterosexual population or that the HIV antibody tests are non-specific.
That the latter is the case is suggested by the fact that by 1988, in the
USA, (73) only approximately 66 white males were reported to have had "heterosexually
acquired AIDS". By 1992 in New York only 11 men were reported to have
AIDS due to heterosexual infection.(74)
Rodriguez and his colleagues (75) found that Amazonian Indians who have
no contact with individuals outside their tribes and have no AIDS have
a 3.3-13.3% HIV WB seropositivity rate depending on the tribe studied.
In another study (76) they found that 25%-41% of Venezuelan malaria
patients had a positive WB, but no AIDS. The above data means either that
HIV is not causing AIDS "even in the presence of the severe immunoregulatory
disturbances characteristic of acute malaria", as Rodriguez et al
concluded, or the HIV antibody tests are non-specific.
The problems associated with the specificity of the WB could be avoided
by use of the only suitable gold standard, HIV isolation. To date this
has not been done and based on the problems associated with HIV isolation,
it may never be feasible.
HIV Isolation
It goes without saying that virus isolation can be used as a gold standard
only if it provides conclusive genetic, virological and molecular evidence
for the existence of a unique virus. For retroviruses, as a first step
towards this goal one must find particles with morphological characteristics
similar to other retroviruses, and demonstrate that these particles have
a unique set of structural components including RNA and proteins which
belong only to these particles and to no other entity.
Peyton Rous (77) is credited with the discovery and isolation of the
first retrovirus. In 1911 he was able to repeatedly induce tumours in a
particular breed of chickens by means of tumour derived, cell free filtrates.
Rous contemplated that either a "minute parasitic organism"
or "a chemical stimulant" might form the basis of his observations;
nevertheless, the tumour inducing filtrates became known as "filterable
viruses" or oncoviruses.
In the 1950s, in animal cultures and in fresh tissue, especially tumour
tissue, particles later attributed to retroviruses were readily detectable
with electron-microscopy (EM).
In 1970, the enzyme reverse transcriptase (RT) which transcribes RNA
into DNA, was discovered in oncoviruses. Because of this, in the 1970's,
oncoviruses became known as retroviruses.
In the preceding decade, density gradient centrifugation was introduced
to separate and isolate sub-cellular particles including viruses.
Because some cellular constituents were found to have the same buoyant
density as viruses, when viruses were isolated from cell cultures, the
best results could be obtained with supernatant fluids which had high viral
concentration, and had low cellular contaminants.
This was best satisfied by non-cytopathic viruses and by culture conditions
which maintained maximum cellular viability. Most animal retrovirus (exceptions
are the so called animal immunodeficiency viruses) satisfy the above conditions.
Taking advantage of the above retroviral properties, by repeated suspension
and sedimentation in sucrose density gradients, one could obtain, at a
density of 1.16 gm/ml, a relatively pure concentration of retroviral particles-that
is, obtain retroviral particles, separate from everything else, and thus
isolate them.(78)
Nonetheless, as many eminent retrovirologists point out, contamination
of the viral preparation with virus-like particles which contain RT, but
could be nothing more than "cellular fragments", microsomes from
disrupted cells, "membraneous vesicles which may enclose other cellular
constituents including nucleic acids", especially when "inadvertent
lysis of cells" was induced, could not be avoided.(79,80,81)
Because of this, to prove that the material which banded at 1.16 gm/ml
contained nothing else but particles with "no apparent differences
in physical appearances", and that the particles were indeed retroviruses,
every retrovirus preparation was further analysed using the following assays:
(1) Physical-electron microscopy (EM) for virus count, morphology and purity;
(2) Biochemical-RT activity, viral and cellular RNA, total protein, gel
analyses of viral and host proteins and nucleic acids; (3) Biological-infectivity
in vivo and in vitro.(78,82)
Unlike animal virus cultures where the particle concentration is very
high (104-105 infectious units/ml), in the AIDS cultures/co-cultures
the particle concentration is low, so low that both Gallo's and Montagnier's
group had difficulty in detecting them.
Unlike most animal retroviruses, HIV is considered to be a cytopathic
virus. If this is so, then cell culture supernatants will contain many
cellular constituents. If, as has been recently proposed, "a single
unique mechanism", HIV induced apoptosis, can account for T4 cell
death, (83) then the supernatant must also contain apoptotic bodies, that
is, membrane bound cellular fragments which, (like many retroviruses),
bud from the cell surface.
