The following research projects and studies were proposed by indicated panelists during the period following the panel meetings.
The proposals that follow (proposals 1,2 and 3) have been put forward by the group that was set up by the Presidential AIDS Advisory panel during their meeting in South Africa in May 2000. The members of the group are Drs H Bialy, P Duesberg, H Gayle and MW Makgoba.
The basic idea in the validation of HIV ELISA Testing in South Africa is to proceed in stages, graded in order of simplicity, and designed so that the results of each stage will determine what, if any, form the next stage will take. This study is based on the fact that:
A random and blinded Quality Assessment study of 2500 samples from different sites will be undertaken. The sites are:
These sites cover the spectrum of high risk and low risk groups as well as high prevalence and low prevalence samples.
The samples will be subjected to time-tested protocols for quality assurance in South Africa and at the Centers for Disease Control in the USA. The correlation between the results in South Africa and the Centers for Disease Control will be analysed to provide the following:
These data should also form the basis of subsequent studies as proposed below in the Preadsorption and Virus isolation experiments.
Time table for the study:
To determine the robustness of the current HIV ELISA tests that are being used in South Africa when the sera that is being tested has been treated to remove antibodies that are reactive to a series of known antigens that have been previously reported to interfere under certain conditions with HIV ELISA tests that depend on either recombinant proteins, or recombinant proteins and synthetic peptides such as V3.
Blood samples from 100 TB patients that have had no prior HIV serology will be obtained by Professor Mhlongo. An additional 100 blood samples from "HIV/AIDS" patients from the most densely affected region in the country will be obtained by Dr Makgoba.
These blood samples will be taken to the major laboratory in South Africa that does HIV ELISA testing, where Drs Makgoba and Roberto Stock (an investigator from the Institute of Biotechnology in Mexico, an expert on immunodiagnostics of all varieties and protein purification and biochemistry), along with South African colleagues of the panel's choosing (scientists, students, technicians) will have prepared a series of ELISA plates that have been coated to contain:
Sera from each of the 200 samples will be added to the ELISA wells of these plates and incubated for two hours, after which the contents of the wells will be transferred to HIV ELISA plates and treated as is normally done. Everything will be done in duplicate.
South African HIV researchers need to be assisted to gain even greater awareness of the power and usefulness of the beacon technology as a general diagnostic tool, but particularly with reference to Multiple Drug Resistant Tuberculosis. It is not being proposed that the beacon assay be used as any form of gold standard.
The first proposed step is to set up and calibrate the ABI Prism Machine as well as teach South African researchers how to operate the machine and how to synthesise, purify and use molecular beacons. Once this is done, decisions will then be taken as to what degree and how the use of the beacon technology on the samples collected for the Quality Assessment of HIV Testing (as in Proposal 1 above) would be productive.
The proposals that follow were suggested by members of the Presidential AIDS Advisory Panel either during the panel meetings in May and July 2000 or during the Internet debate between the two meetings.
Proposer: Prof Peter Duesberg
One Thousand and Five Hundred (1500) healthy HIV-positive and 1500 matched
healthy HIV-negative men from the South African army and/or mining industry,
or some other governmental institution would be required for this study.
This experiment will exclude people who suffer poverty, malnutrition, poor
sanitation. Since the time of infection of these men is not known,
and since they are currently healthy, their times to AIDS would be randomly
distributed from a maximum of 5-10 years to a minimum of one day to AIDS.
On average they are half way into their HIV to AIDS latent period of 5-10
years, or 750-1500 days (1/2 of 5-10 years) from getting AIDS. Therefore
in the HIV positive group there should be 1 or 2 AIDS cases per day, and
in the negative group there should be no AIDS cases. We would know
much of the answer in a few months and certainly within a year if we had
1500 men in each group. It would take longer if the groups are smaller.
The cost would be one conventional HIV test per person, and perhaps a second
one if a AIDS disease co occur and a phone call per person or to their supervisor
every 2 months to find out how they are.
9.6 Proposal 5: Preadsorption and Virus Isolation Experiments
- The need for a Gold Standard in the diagnosis of HIV infection.
The proposal on the preadsorption studies has strong similarities with proposal 2 above, but is included in this document as it appears as a package with the proposed experiments on virus isolation.
