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  • The Perth Group

  • Eleni Papadopulos

  • Valendar Turner

  • John M. Papadimitriou

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  • Bio/Technology

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  • HIV Tests

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  • HIV Test Accuracy

  • ELISA

  • Western Blot

  • HIV Antibody

  • False Positives

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  • Scientific Paper

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  • 1993

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  • The content discusses the reliability of Western Blot tests for HIV infection, highlighting potential false positives and the impact of various factors on results.

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  • This is a scientific paper that questions the reliability of HIV antibody tests as a definitive proof of HIV infection. The authors argue that these tests are not standardized or reproducible, and the proteins used in the tests may not actually be specific to HIV. They emphasize the need for a gold standard, such as the HIV virus itself, to validate the accuracy of these tests. The paper calls for a careful reevaluation of the use of HIV antibody tests in predicting, diagnosing, and studying HIV infection.

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  • HIV

  • Western Blot

  • Infection

  • ELISA

  • Reproducibility

  • Laboratories

  • Antibodies

  • Specificity

  • Sensitivity

  • Predictive value

  • Gene products

  • False positives

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Eleni Papadopulos-Eleopulos, Valendar F. Turner and John M. Papadimitriou

Original Publication
BIO/TECHNOLOGY VOL.11 JUNE 1993
https://pubmed.ncbi.nlm.nih.gov/7763673/

View file
nameEPENatBioTech1993.pdf

...

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..."

...

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).

...

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);

...

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)

...

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.

...

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.

...

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 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.

...

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.

...

(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)

...

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.

...

As far back as 1989 researchers at the Pasteur Institute concluded that "the task of defining HIV infection in molecular terms will be difficult".(145) They confirmed their conclusion in a recent study where they "described the enormous heterogeneity found in vivo within HIV-1 populations" and the possibility "that an HIV carrier may harbour easily in excess of 1010 proviruses, most of which will be genetically unique". They conclude: "It is therefore possible that the sheer size of variants within an infected individual will allow HIV to explore totally new genetic possibilities". The appearance of "radically different genetic" retroviral structures may be the result of "rearrangement, duplication, deletion or hypermutation. The transduction of host cell DNA represents possibly the most startling genetic trait of retroviruses".(161)

Conclusion

It is axiomatic that the use of antibody tests must be verified against a gold standard. The presently available data fail to provide such a gold standard for the HIV antibody tests. The inescapable conclusion from the above discussion is that the use of HIV antibody tests as predictive, diagnostic and epidemiological tools for HIV infection needs to be carefully reappraised. *

Acknowledgements

We wish to thank all our colleagues and especially Udo SchEklenk, Barry Page, Bruce Hedland-Thomas, David Causer, Richard Fox, John Peacock, David Prentice, Ronald Hirsch, Patricia Shalala, Keith Jones, Alun Dufty, June Rider Jones, Coronary Barrow, Dorothy Davis, Julian Smith, Mark Strahan, Vincent Turner, Wallace Turner and Graham Drabble for their continued support and assistance.

Dedication

This work is dedicated to the memory of Methodios Papadopulos and Margaret Joan Turner.

Authors:

Eleni Papadopulos-Eleopulos, Physicist
Department of Medical Physics
Royal Perth Hospital

...

John M. Papadimitriou Professor of Pathology
Department of Pathology
University of Western Australia

Correspondence to:

Eleni Papadopulos-Eleopulos
Department of Medical Physics
Royal Perth Hospital
Box X2213 GPO Perth

Captions for figures 0-4.

Figure 0.(left out with publication)

...

Structural model of HIV. From reference 107.

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