Since the size and composition (some contain pyknotic chromatin) of
the apoptotic bodies vary widely, (84) one would expect that some of these
fragments will also band at 1.16 gm/ml.
It is significant that the AIDS cultures/co-cultures do not have maximum
viability, and most if not all claims of "HIV isolation" have
been from cellular lysates. Furthermore and most importantly, in an extensive
search of the AIDS literature no electron micrographs were found from
the material which bands at 1.16 gm/ml; all the electron micrographs
are of particles found in the cell cultures.
Thus it is impossible to be know whether the material-lipids, proteins
and nucleic acids, which bands at 1.16 gm/ml, (the "pure HIV particles"),
contains any such particles whatsoever, and if such particles are present,
what is their purity.
The presently available evidence indicates that only about 20% of the
proteins which band at 1.16 gm/ml are "HIV proteins", the rest
are cellular, including beta-2 microglobulin and HLA-DR proteins (4.4%).(12,85)
Thus, even if particles are present at 1.16 gm/ml and all the proteins
assumed to be HIV are embodied in the HIV particle, the material which
bands at 1.16 gm/ml cannot be considered "pure HIV".
Conversely, "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 per cent of the total p24 and gp120 present [in the
culture] is in virions".(86) In fact, p24 is released from "infected
cells independently of infectious virus particles" and RT.(87,88)
It must be pointed out that the terms in the AIDS literature "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: (a) "RNA wrapped in protein";
(89) (b) material from the cell culture supernatants which passes through
cell tight filters but through which organisms such as Mycoplasmas may
pass; (90) (c ) the pellet obtained by simple ultracentrifugation of the
culture supernatant (91); (d) recently, very often, detection in AIDS cultures
of p24.(92,93)
In the first report of "HIV isolation", Montagnier's group
detected in a mitogenically stimulated culture derived from lymph node
biopsies of gay men with lymphadenopathy, "a transient", "reverse
transcriptase activity". In mitogenically stimulated umbilical cord
lymphocytes cultured with supernatant from the above cultures, they reported
type-C retroviral particles (RVP) in the cultures and RT and antigens which
reacted with pre-AIDS sera in the material which banded at 1.16 gm/ml.4
Gallo's group did not consider the detection of the above as representing
"true isolation", "...the virus has not been transmitted
to a permanently growing cell line for true isolation and therefore has
been difficult to obtain in quantity".(94)
However, although Gallo's group used a permanent cell line for "HIV
isolation", they reported nothing more than the same phenomena as
Montagnier's group.
Nevertheless, at present, the detection of the above phenomena are considered
to represent "true isolation" and their finding in a similar
culture is regarded as proof of infectivity. However, isolation is defined
as separating the virus from everything else and not detection of some
phenomena attributed to the virus (RT, antibody/antigen reactions [WB]);
or similar to it, (particles).
Phenomena can only be used for viral detection-even then, if and only
if, the phenomena have been identified as being specific for the virus,
by using the isolated virus as a gold standard.
Although this has not been done, the presently available indirect evidence
(that is, evidence that has been obtained without a gold standard) from
both general retrovirology and AIDS research, indicates that RT, RVP and
the antigen/antibody reactions are not specific for HIV, (or even retroviruses).
The specificity of the antigen/antibody reactions has already been discussed
and will not be further mentioned. In any case, this reaction cannot be
used as a gold standard for the WB, since a test cannot be its own gold
standard.
Reverse transcriptase
In all HIV research, the copying of the template-primer An.dT15 when
incubated with the supernatant or the material which bands at 1.16 gm/ml
from the AIDS cultures/co-cultures is considered proof of HIV RT activity.
In many instances this activity is considered synonymous with "HIV
isolation" and is used to quantify the virus.