Proposers: Dr Eleni Papadopoulos-Eleopoulos and Dr Val Turner
At present, all the HIV experts admit that:
Since the late 1970s, all the HIV experts claim that:
This means that once the existence of HIV is accepted, it is not possible to refute the HIV theory of AIDS by claiming that:
Each of the above arguments against the HIV theory can be easily refuted :
The only way to prove or disprove the HIV theory is by experiments. Several experiments have been proposed including the following:
Although these experiments are useful, they never can prove or disprove the HIV theory of AIDS. Regarding the first experiment listed above, finding for example that only 50% of African patients who satisfy the clinical definition of AIDS in Africa have a positive antibody test will show that the African clinical syndrome has a poor positive predicting value for HIV infection and will reduce the number of AIDS cases by 50% but will not disprove the HIV theory. With respect to the second experiment listed above, even if a very small proportion, for example 10%, of military recruits had developed AIDS in ten years, it does not prove that HIV is not the cause of AIDS. There are many infectious diseases in which only a small proportion of infected patients end up with the clinical manifestation. Finally, the third experiment listed above, even if it shows that no correlation exists at all between retroviral-like particles observed in the plasma, it does not prove that HIV is not the cause of AIDS or even that the patients are not infected with HIV.
From the very beginning when the HIV theory was introduced, we wanted to perform pre-adsorption experiments. These experiments like the ones outlined above even if they show that all the antibodies present in AIDS patient's sera can be adsorbed by antigens other than HIV are not going to disprove the HIV theory or that the patients are infected with HIV. However, they will show that it is not possible to claim that a positive antibody test proves HIV infection unless HIV isolation (purification) is used as a gold standard to prove the specificity of this test.
The reasons why we have been proposing, again from the beginning of the HIV era, for the HIV isolation (purification) experiments are:
Claims for HIV isolation (purification) have been made by Montagnier's group in 1983 and by Gallo's group in 1984. However, in 1997 Montagnier acknowledged that his group did not obtain proof for isolation (purification) and in his view neither did Gallo's group. In the same year, some of the best known retrovirologists noted that no one had presented proof for isolation (purification) of a unique retrovirus, HIV.
The isolation experiments, as proposed by us, will prove once for all if HIV has been isolated (purified) and thus if there is such a thing as a human retrovirus, HIV. There are several indications that this has not been achieved so far:
From this, it follows that the antibody test cannot be used to prove "HIV" infection unless "HIV" isolation (purification) is used as a gold standard to prove (a) is the reason and not (b).
One of the physical characteristics of retroviruses is their density. In sucrose density gradients, they band at the density of 1.16gm/ml. So for this experiment:
Proposers: Prof. Gordon Stewart, Prof. Sam Mhlongo, Dr. Christian Fiala, Prof. Charles Geshekter and Dr. Roberto Giraldo
The proposers will need to spend some 2 - 3 days in Geneva re: UNAIDS data.
Proposers: Prof. Gordon Stewart, Dr. Roberto Giraldo, Dr. Harey Bialy and Prof. Sam Mhlongo
Principal Proposer: Prof Gordon T. Stewart
The investigations should be arranged in consultation with Professor Schoub or Dr Gray, and performed in the laboratory or laboratories responsible for routine serological tests for HIV by the ELISA method or Western Blot or both.
For clinical purposes and for surveillance, a diagnosis of AIDS (AIDS/HIV, HIV Disease) is made by the demonstrations of antibodies to antigens of HIV obtained from original LAV-BRU, HTLV3 or similar complex cellular co-cultures. This is usually done by the ELISA method of immunofluorescence with or without "Confirmation" by a chromatographic test for identification of the same reacting antibodies by Western blot.
There is a consensus among expert advisers and health authorities internationally supporting this procedure and indeed denying, as in the Durban Declaration, that there is any reason to doubt the reliability of diagnoses, surveillance or clinical decisions made on this basis. But, irrespectively of the state of health of an individual or community, a positive result by either method supports and in many cases mandates a diagnosis of AIDS. The latest revision of the ICD assumes that all seropositive persons are at risk of AIDS and that the majority will proceed to develop signs, sooner or later. Although false-positive, false-negative, cross-reactive and indeterminate results frequently occur for various reasons or for no obvious reasons, it is further assumed that a "True" positive result can be identifies as a reliable indicator of infection with live HIV and therefore of active disease which will progress to AIDS or AIDS-related conditions (ARC’s). This belief prevails despite the fact that direct isolation of HIV as proof of infection is difficult or impracticable, and that other surrogate tests such as the PCR/RNA and lymphocyte counts are not appropriate for routine diagnosis.
Presymptomatic screening of all pregnant females is deemed to be necessary for prevention or treatment of HIV disease in them and in their infants. However, it is known that many results are indeterminate, that false positive and negative results can occur and that many other disorders and physiological changes can give indeterminate or non-specific results. In situations where the incidence of "True" seropositivity is low, the likelihood of false or indeterminate results may be higher. This leads to uncertainties, anxieties, and unnecessary interventions like cessation of breast-feeding or termination of pregnancy. Errors in either direction are frequent and can cause catastrophes in relationships, families and communities, especially in countries where stigmatisation, social exclusion and expulsions occur when positive results are known or suspected
Reliability of sero-diagnosis is therefore the critical element in the identification and management of all forms of HIV/AIDS, and for assessment and prevention of vertical as well as horizontal transmission. To improve quality control in diagnosis and surveillance, it is suggested that the following method and precautions be adopted This will measure the overlap between HIV/AIDS and other prevalent disorders, give ongoing estimates of sensitivity and specificity of serological results, and provide a data-base for checking projections. Since the object of the exercise, is to check and improve the reliability of diagnosis and prognosis by these tests, which often precede clinical diagnosis or development of disease, especially in pregnant women and infants, the test is regarded as the independent and the outcome as the dependent variable.
