sieroPositivita’ (s+) al TEST Western Blot e’ la prova di una infezione ? (English)
BIO/TECHNOLOGY VOL.11 JUNE 1993 – vedi: Test fraudolento + L’altra storia dell’Aids + Hiv virus inventato
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).
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.
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.
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.
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“.
“Il paziente malato di Aids NON muore a causa del virus dell’HIV ma per alterazioni dell’assorbimento intestinale e quindi per ipoalimentazione (malNutrizione), dovuta a una grave micosi.” (By Dott. Gerhard Orth, Leuthkirch)
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