However: (a) The same template-primer is also copied when incubated
with material which bands at 1.16 gm/ml from leukaemic T-cell cultures
(95) and normal non-infected spermatozoa.(96) Both An.dT15 and Cn.dG15
are copied by material which bands at 1.16 gm/ml originating from normal
non-infected but mitogenically stimulated lymphocytes.(95,97) (b) An.dT15
is copied not only by RT but also by two (beta and gamma) of the three
cellular DNA polymerases. In fact, in 1975, an International Conference
on Eukaryotic DNA polymerases defined DNA polymerase gamma as the cellular
enzyme which "copies An.dT15 with high efficiency but does not copy
DNA well".(98) Thus, the copying of the template-primer An.dT15, cannot
be considered synonymous with the presence of HIV RT.
Particle detection
Retroviruses are enveloped infectious particles about 100-120nM in diameter
with a core comprising a protein shell and a ribonucleoprotein complex.
Retroviruses are classified into three Subfamilies-Spumavirinae, Lentivirinae
and Oncovirinae. Retroviruses belonging to the latter Subfamily are divided
into Type-A, B, C and D particles.
Nevertheless, some of the best known retrovirologists do not consider
the finding of "virus-like particles morphologically and biochemically
resembling", retroviruses, proof of the existence of such viruses.(99)
In the 1970s, such particles were frequently observed in human leukaemic
tissues, (99) cultures of embryonic tissues, (100,101) and "in the
majority if not all, human placentas".(102) However, they continue
to be "an intriguing and important problem that remains to be solved".(103)
The particles detected in AIDS cultures/co-cultures are considered by
all AIDS researchers as being HIV. However:
(I) There is no agreement as to which Genus or even Subfamily of retroviruses
they belong. Sometimes agreement is not found even within the same group.
For example, Montagnier's group initially reported HIV as a Type-C oncovirus,
(4) then a Type-D oncovirus (104) and subsequently as belonging to a different
Subfamily of retroviruses-Lentivirinae.(105) Moreover, the "HIV particles"
in monocytes differ from both the Type-C Oncoviruses and Lentiviruses.(106)
(II) Despite the above, Gelderblom et al put forward an HIV model (fig.
4) which has a well defined morphology and composition, including surface
knobs made of p120, a protein considered to play a crucial role in cytopathogenesis
and to be indispensable for HIV infectivity.(107) The model has been accepted
and is well known. However, the same group using EM and immune electron-microscopy
has shown that: (a) knobs are found only in immature (budding) particles.
Immature particles are "very rarely observed", and are seen only
"on metabolically impaired cells";(2,108) (b) mature particles
are "hardly, if at all, labelled" by AIDS and ARC sera. Immature
particles are "highly labelled", but so is the rest of the cell
from which they are budding, which "might be due to the fact that
natural immune sera are indeed polyspecific";(2,109) (c ) like sera,
antibodies to p120 react preferentially with immature particles.(107) MCA
against gag proteins label the mature particles, but they also label HIV-2
particles and simian immunodeficiency virus particles;(110) (d) in the
HIV particles, including its membrane, they (111) as well as others, (112)
detected many cellular proteins, but with the possible exception of the
"lateral bodies", these proteins are not included in the idealised
HIV model.
(III) The T-cell and monocyte "HIV infected cultures" contain
in addition to particles with the morphologies attributed to HIV, many
other "viral particles" unlike any of the "HIV particles".
(106,111,113,114) Non-HIV infected H9 cells, from which most of the published
EM have originated as well as other cells used for "HIV isolation",
CEM, C8166, EBV transformed B-cells, and cord blood lymphocytes, express
budding virus-like particles albeit they are somewhat different from particles
accepted as HIV.(115) The above data raises questions not only in regard
to the origin and role of the "non-HIV particles", but also the
"HIV particles", and as to which, if any of these particles,
band at 1.16 gm/ml.
(IV) Budding and mature type-C particles appear in metabolically impaired
but non-HIV infected lymphoma cells.(116) "Retroviral particles"
antigenically related to HIV have been found in cultures of salivary gland
extracts from patients with Sjorgen's syndrome.(117)
The independent finding of "virus-like" particles in the lymph
nodes of AIDS patients with lymphadenopathy (118) and of proteins in the
lymph nodes which reacted with MCA to p55, p24 and p18 (119) were interpreted
as proof that the " virus-like particles" were HIV. However:
(I) MCA to p18 react with lymphatic tissues of patients who suffer from
a number of non-AIDS related diseases, and also healthy individuals;(20,21)
(II) As in the AIDS cultures/co-cultures, in the lymph nodes of patients
with AIDS and persistent generalised lymphadenopathy, in addition to the
"HIV particles", particles unlike those of HIV are also found;(120)
(III) Most importantly, in the only EM study (121), either in vivo
or in vitro, in which suitable controls were used and in which extensive
blind examination of controls and test material was performed, virus particles
indistinguishable from HIV were found in a variety of non-HIV associated
reactive lymphadenopathies leading the authors to conclude: "The presence
of such particles do not, by themselves indicate infection with HIV".
Comments on "isolation"
One can conclude then that neither the antigen/antibody reaction, nor
the particles nor RT can be considered specific for retroviruses. Even
if they were, their finding cannot be considered as synonymous with the
detection of an externally acquired retrovirus, as is claimed to be the
case for HIV. Such findings may represent the expression of endogenous
retrovirus (vide infra) or other exogenous retrovirus. Lately, "several
laboratories reported retroviral activity [RT, particles] in cells of patients
who appear not to be infected by HIV", an activity said to be "from
endogenous retrovirus".(122)
The cell line most often used in AIDS research is the leukaemic cell
line H9. It is known that H9 is a clone of HUT78, which was derived from
a patient with adult T-cell leukaemia. Since the causative agent of this
leukaemia is accepted to be HTLV-I, another exogenous retrovirus, the H9
cultures should have both RT and retroviral particles even in the absence
of HIV.
Because about 25% of AIDS patients have antibodies to HTLV-I, about
25% of cultures should have in addition to particles and RT, a positive
WB to HTLV-I. However, since the proteins from HIV and HTLV-I share the
same molecular weights, the HTLV-I WB bands will appear to be positive
for HIV.
A more direct problem associated with the use of "HIV isolation"
as a gold standard is the fact that, irrespective of the various phenomena
accepted by AIDS researchers as representing "HIV isolation",
and despite the fact that no effort has been spared, it is not possible
to "isolate HIV" from all antibody positive patients. The success
rate varies between 17% and 80%.(92,93,123)
Conversely, when the same effort is made, HIV can be isolated from some
non-AIDS seronegative patients, and even from normal seronegative individuals
at no risk for HIV infection.(124,125) With a more recent method used for
"HIV isolation", detection of p24 in cultures with whole unfractionated
blood, (126,127) positive results have been reported in 49/60 (82%) of
"presumably uninfected, but serologically indeterminate" individuals
and in 5/5 "seronegative blood donors".(128)
As far back as 1988, researchers at the CDC in the USA realised that
no correlation exists between "HIV isolation" and a positive
antibody test (which they call documented infection), and more importantly,
between "HIV isolation" in vitro and its presence in
vivo-"correlation between these two methods is limited; they are
inconsistent, in that virus cannot be detected in every person with a documented
infection. Furthermore, the culture technique determines the ability of
infected cells to produce virus in vitro but does not necessarily
indicate the status of virus expression in vivo".(129)
Genomic Investigations
In the decades following Rous' experiments, Rous as well as other researchers
performed similar investigations with several animal species. However,
although neoplasia could be induced by injection of filtrates from tumour
tissues, (infectious retroviruses, exogenous retroviruses), no epidemiological
evidence existed to suggest an infectious origin of cancer.
In 1939 Andrews "speculated on the possible activation of latent
viral infectious particles in cancerous tissues", and in 1948 Darlington
postulated "that such viruses [endogenous viruses] could arise from
cellular genetic elements which he named proviruses".(80)
In the 1950s and 1960s the following experimental evidence was considered
proof of the proviral hypothesis: (a) healthy animals in which no complete
virus could be detected had viral antigens similar to those of exogenous
virus; (b) DNA genomes or partial genomes of the infectious retroviruses
were found to be integrated into the genomes of normal non-virus producing
cells; (c ) "Final proof came with the isolation of infectious viruses
from uninfected cells". Healthy non-virus producing cells when cultured
were found to spontaneously produce viruses.(80) Their appearance and yield
could be increased a millionfold by (i) mitogenic stimulation;(130) (ii)
co-cultivation techniques;(131) (iii) cultivation of cells with supernatant
from non-viral producing cultures.(132) (Note:For HIV isolation, mitogenic
stimulation is an absolute requirement, and in fact, in most cases, all
of the above are employed).
At present it is generally accepted that "one of the most striking
features that distinguishes retroviruses from all other animal viruses
is the presence, in the chromosomes of normal uninfected cells, of genomes
closely related to, or identical with, those of infectious viruses".(80)
Depending on conditions, the provirus genome remains unexpressed or
part or all of it may be expressed. The latter may or may not lead to the
assembly of viral particles (endogenous retrovirus). (80) In other words,
the finding of a viral genome (DNA) or even of RNA, antigens and antibodies
to them, is not proof of the presence of infectious particles.
Although most of the above findings are from animal experiments, at
present, evidence exists that "The human genome carries DNA sequences
related to endogenous retroviral genomes that are subdivided into families
according to sequence homology. Some are present in only a few copies,
whereas others are present in hundreds to thousands of copies".(133)
Animal data also shows that new retroviruses may arise by phenotypic
mixing, and genetic recombination and deletion.
When a cell contains two proviruses, progeny may be found that possess
the genome of one but the structural proteins of either or both viruses
present. Conversely, the RNA may be viral but at least some of the proteins
may be cellular.
In other instances, the particles do not have a genome at all, or one
or more genes are missing (genetically defective viruses). The genetic
mixing can be between viral genomes or between viral and cellular genes.(80,134)
According to distinguished retrovirologists such as Weiss and Temin,
new retroviral genomes may arise by rearrangement of cellular DNA caused
by many factors including pathogenic processes, a view that proposes retroviruses
as an effect and not the cause of disease.(135,136)
The time and appearance of the viral genome "may be millions of
years in germ-line cells and days in somatic cells".(136)
In addition to the above, the retroviral replicative cycle "involves
three distinct steps: reverse transcription, DNA polymerization, and the
synthesis of RNA from a DNA template (transcription). Any errors made by
the polymerase enzyme during the first and the third steps are not subjected
to proof reading, the result being pronounced sequence variability".(137)
Hence, as long ago as 1973, it was concluded that the above phenomena
"will prove a stumbling block to any genetic analysis of RNA tumour
viruses" (138) (RNA tumour viruses=retrovirus). To date, the data
on the HIV genome has not altered the above prediction and shows that many
problems may exist with the use of the genomic studies in efforts to prove
infection of AIDS patients with a unique exogenous retrovirus, HIV.
Some of these problems can be summarised as follows:
(I).No two HIV genomes are the same.(a) No two identical HIV
have been isolated even from the same person. In one case where two sequential
isolates were made 16 months apart, none of the provirus in the first isolate
was found in the second (139) leading one HIV researcher to conclude "The
data imply that there is no such thing as an [AIDS virus] isolate"
(140); (b) from the same person at a given time more than one HIV can be
isolated (141,142); (c ) many, if not all of the proviruses detected in
vivo and in vitro are defective; (143) (d) In one and the same
patient, the genomic data in monocytes differs from that in T-lymphocytes;
(144) (e) the genetic data obtained in vitro does not correlate
with the data obtained in vivo-"To culture is to disturb"
(145); (f) The type of virus isolated is determined by the cell types used
for HIV isolation.(142,146)
(II) There is no correlation between "isolation" of HIV
and detection of the HIV genome. Cultures positive for "infectious
virus", may be "polymerase chain reaction-negative".(147)
(III) HIV sequences cannot be found in all AIDS patients. Gallo
and his colleagues, summarising the first hybridisation studies with fresh
tissue concluded: "We have previously been able to isolate HTLV-III
from peripheral blood or lymph node tissue from most patients with AIDS
or ARC" [approximately 50% of patients referred to by Gallo]. "However,
as shown herein, HTLV-III DNA is usually not detected by standard Southern
Blotting hybridization of these same tissues and, when it is, the bands
are often faint...the lymph node enlargement commonly found in ARC and
AIDS patients cannot be due directly to the proliferation of HTLV-III-infected
cells...the absence of detectable HTLV-III sequences in Kaposi's sarcoma
tissue of AIDS patients suggests that this tumor is not directly induced
by infection of each tumor cell with HTLV-III...the observation that HTLV-III
sequences are found rarely, if at all, in peripheral blood mononuclear
cells, bone marrow, and spleen provides the first direct evidence that
these tissues are not heavily or widely infected with HTLV-III in either
AIDS or ARC".(148) These studies were confirmed by many other researchers.