Under present procedure, a person who is seropositive to HIV (i.e. whose blood contains antibodies to HIV) is diagnosed as having AIDS or a related condition (ARC, or AIDS-defining Disease (ADD)), or being at risk of it. But it is known that many conditions unrelated to HIV/AIDS can also give positive or indeterminate results for shorter or longer periods. These conditions include tuberculosis and malaria, recent vaccinations, certain tumours, pregnancy and other altered states of health which are commonplace in populations where AIDS is prevalent, especially in Africa. For accurate diagnosis and to enable appropriate advice to be given to patients, contacts and families, it is important to recognise this overlap. For doctors and health authorities, it is essential to know the full implications and extent.
Since seropositivity in itself mandates a diagnosis of AIDS, the overlap has to be ascertained by recording details of any other conditions present at the time in samples of blood sent to designated laboratories from clinics, hospital wards and surveys. Results, whether positive, negative or indeterminate, are then tabulated for comparison with the information held by the senders, each set of data being “Blinded”. This produces data sets in which the frequencies of these three grades of results are shown in relation to clinical diagnosis of presumed AIDS, at risk of AIDS from behaviour or contact, AIDS-defining diseases (ADD’s), AIDS or ADDS plus other named diseases or conditions, and other diseases or conditions without AIDS.
With thousands of samples of blood being routinely tested as at present, this procedure will yield data sets from which the frequency of true positive (AIDS only) results can be measured against those in the other categories. If, for example, 10 out of a hundred samples are true positive but 10 positive results are obtained also from those with other diseases without unequivocal clinical signs of AIDS, a person with a positive result in that sample is as likely to have some other condition, and so on according to the alternates indicated in the text of the full proposal.
Because direct identification of HIV itself is not required and is indeed impracticable at present for routine diagnosis, indirect serological tests are the measures used for decisions about all aspects of HIV/AIDS, and especially for assessing and controlling vertical, perinatal and puerperal transmission. Failure to detect false positive and false negative reactions leads to errors not only in diagnosis, treatment and other interventions, but also to erroneous projections and fear – or alternatively irresponsible disregard – of dangers to persons, families and entire communities. These dangers apply to underestimates no less than to overestimates of AIDS and also to risks of overlooking other diseases submerged in the over-riding classification of HIV/AIDS. The present proposal, which should be discussed and implemented co-operatively with existing clinical and laboratory services, is designed to minimise these dangers.
This investigation could be extended to Sentinel surveillance and all cases of AIDS (with controls) admitted to hospitals. Samples giving positive and indeterminate results should be, as often as is practicable, subjected to tests for antibodies to other agents, for example: CMV, HSV, VZ, EBV, and to tests for auto-immune and non-specific antibodies, in such conditions as pregnancy, disseminated lupus erythematosis and other auto-immune disorders to see if patterns of cross reactions can be identified. These data might then be used for more critical analysis of the hypothesis that HIV is the essential cause of AIDS.
This proposal is submitted in outline so that it can be circulated for comment and revision.. It is accepted that further detail will be required for implementation which should be arranged if possible with the National Institute of Virology, with those responsible for testing and surveillance, and for compilation of registration data in South Africa.
Although Sentinel Surveillance as organised by the WHO requires serodiagnosis by Elisa, using recombinant antigens prepared from co-cultures of HIV, it should be noted that the Bangui definition of AIDS agreed by the WHO and member States in 1987, is regarded as sufficient to warrant a diagnosis of AIDS or AIDS-related conditions without any serological test. National and international data do not normally indicate the proportion or location of diagnoses or projections made on this basis but it is obviously important to include in the programme described above some provision for identifying this proportion.
Principal Proposers: Dr Harvey Bialy and Dr Roberto Giraldo
To take blood from four groups of people and run the tests highly diluted, undiluted and at a wide spectrum of dilutions in between.
All groups would be subjected to both ELISA and Western blot tests. Additionally, all plasma samples will be subjected to the viral load test for HIV.
The result of such experiment could determine whether these tests measurements bear any relationship to an individual's level of exposure to stressor or oxidizing agents. If so, the tests could be salvaged as a measure of individual's level of intoxication.
Bases and references for these experiments can be seen in my postings "Tests for HIV are highly inaccurate" and "Everybody is HIV-positive".
