Is a Positive Western Blot Proof of HIV Infection?
Bio/Technology Vol.11 June 1993
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. *
References-
Second Part of Paper
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