To improve detection, the polymerase chain reaction (PCR) method was
introduced. However, "a striking feature of the results obtained so
far" with this method, as with the standard hybridisation technique,
"is the scarcity or apparent absence of viral DNA in a proportion
of patients"(149) and, when viral RNA or DNA is found, the "signal"
is very low.
For example, HIV is thought to be transmitted primarily by sexual intercourse
yet with the PCR the "HIV genome" can be detected in a minority
of semen samples (1/25).(147) It must be pointed out that a positive PCR,
even if found in all patients as is claimed in some publications, (149)
cannot be regarded as signifying the presence of the whole HIV genome.
With the PCR "only small regions may be amplified, a gene at best"
(143) that is, one does not detect the whole viral genome, and, since most
HIV "isolates" to date are defective, detection of part of or
a whole gene, or even several genes, cannot be considered synonymous with
the whole HIV genome.
Furthermore, the PCR is not standardised and to date, there has been
only one study in which the reproducibility, sensitivity and specificity
of PCR were examined. In this study, the gold standard used was serological
status. Specificity was determined by measuring the percentage of negative
PCR results in seronegative (ELISA), healthy, low risk individuals (blood
donors).
The PCR was found not to be reproducible and "false-positive and
false negatives results were observed in all laboratories (concordance
with serology ranged from 40% to 100%). In addition, the number of positive
PCR results did not differ significantly between high- and low-risk seronegatives".(150)
(IV) The positive hybridisation results may not be HIV specific.
In 1984 when Gallo and his associates conducted their first hybridisation
studies, they found that when the results were positive, the hybridisation
bands were "faint", "low signal".
The "low signal" was interpreted as proof that HIV infected
individuals contain provirus in small numbers of peripheral blood mononuclear
cells and at low copy numbers. However, according to Gallo and his associates,
"theoretically this low signal intensity could also be explained by
presence of a virus distantly homologous to HTLV-III in these cells".(148)
Data which has come to light since then suggest this theoretical possibility
may be a fact: (a) Although it is no longer accepted that HIV is transmitted
by insects, in 1986 researchers from the Pasteur Institute found HIV DNA
sequences in tsetse flies, black beetles and ant lions in Zaire and the
Central African Republic.(151) (b) In 1984 Gallo's group reported that
the genome of HIV hybridises with the "structural genes (gag, pol,
and env) of both HTLV-I and HTLV-II".(152) Presently available evidence
shows that normal human DNA contains retroviral genomic sequences related
to HTLV-I and II.(153,154) (c ) In 1985 Weiss and his colleagues reported
the isolation, from the mitogenically stimulated T-cell cultures of two
patients with common variable hypogammaglobulinaemia, a retrovirus which
"was clearly related to HTLV-III/LAV"; evidence included positive
WB with AIDS sera and hybridisation with HIV probes.(155) (d) DNA extracted
from thyroid glands from patients with Grave's disease hybridises with
"the entire gag p24 coding region" of HIV.(156) (e) Horowitz
et al, "describe the first report of the presence of nucleotide sequences
related to HIV-1 in human, chimpanzee and Rhesus monkey DNAs from normal
uninfected individuals". They have "demonstrated the presence
of a complex family of HIV-1 related sequences" in the above species,
and concluded that "Further analysis of members of this family will
help determine whether such endogenous sequences contributed to the evolution
of HIV-1 via recombination events or whether these elements either directly
or through protein products, influence HIV pathogenesis".(157)
That the positive hybridisation signals may be due to such events induced
by the oxidative agents (mutagens and mitogens) to which the AIDS risk
groups and the cultures are exposed is suggested by the following: A positive
PCR reverts to negative when exposure to risk factors is discontinued (158,159),
and monocytes from HIV+ patients in which no HIV DNA can be detected, even
by PCR, become positive for HIV RNA after cocultivation with normal ConA-activated
T-cells".(160)
As far back as 1989 researchers at the Pasteur Institute concluded that
"the task of defining HIV infection in molecular terms will be difficult".(145)
They confirmed their conclusion in a recent study where they "described
the enormous heterogeneity found in vivo within HIV-1 populations"
and the possibility "that an HIV carrier may harbour easily in excess
of 1010 proviruses, most of which will be genetically unique". They
conclude: "It is therefore possible that the sheer size of variants
within an infected individual will allow HIV to explore totally new genetic
possibilities". The appearance of "radically different genetic"
retroviral structures may be the result of "rearrangement, duplication,
deletion or hypermutation. The transduction of host cell DNA represents
possibly the most startling genetic trait of retroviruses".(161)
Conclusion
It is axiomatic that the use of antibody tests must be verified against
a gold standard. The presently available data fail to provide such a gold
standard for the HIV antibody tests. The inescapable conclusion from the
above discussion is that the use of HIV antibody tests as predictive, diagnostic
and epidemiological tools for HIV infection needs to be carefully reappraised.
*
Acknowledgements
We wish to thank all our colleagues and especially Udo SchEklenk, Barry Page, Bruce Hedland-Thomas, David Causer,
Richard Fox, John Peacock, David Prentice, Ronald Hirsch, Patricia Shalala, Keith Jones, Alun Dufty, June Rider Jones,
Coronary Barrow, Dorothy Davis, Julian Smith, Mark Strahan, Vincent Turner, Wallace Turner and Graham Drabble for
their continued support and assistance.
Dedication
This work is dedicated to the memory of Methodios Papadopulos and Margaret Joan Turner.
Authors:
Eleni Papadopulos-Eleopulos, Physicist
Department of Medical Physics
Royal Perth Hospital
Valendar F. Turner, Staff Specialist
Department of Emergency Medicine
Royal Perth Hospital
John M. Papadimitriou Professor of Pathology
Department of Pathology
University of Western Australia
Correspondence to:
Eleni Papadopulos-Eleopulos
Department of Medical Physics
Royal Perth Hospital
Box X2213 GPO Perth
Captions for figures 0-4.
Figure 0.(left out with publication)
WB patterns with patient sera "and reaction with a strong, weak and non-reactive control". (Reproduced from Bio-Rad
Laboratory Manual).
Figure 1.
(A): "Cord blood T-lymphocytes infected with virus" (HIV-1) were lysed and the supernatant of a 10,000g centrifugation of
the cell lysate was immunoprecpitated with sera from patients with lymphadenopathy (P); a healthy donor (h); goat
antiserum to HTLV-I p24 (G); normal goat serum (g).
(B): As 1A but cells infected with HTLV-I instead of HIV-1. 2C: The cell free supernatant from the cultures of "cord blood
T-lymphocytes infected with virus" (HIV-1) was ultracentrifuged for one hour at 50,000 rev/min.The pellett was banded in
sucrose density gradients. Material which banded at 1.16gm/ml (the complete virus) was immunoprecipitated with the above
sera but instead of normal goat serum, serum from another healthy donor (h) was used. Although in the published strips it is
hard if not impossible to distinguish any bands, in the text, it is stated that "three major proteins could be seen: the p25
protein and proteins with molecular weights of 80,000 and 45,000" (Modifed from BarrG-Sinoussi et al. Science Vol
220:p870).
Figure 2.
(A): "Lysates of HTLV-III producer" H4 clone cells, derived from the HUT78 cell line immunoprecipitated with various
sera.
(B): "Lysates of HTLV-III producer" H17 clone cells also derived from the HUT78 cell line, immunoprecipitated with
various sera; (the serum in B lane 2 is identical to (A) Lane 4).
(C): Lysates of H17 and H4 clones (b) "before" and (a) "after infection", immunoprecipitated with serum from a male
heterosexual drug user with lymphandenopathy and thrombocytopenia (pre-AIDS). This is the same serum as (B) Lane 5.
(D): "Lysates of H4/HTLV-III... cells" (C), or "virus purified from the cells culture fluids", (V), using (I)-same serum as (B)
Lane 5; (II)-serum from a patient with pre-AIDS; (III) serum from a patient with AIDS. This is the same serum as (B) Lane
4.
Key to sera: (A) AIDS patient; (P) pre-AIDS patient; (h) healthy control; (U) drug user; (H) homosexual control; (Modified
from Schubach et al 1984. Science Vol 224:p504).
Figure 3.
WB of one and the same serum specimen tested by 19 laboratories. (From Lundberg GD 1988. JAMA Vol 260:p676).
Figure 4.
Structural model of HIV. From reference 107.
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