Proposer: Prof. Etienne de Harven:
To test the reliability of one of the main laboratory method currently used to quantify so-called HIV in the blood of sero-positive individuals. More specifically, to use electron microscopy (EM) to verify that the blood plasma of patients identified as having a high "viral load" by PCR does indeed contain retroviral particles, and that, therefore, such samples could be used to isolate and purify HIV, free from cell debris and adventitious material from co-cultures.
Using the Roche Diagnostics Corporation "Amplicor HIV-1" monitor test:
Readily access to 5 patients with very high PCR counts. (Group A)
Readily access to 5 patients with undetectable PCR counts. (Group B)
Low speed centrifugation to separate and discard erythrocytes, leukocytes and platelets. Plasma samples (10 ml) diluted 1/1 with cold heparinized Ringer solution. Filtration by aspiration through a Millipore 0.6u membrane. Collecting filtrate #1, and filtering it this time using a Millipore 0.2u membrane. Collecting filtrate #2 and placing it in appropriate Beckman tubes for ultracentrifugation in either a fixed angle or a swinging bucket rotor, using the refrigerated ultracentrifuge to spin the sample under 30.000g for 2 hours. Inspect the tubes for the likely presence of extremely small pellets. Avoiding any risk of resuspending the pellets, cover them with 1.5 % glutaraldehyde in 0.1M cacodylate buffer (pH7) overnight at 0-4°C, rinsed with buffer and post-fixed with 1% osmium tetroxide for 90 min. After rinsing, the pellets will be kept for several hours in 0.5% uranyl acetate at 0-4°C, dehydrated in ethanol and propylene oxide and embedded in Epon. Thin sectioning with diamond knives, staining with uranyl acetate and lead citrate will be followed by examination under the transmission electron microscope at initial magnification ranging between 10.000 and 40.000x.
If the current interpretation of PCR "viral load" result is correct, EM should show plenty of retroviruses in Group A, none in Group B. Otherwise, the existence of a viremia in "high viral load" patients will have to be fundamentally re-appraised.
Samples from the same patients will be used to perform a classical PCR test by the Roche Amplicor HIV-1 routine method, following rigorously the test kit manufacturer’s recommendations.
The pellets obtained in the microfuge, with the 5 samples from Group A, will be analyzed by EM using a methodology comparable to the one described above. This could, eventually, confirm the presence of retroviral particles and will certainly permit to evaluate the presence and the amounts of cell debris.
The absence of HIV particles from the discarded supernatant should be verified, simply by submitting it to high speed centrifugation at 30.000xg for 2 hours and subsequent analysis of the resulting pellet, as described above.
Control samples should be prepared by the double Millipore filtration method that is known to eliminate most cell debris and to concentrate retroviruses in an almost pure form. The final viral pellet will be compared to a "microfuge" pellet from the same patients, both pellets being then processed identically for RNA amplification.
Technical assistants, trained in routine virology, knowledgeable in ultrafiltration and ultracentrifugation methods, and fully trained in routine Roche Amplicor PCR method.
Technical assistant knowing routine transmission EM procedures, with special proficiency in chemical fixation, plastic embedding, and ultrathin sectioning.
Major equipment needed:
Laminar flow hood, Millipore filtration equipment, refrigerated ultracentrifuge, PCR test kit, ultramicrotome fitted with diamond knives, high resolution transmission electron microscope.
Estimated cost:
If trained technical assistance and all the major equipment are available, the cost of running these experiments can be anticipated to be approximately 500 US dollars for each patients. Total cost for 10 patients would run around 5,000 US dollars. If the results of the proposed experiment are clear-cut and unambiguous, the n umber of 10 patients should suffice to make the point. If they are not, another group of 10 patients should be considered for an extension of the study.
Time required:
If the appropriate patients can be reached without delay, the total time frame of these experiments should probably not exceed a matter of 4 to 8 weeks. My presence would be essential in setting up the sampling procedures and to perform all the EM examinations.
At this time, priority should be given to the identification of ZA labs were this type of work could efficiently be performed, efficiency depending on 3 main factors: 1) a friendly welcome, 2) satisfactorily trained and available assistants, and 3) appropriate equipment. If this would appear difficult, we should then consider doing part of the work either in Europe or in New York.
Finally, it would be of considerable importance for me to be informed of the experimental proposals presented by Dr. Peter Duesberg and by the Perth group, in order to coordinate the entire project in a coherent fashion.
Proposers: Dr. David Rasnick and Dr. Claus Köehnlein
Brief Description:
Chimpanzees are known to be susceptible to HIV infection (i.e., develop antibodies to HIV), however, none to date has come down with AIDS. We propose to take 60 chimpanzees divided into three groups of 20 each as follows:
There are two outcomes of the study: