From: Subject: In search of virus HIV Date: Sun, 30 Mar 2008 20:48:05 +0200 MIME-Version: 1.0 Content-Type: multipart/related; type="text/html"; boundary="----=_NextPart_000_00A0_01C892A7.5AA1DC50" X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.3198 This is a multi-part message in MIME format. ------=_NextPart_000_00A0_01C892A7.5AA1DC50 Content-Type: text/html; charset="iso-8859-1" Content-Transfer-Encoding: quoted-printable Content-Location: http://www.cesil.com/0898/enfrah08.htm In search of virus HIV ......... =20
 Fabio Franchi
 =20
Abstract         = Curriculum         =20 Bibliografia  
=93The task of researchers is to = try to falsify=20 as many theories as = possible=94  =20
Karl Popper 
=93Is = there a danger,=20 in molecular biology, that the accumulation of data will get so = far ahead=20 of its assimilation into a conceptual framework that the data will = eventually prove an encumbrance? Part of the trouble is that = excitement of=20 the chase leaves little time for reflection. And there are grants = for=20 producing data, but hardly any for standing back in=20 contemplation=94. =20
John Maddox=20 (1)
 According to the AIDS = infectious=20 theory, the HIV infection is active since from the first phases = and during=20 the years and it is very difficult for the organism to get rid of = it.=20 Proper tests reveal its presence. Those which at the moment are at = our=20 disposal are essential not only for the diagnosis (2), but they = are used=20 as indicative elements with prognostic and therapeutical purposes=20 (3).  
As science for definition is open to every control and = all its=20 demonstrations must be proved on the basis of logical rules, this = study=20 has the object of verifying if there is effective relation among = the=20 different =93AIDS tests=94 and the meaning attributed to them. We = shall=20 examine the most important: the tests of the antibodies (the = ELISA, the=20 Western Blot and the p24), the viral culture, the genetic = identification=20 with amplification methods, the viral isolation, the micrographs = of HIV.=20 We shall analyse and criticize the procedure followed at present, = as=20 presented in the updated specialized texts including =93AIDS, = Biology,=20 Diagnosis, Treatment and Prevention=94 (2) published in 1997, = edited by=20 several top researchers including A. Fauci, former manager of the = National=20 Institute of Health.  
In other words we tried to = answer to=20 the crucial question as the great clinician and science = methodology=20 scholar, prof. Augusto Murri would have formulated it: =93Why have = I to=20 believe that the AIDS test meaning is equivalent to an infection = with the=20 HIV virus?=94 He thought it was essential, during one's life, to = be in the=20 habit of criticizing before of accepting = information. =20

ANALYSIS OF THE = ANTIBODY=20 TESTS =20

The tests of the = antibodies we=20 shall examine are the Elisa, the Western Blot, the p24 antigen = capture=20 assay. =20

ELISA (Pict.=20 1) =20

In Italy, the = screening test=20 (ELISA), =93provided with a good sensibility (near 99%) and a = lower=20 specificity, valued about 90-95% in 1988=94(4), is used at first = to identify=20 the HIV infection. However these percentages must not deceive, in = fact the=20 test predictive value changes depending on the infection = prevalence in a=20 certain population. According to a theory expressed in 1988 by the = AIDS=20 National Commission (4), in case of screening of the Italian = prison=20 population, in comparison with 6,902 real positive individuals = there=20 should have been 1.751 false positive; on the contrary, in case of = blood=20 donors, in comparison with 198 real positive, a good 999,990 = should have=20 been false positive (505 times more!). In the over mentioned text = (2) the=20 authors assert that the progressive improvements took sensibility = of the=20 test to 95% and its specificity to 99.8%, in high-risk population. = Nevertheless they admit that =93the positive predictive value can = greatly=20 fluctuate depending on the population they study=94. Actually this = happened.=20 Depending on the information published on the English = authoritative=20 medical magazine The Lancet (5), in 1990 on 20.2 million ELISA = tests made=20 in Russia, 20,000 were positive, but only 112 were confirmed by = the WB; in=20 1991, on 30 million ELISA tests, a good 30,000 were positive, but = of these=20 only 66 were confirmed by the Western Blot, that is a minimum = percentage=20 (0.002%). 
=93Then as the ELISA test has been planned to optimize = the=20 sensibility to the detriment of the specificity, it should not be = used by=20 itself for the HIV-1 infection diagnosis without a confirmation = test (2),=20 although in some Countries is used alone. =20

Western Blot = (pict.=20 2) =20

The Western Blot = is commonly=20 used as a confirmation test of HIV infection; the combination of = the Elisa=20 and the WB should have a =93positive predictive value higher than = 99% for=20 low-risk and high-risk population=94 (2).  =
However even the WB does = not seem to be=20 so specific as sustained if we analyze some data at our=20 disposal.  
A) Even official sources acknowledge that =93for = reasonable=20 disagreements it was very difficult to code the definition on what = a=20 positive Western Blot should be considered=94 (2) . Since 1993, in = United=20 States there were just 5 official criteria, and only one = (indicated in Du=20 Pont's diagnostic kit) was approved by FDA (Food and Drug = Administration).=20 This was the most restrictive one and was used by a few = laboratories. If=20 only this criterion had been used , in United States only the 50%=20 seropositive individuals would have been confirmed as really = infected (6)!=20 In Italy in a publication of the Health Ministry directed towards = G.P.=20 (7), the authors denied there had ever been such a problem. On = this=20 subject they affirmed: =93in most cases, quite apart from the = criterion=20 used, the test results correctly coded when the individual is=20 infect=94.  
The remedy to such a situation was found simply = standardizing the=20 different criteria without reconsidering the past and without = explaining=20 the scientific reasons for which they opted in that=20 way.  
Afterwards the CDC identified their criteria with the = ASTPHLD's=20 ones and in 1993 the United States Red Cross conformed adopting = the=20 ASTPHLD/CDC's advice=94(2). =93According to the American authority = ASTPHLD, a=20 positive result must present at least two of the three main bands = of=20 diagnostic meaning (antigp-41, anti-gp120/160,=20 anti-p24)=94. 
B)The reading of the WB is visual and therefore is = liable to=20 interpretation from the operator, a variability factor not = negligible.=20 =93Its technique (note: the Western Blot) was not standardized, = the=20 importance and the consequences occurred in the laboratories have = not been=20 yet measured.  
Their results must be = interpreted; the=20 criteria for these interpretations vary not only from laboratory = to=20 laboratory, but even from month to month..=94 (8). The Consortium = for=20 Retroviral Serology Standardization (CRSS) made a quality control=20 study*and sent 19 aliquots of the same serum to 19 different USA = reference=20 laboratories. Results comparison has shown an incredible = differences of=20 the number of bands and of their intensity (6) (pict.=20 3). 
C)=20 The antigenic preparations are not purified.  =
=93The presence of bands in = positions not=20 corresponding to well-known virus antigens is a rather common = finding and=20 we suppose it is the consequence of contaminants in the = preparation...=94=20 =93The molecular weight of these aberrant bands may vary either = from company=20 to company or even from lot to lot of the same producing = company=94=20 (2). 
Here=20 is Zolla-Pazner's comment in 1989: =93A confusion on the = identification of=20 these bands (note: the results of the Western Blot test ) resulted = in=20 incorrect conclusions in experimental studies. [...] A = re-interpretation=20 of the already published results could be necessary=94(9). = Re-interpretation=20 that has never been made. 
D) The bands considered = =93specific=94 in=20 many cases does not indicate an HIV infection. Some examples=20 follow. 
-=20 Even if the more restricting criteria were employed, Lundberg = noticed that=20 10% control sera, which included blood samples from blood banks, = had a=20 positive WB (6).  
-  A study(10) shows = that from 2=20 to 49% tested patients can have a reaction to the WB (unspecified = WB),=20 more frequently caused by cross-reacting = antibodies. =20
- A simple = vaccination=20 against flu can give a positive result too = (11). =20
 - = Transfusion of own=20 irradiated blood induces the formation of the same antibodies=20 (12). 
-=20 Among the homosexuals and bisexuals who resulted negative for = ELISA and=20 PCR,  20%-30% can have either one or more bands present in = the=20 Western Blot.(2) 
- There are even more amazing data concerning dogs and = published=20 in 1990. Writing on Cancer Research, Strandstrom at al. reported = that=20 72/144 (50%) dog blood samples obtained by the Veterinary Hospital = of the=20 University, California, and tested with the Western Blot reacted = with one=20 or more HIV  recombinant proteins [gp120--21.5%, gp41--23%,=20 p31--22%.p24--43%]=94(13). As the Authors think that dogs can not = be=20 infected with HIV, one must conclude that these data are a further = prove=20 of the frequent antibody cross-reaction with many proteins = different from=20 HIV ones=94. 
Remarks: The fact that the bands are considered = unspecific if they=20 are found separately, but virus-specific if they are present in a = number=20 of two or more, seems to refer not to considerations of a = biological kind,=20 but to a convention, a legal agreement changeable in time and = space (in=20 fact it is different from nation to nation). According to a = convention=20 accepted since a few time ago by many authors, three bands in a = clinically=20 healthy blood donor's WB were considered false positive reactions. = According to the same convention, three bands of the same blood = donor,=20 this time presented to an Insurance Company for a life policy,=20 should  be considered true positive.  =
The researchers group from = Perth who=20 analyzed in depth the WB meaning, concluded that the proteins = considered=20 viral proteins are not peculiar to HIV, but one cannot distinguish = them=20 form proteins of a cellular source. In particular, according to = the review=20 by Eleopulos et al.(14) which has not yet been refuted up to now, = the gp41=20 should correspond with actine; the p18 and p24/25 with the two = myosin=20 sub-units (p24 is the unspecific band found more frequently in=20 =93uninfected=94 individuals); the p32 should be identical to the = beta chain=20 of the antigen DR of the histocompatibility class II; the = gp120-160 should=20 not be other than gp41 oligomers. =20

The p24=20 antigen =20

In U.S.A. the = evaluation of=20 the p24 antigen capture test was included among the assays which = are=20 carried out to each blood unit (and it is considered as an =93HIV = direct=20 evidence=94). Fauci et al.(2) think that: a) the p24 corresponds = to virus=20 capsid antigen (core) which is highlighted by a serum containing = the=20 related monoclonal antibodies (anti-p24) associated to a detector = system;=20 b) however the test is not useful like a primary screening mean in = establishing a precocious HIV infection diagnosis; c) the p24 = level=20 increasing corresponds presumably to the blast in virus = replication, which=20 can be observed by other methods like plasma viraemia; d) the p24 = level=20 usually falls under the determination threshold with conventional = methods=20 and like the antibody anti-p24 starts developing during = seroconvertion and=20 can remain in that state during the following asymptomatic = infection=20 years. Only 20-30% of the individuals with asymptomatic infection = has=20 detectable levels of p24 serum.    =
However there are many = perplexities on=20 its real meaning. Quantity data indicate that 50 pg and sometimes = more of=20 core protein (p24) may be found in seropositive patients. If a = retrovirus=20 weight is of 10-3 pg and half of its weight is constituted  = by p24=20 (15), then this quantity corresponds to about 100,000 viral = particles,=20 according to other authors and on other premises even to 500,000 = particles=20 (16)! Then if all this protein came from virions, these patients = should be=20 highly viraemic (17, 18). In other words there should be a direct=20 correspondence among the p24 levels, =93virus isolation=94, = =93viral RNA=94 levels=20 (said also =93viral load=94). On the contrary it is not so. = Results are=20 irremediably different from the ones one should expect. For = example a=20 paper reports the presence of antigenaemia, but that no virus = could be=20 isolated by 31 antigenaemic patients, even after an extended = culture=20 (19). 
Other studies report similar remarks. We shall mention = some of=20 them:  
1) in half of the cases when a person had a positive = test,=20 afterwards he had a negative one without making any treatment = which could=20 alter the p24 levels... the test is clinically  erroneous and = should=20 be interpreted with much caution=94.(20)  =
2) the p24 antigen is found = neither in=20 all seropositive subjects nor in individuals with clear AIDS. In a = study ,=20 the PCR (Polymerase Chain reaction) and p24 were used to detect = HIV in=20 patients in in various stages, from asynptomatical to AIDS = individuals.=20 They found the p24 in 24% and  HIV-RNA in 50% of patients=20 (21). 
3)=20 More than 29% of the patients who receive a transfusion from = seronegative=20 individuals, become positive for the anti-p24 antibody (22), even = =93on=20 infection absence=94.  
4) The unspecificity of the = p24 antigen=20 assay is so obvious to be accepted by an authority recognized in = the=20 field, Philip Mortimer.et al- from Public Health Laboratory = Service in=20 England.=94 =93Experience proved that neither virus culture, nor = p24 tests are=20 of great value in diagnostic tests. They can be either insensible = or=20 unspecific=94 (23).  =20

Conclusion =20
The antibody = tests (the=20 ELISA, Western Blot and p24 antigen capture assay) for the many = reasons=20 over explained, are non able to indicate with reasonable assurance = they=20 can face an HIV infection and so the assertion that WB positive = predictive=20 value (24) can be considered higher than 99% does not correspond = with=20 reality. These tests show reactions of a variable or rather = undetermined=20 specificity, so that they cannot allow a distinction between=20 cross-reaction and =93HIV infection=94. =20

THE DIRECT=20 IDENTIFICATION  
OF THE = VIRUS =20

It is a common = conviction that=20 the antibody tests should be ratified and in a certain way = verified from=20 other ones which are able to highlight the virus itself, tests = able to=20 identify it directly. However, this direct determination is such = only=20 apparently. It can only record some phenomena which are considered = associated to the HIV in an unequivocal way. Virus direct = identification=20 is divided into 2 main categories (2): the virus culture and the = direct=20 identification of the viral nucleic acids (Southern Blot, = amplification=20 techniques like the PCR,TR PCR, bDNA, NASBA). =20

The viral=20 culture =20

Usually virus = isolation=20 involves a patient's cell co-culture with a donor's uninfected = cells which=20 have been stimulated with phitoemoglutinine (PHA) for 3 days. = These=20 co-cultures are controlled about every 3 days for 28 days or more = to=20 control either the syncitia formation and p24 presence or the = reverse=20 transcriptase (RT) in culture supernatant (2). The presence either = of=20 syncitia (a) or the p24 antigen (b) or the RT (c) is considered a = virus=20 replication evidence (2). According to Gallo et other authors even = the=20 finding of virus-like particles in culture must be considered a = sufficient=20 criterion.  =20

a) The appearance = of=20 sincytia.  
Sincytia appearance is observed only in considered = infected=20 cultures, but these are present even in =93uninfected=94 cell = lines and are=20 similar to the ones used for HIV (25). Then such morphological = feature=20 could be a characteristic either of the same cell lines, or of the = result=20 of the culture conditions or both these factors = (14). =20

b) The p24=20 antigen.  
Its specificity lack has already been mentioned. The = objections to=20 use it like a test on the patient's serum are efficacious even for = its use=20 in cultures. In a study where this =93virus isolation=94 method = was employed (=20 the determination of the p24 in cultures with unfractioned entire = blood),=20 positive results were obtained in 49 of 60 =93presumably = uninfected but=20 serologically undetermined individuals=94(82%) and in 5 of 5 = seronegative=20 blood donors (26). =20

c) The reverse = transcriptase=20 (RT).  
In all the HIV research, the ability of copying = template-primer=20 A(n)dT15 - when it is incubated with the supernatant or material = coming=20 from infected material co-cultures with the AIDS virus which = separates at=20 a 1.16 gm/ml level (sucrose density gradient) - is considered the = proof of=20 a reverse-transcriptase activity of HIV. In many cases this = activity is=20 considered synonymous of =93HIV isolation=94 . However: also the=20 template-primer itself is copied when it is incubated with = material which=20 separates in 1.16 band from leukemic cell cultures (27) and = uninfected=20 spermatozoa(29); b) the template-primer A(n)dT15 can be = transcripted not=20 only by RT, but also by other DNA cell polymerases. All the DNA = cell=20 polymerases, alfa, beta and gamma are able of copying the A(n)dT15 = (29).=20 In fact. in 1975, an International Conference on DNA which = included=20 Baltimore and Gallo (30) on eukariotic polymerases defined the DNA = polymerase gamma, =93a normal cell component=94 (31) which = activity can be=20 increased by many factors including the PHA stimulation (32), an = enzyme=20 which =93copies the A(n)dT15 with high performance, but does not = copy well=20 the DNA=94 (33). From what over explained we may deduce that it is = wrong to=20 affirm that such an enzyme activity is peculiar of retroviruses, = and less=20 that this is an unequivocal index of the presence of HIV. = Particles and=20 proteins which separate at 1.16 mg/ml band level could reflect = fully=20 unviral material, most of the same material is certainly cellular = and=20 coming from cultures stimulated with PHA (as we shall see better = later),=20 since transcriptase activity could derive from = it. =20

d) Objects with a = retrovirus=20 appearance.  
Gallo considered the finding of retrovirus-like = particles in=20 culture like the proof of the HIV isolation (at that time = HTLV-III) (34).=20 However Gallo used H9 (HUT78) cell line to isolate HTLV-IIIB, and = this=20 cell line releases retrovirus-like particles even when it is not = infected=20 with HIV=94 (35). Weiss obtained his =93isolate CBL-1 from the = leukaemic cell=20 line CEM, a cell line which hosted retroviruses even when they = were not=20 infected with =93HIV=94(36). In 1970, these particles were = frequently observed=20 in leukaemic tissues (37), in embryonic tissue cultures (38, 39) = and in=20 most if not in all human placenta (40). Mature and *extrobended* = particles=20 of the C pattern appear in linphoma cells which are metabolically = damaged=20 but are not infected with HIV (41). =93Retrovirus particles=94 = antigenically=20 similar to HIV were found in extracts from Sjorgen syndrome = patient's=20 salivary glands(42). And more important thing, in the only study = of=20 electron microscopy(43), both in vivo and in vitro where suitable = controls=20 were used and where an extensive assay in cieco of controls and = test=20 material was made, =93virus particles indistinguishable from HIV = were found=94=20 in a variety of reactive linphoadenopathies which are not = HIV-associated,=20 so that the authors concluded: =93The presence of such particles = does not=20 indicate, for itself, an HIV infection=94(pict = 4). =20

Conclusion: it is = inevitable=20 to agree that neither syncitia presence, nor p24 finding, nor = reverse=20 transcriptase activity, nor virus-like particle presence (well, = each=20 element which settles that an HIV culture is positive) are =93a = virus=20 replication evidence, though just this is the significance given = to=20 it. =20

Direct = identification of the=20 nucleic acids   =20

For direct = identification of=20 the nucleic acids one intends the Southern Blot, PCR techniques, = the=20 quantitative amplification techniques (RT PCR, bDNA, NASBA). The=20 identification is based on the identification and amplification of = little=20 nucleotide sequences attributed to HIV, not of the entire viral = genome.=20 This research is made not on cultures, but direclty on patient's = tissues=20 (including blood). =20

a) Southern blot = technique=20 (technique which allows to separate and highlight nucleic acids of = the=20 searched kind).  
In 1984 Gallo carried out = the first=20 study. Using the Southern blot hybridization technique (capable=20 theoretically of identifying only one infected on ten cells), AIDS = patient's tissues were examined, but weak bands were found only in = a=20 minority of them (44). =20

b) The PCR = (polymerase chain=20 reaction).  
The PCR sensibility is valued 92-100% (45) and is able = to detect=20 one =93infected=94 cell on 100.000 (2). However, =93for its high = sensibility, it=20 is greatly liable to false positivity either for even minimum=20 contaminations or other common laboratory mistakes=94 (2). = Retrovirus-like=20 sequences can be found in human genome in every cell (46). Already = in=20 1991, Imagawa and Detels pointed out a  potential fallacy of = the PCR.=20 =93The determination of a part of the genome- they affirmed- does = not mean=20 to detect an entire virus=94 (47). =20

The problem of the = false=20 positives.  
Many researchers challenge the PCR reability for the = great number=20 of false positive that this test should produce(48). The PCR did = not prove=20 reproducible and false positives and false negatives were observed = in all=20 the 7 reference laboratories involved in an important French study = (the=20 concordance with serology varied from 40% to 100%). Moreover, the = number=20 of positive results was not significally different among high and = low risk=20 seronegatives (49). There are many other confirmations to these=20 remarks:  
1) A validity study in evaluating the HIV PCR = performance and=20 in  identifying free-cell viral  DNA showed =93a = disturbing high=20 rate of unspecific positivity=93 employing commonly used primers = (SK38, both=20 for the p24 and the gag gene). Really, similar positivity rates = were found=20 either for antibody negative samples than for antibody negative=20 samples(18% against 26%) (50).  
2) the PCR (qualitative = DNA-PCR) made=20 on uninfected infants appeared positive on many occasions=20 (51).  
3) In a PCR validity study coordinated by the OMS Work = Group on=20 the PCR, 54% of involved laboratories had problems with false = positive=20 results; 9.3% of uninfected samples were reported as=20 positive(52).  
4) The authors of a = multicenter study=20 of quality evaluation stated =93this study showed that false = positive=20 results, in spite of rigorous procedure algorithms, happen among=20 uninfected individuals with such a frequency to remain a serious=20 problem=94(53). The discover of a considerable number of =93false = positives=94=20 gives evidence of a remarkable PCR specificity = lack. =20

c) Quantitative=20 techniques.  
The =93quantitative=94 techniques=94 should be able to = calculate, in an=20 approximate way, the number of virions per  milliliter of = plasma=20 using opportune modifications of some amplification techniques = (either=20 substrate amplification, like RT-PCR and NASBA, or signal = amplification,=20 like bDNA.The substrate consists in =93preserved regions=94 of a=20 virus-presumed gene. The calculated sensibility is: for the RNA = RT-PCR of=20 100 HIV RNA copies per milliliter of plasma(2); for NASBA of 1,000 = molecules per milliliter (54); for bDNA (branched DNA) of 10,000 = molecules=20 per milliliter (2). Up to 1994 HIV was considered to be present in = small=20 quantities in asymptomatic and AIDS subject's tissues (1 = lymphocyte on=20 1,000-100,000). In 1995 two studies (55, 56) seemed, on the = contrary, to=20 show that HIV replicated with much activity since from the first = phases of=20 the infection. The over-mentioned techniques in appearance allowed = huge=20 virus quantities, which in a first time had escaped from = observation, to=20 be detected. 
However if a massive infection were present in infected=20 individuals, as some of the most known experts claim, the Southern = blot=20 hybridization would have been more than sufficient to detect it. = Don't=20 forget that =93either virus DNA scarcity or lack in a considerable = patient's=20 part was a surprising result=94(57). A John Maddox' significant = Freudian=20 slip confirms suspects: according to Maddox =93both Ho than Wei = and their=20 colleagues were able to come to their alarming conclusions only = after a=20 ten years research of the HIV as they teamed up with = mathematicians and=20 because they were able to use new techniques to detect the low = levels of=20 virus involved =93(58)! 
Logical incompatibilities = of results=20 are macroscopic and evident.  
A) In fact there is a lack = of=20 correlation between p24 and gene amplification techiniques. (We = have=20 already mentioned the fact that the p24 is considered to be =93 = the direct=20 proof of HIV identification=94). =20

A) No relation was = found=20 between the p24 level and the RT-PCR (59). The determination of = the viral=20 charge was highly sensible when it was compared with the p24 = measurement=20 (more than 95% of patients had detectable virus charge while only = 40% had=20 detectable p24), but in positive samples antigen and viral charge = level=20 had only a borderline significance (60).   =

B) No relation was = observed=20 between viral charge quantified either with the NASBA or the = Amplicor and=20 the concentration with the p24 antigen. No relation was observed = between=20 the infectious dose, the viral virl load found either with the = NASBA or=20 the Amplicor(RT-PCR) (61). =20

C)There is a = relation lack=20 between RT-PCR and other assays. In a study on vaccinated = subjects, a=20 group of four serum samples resulted positive with RT PCR during a = 11=20 months period, including a result of 104-105 copies/mL, and an = year later=20 a fifth positive result in a vaccinated subject, but not = infected=94 (62).=20 For this reason, the authors advanced =93doubts concerning = specificity of=20 HIV RNA measurement as a golden standard for the screening and the = infection confirmation=94. =20

D) There is = finally a lack of=20 correlation between =93virus=94 and =93viral = load=94.  =20
1) According to = Piatak, the=20 high =93virus=94 level in plasma was 999 per 1,000 = =93unculturable=94=20 (63).  
2) Ho et al. showed that 10,000 plasma =93virions=94 = counted with the=20 branched PCR correspond with less than one =93infectious=94 virus = per ml (64).=20 3 
) In a=20 more recent study by Ho (65) one may notice that 0 (zero!) = =93cultured=20 viruses=94 corresponded to a load of 500,000 units! According to = the=20 reported data, to patient 105 before the treatment (with = Ritonavir)=20 643,000 =93virions=94/ml were detected, where the number was = calculated with=20 the branched DNA assay, more than 1,000 TCID-50/ml corresponded to = it (50%=20 tissue culture infectious doses). After a two day therapy and in = following=20 days the =93cultivable virus=94 was 0(zero). In the meantime, at = 2, 3 and 4=20 days =93free virions=94 (that is the so-called =93viral charge=94) = remained over=20 500,000. Till 7 day =93virions=94 were again over 100,000. A = virion is defined=20 as a complete viral particle constituted with the viral nucleic = acid,=20 capsid and pericapsid (when present). The possible objection for = which a=20 defective virion is =93seen=94 by branched DNA assay, but is not = detected by=20 culture samples, does not resolve the problem of = non-correspondence with=20 the other tests and the impossibility of seeing these particles at = the=20 microscope directly from patient's tissues, without culture = transfers. If=20 it were so, Gallo would not need for applying to frauds (67, 68), = he would=20 not call for three year tireless researches in order to find = something=20 which had the appearance of a virus. He would have come to it = immediately=20 with other researchers. 
Mark Craddock, professor at = the School=20 of Mathematics and Statistics, The University of Sidney, Australia = commented: =93What is this viraemia of billions of RNA particles = that can=20 only be seen with an undocumented branch-PCR or PCR but not with a = functional infectivity test?=94. (68) =20

VIRAL=20 GENOME 
  
As already stated, genomic amplification techniques = concern small=20 segments, not surely the complete =93viral=94 genome and the = results which are=20 obtained do not give the assurance to have the true =93HIV=94, = besides they=20 are frankly discordant. At this point it's legitimate to ask what = is=20 really and how it's made the original viral genome. The reserch = group from=20 Perth (69) notes that if an unique AIDS retrovirus existed, then = less than=20 1% genomic differences should be the rule, not the exception. For=20 instance, the type 3 Sabin poliovirus vaccine differed from its=20 neurovirulent progenitor at only 10 nucleotide positions after 53 = in vitro=20 and 21 in vivo passages in monkey tissues. In 1977, H1N1 influenza = A virus=20 reappeared in the human population after 27 years of dormancy with = sequences mainly identical to those of the 1950s virus=94. = Although Eigen's=20 quasispecies model has been used to describe the genome of RNA = viruses,=20 even 1% sequence differences in these genomes are considered to = represent=20 =93extreme variability=94. =93Many selective forces may stabilize = virus=20 populations. These stabilizing factors may include the need for=20 conservation of protein structure and function, RNA secondary = structure,=20 glycosylation sites, and phosphorylation sites=94. Let us see what = is the=20 picture offered by HIV research. Not long afterwards it was = discovered=20 that =93If you were to test two HIVpositive people at random and = analyse the=20 genetic material of their strains, they would differ, on average, = by about=20 13 percent=94 (70). Besides what is considered to be the genotype = of HIV=20 comes from sequence analysis of subgenomes that   HIV = genotype=20 consignments are derived from sequence analysis of subgenomes = measuring 2%=20 to 30% of the total (71). The data is that such =93genomes=94 vary = between=20 3-40% (72,73). =93If 30% of the HIV genome varies as much as 40%, = how much=20 does 100% of the HIV genome vary? This is the legitimate question = of=20 Eleopulos and colleagues (69). 
Among researchers there is = disagreement=20 also about the number of HIV genes. In 1988 they said HIV genome = had eight=20 genes (74), in 1990 ten (75). In 1996 Montagnier referred that HIV = had=20 eight genes (76), and according to Barr=E9-Sinoussi, HIV has nine = genes (77)=20 Not even the number of nucleotides of HIV genome seems to be = constant=20 (69) =20

HIV=20 quasi-species  =20

Because great = genomic=20 differences were found - even the same person can harbor more than = 106-108=20 genetically distinct variants (78) - to try to find a = justification for=20 this phenomenon, the concept of quasi-species has been introduced. = Prior=20 to the 1990s, the HIV sequences were classified as African and=20 USA/European with sequence differences of 20-30 percent between = these two=20 groups. (79). In the 1990s, HIV researchers started to divide the = =93HIV=20 genome=94 into subtypes A, B, C, D, E, etc. The basis for this=20 classification system is: =93(a) subtypes are approximately = equidistant from=20 one another in env; (b) the env phylogenetic tree is for the most = part=20 congruent with gag phylogenetic trees; (c) two or more samples are = required to define a sequence subtype=94. However, =93Subtype = naming problems=20 have arisen. A small but not insignificant number of viral = sequences are=20 hybrid, clustering with one sequence subtype in gag and another = sequence=20 subtype in env, for example;... Naming becomes problematic when = highly=20 divergent forms of a given subtype arises: such forms are = sometimes=20 designated A', B', F', etc=94. It is increasingly necessary to = have sequence=20 data from both gag and env coding sequences when a new form or = subtype is=20 being claimed=94 (80). By the middle of 1996 =93at least ten=94 = (A-J) prevalent=20 major (M) and a low prevalence, O, HIV-1 genotypes were described = and new=20 genotypes are still reported (81,82).  =
Is it possible then to = describe the=20 =93HIV DNA=94 even if it has variation of 10% , not to mention 20 = or 30 or 40%=20 as is the case, as a ...=94population of closely related genomes, = referred=20 to as a quasispecies=94 (69)? The extreme variability of HIV = genome make us=20 ask what is the sense to refer to HIV as a clear distinct entity. = The=20 genomic difference among human being of different race is about 1 = per=20 1.000, between man and orang-utan about 2%, but to find a 30% = difference=20 is necessary to compare non the less man and mouse, man and ass, = man and=20 elephant! Can one consider mouse, ass and elephant as = quasi-species of=20 man? (pict. 5). 
Particle detection Retrovirus are defined as particles = of about=20 100-120nM of diameter with a core which includes a proteic coat = and a=20 ribonucleoprotein complex. Retroviruses are classified in three=20 subfamilies:  Spumavirinae, Lentivirinae and Oncovirinae.=20 Retroviruses belonging to the last subfamily are divided in type = A, B, C,=20 and D particles. To which of these should belong HIV, is not very = clear.=20 According Gallo in 1984, it was a type C particle, but in 1985 he = admitted=20 it could be also a type D (83). Montagnier and coll. initially = reported it=20 was a type C particle (84), then a type D (85), and then a = lentivirus=20 (86), that is to say a different subfamily. According to Munn and = others=20 AIDS virus shows characteristics of type C, of type D and of = lentivirus=20 (87). Fauci and others described HIV in monocyte/macrophages = cultures as=20 retroviral particles with characteristics of type A (88). HIV = identity=20 seems to be so vague, so =93shot=94, that it adds further reasons = of=20 perplexity. Photographs Several researchers in the AIDS field have = shown=20 HIV photographs, but those that can be seen are only virus-like = viral=20 particles indistinguishable from normal cellular microvescicles. = These=20 particles, in contrast with viruses, are very unstable when = removed from=20 their contest, and it's difficult to isolate them and photograph = them in a=20 condition of a true isolation. On the contrary, viruses are stable = because=20 they have to leave again the cells and host organisms. The use of=20 centrifugation techniques doesn't constitute a problem to separate = viruses=20 from various contaminants and so get their isolation - then = photograph=20 them, to put in evidence their proteins and their genetic = constituents in=20 a direct way. True viruses are so stable that it's easy to = photograph=20 directly them as tridimensional particles by an electronic = scanning=20 microscope, without the need to first fix them chemically. On the=20 contrary, the microvescicles are so unstable that can be = photographed only=20 by a transmission electronic microscope which requires that they = be=20 previously fixed chemically in ultra-thin sections. What has been = shown to=20 the world as HIV micro graphs are ultra thin sections which = include=20 particles indistinguishable from the cellular ones (89) (pict=20 6-8). 
The=20 evidence of HIV (non-)existence A noteworthy confirmation to the = argued=20 critics of the researchers from Perth came in march 1997: two = groups, one=20 French/German (90) and one from the American NCI (91), have = published the=20 photographs of density gradients in which pure HIV virus had to be = present=20 (pict. 9). These data should have been available by the first = isolation of=20 the virus (1983-84), but nobody showed them before. The most = likely reason=20 is the lack of correspondence between what they found and what = they=20 expected. 
In the Franco/German study the  pictures are from = the 1.16=20 gm/ml band. It is impossible to tell from which density the = pictures in=20 the American study are taken but presumably it's the same 1.16 = density for=20 retroviral particles. Eleopulos and colleagues note that =93. The = authors of=20 both papers concede that the particles which are present in the=20 banded  material and which are said to be HIV represent only = a very=20 small fraction of the total  material. Gelderblom et al. = state that=20 the material contains =93an excess of [cellular] vesicles with a = size range=20 50-500nm, as opposed to a minor population of virus particles... = cellular=20 vesicles appear... to be a major contaminant of HIV preparations = enriched=20 by sucrose gradient centrifugation=94. =93For the small number of = particles=20 deemed to be =93HIV=94 no evidence is given that they are even a=20 retrovirus-like particle. Indeed, to the contrary: (a) the = particles do=20 not appear to have surface spikes (knobs)... In other papers = published by=20 many researchers including Gelderblom and his associates such = projections=20 are noted to be absent (93, 94); (b) the particles referred to as = =93HIV=94=20 are not spherical and have diameters exceeding 100-120 nM. In the = EM in=20 Gluschankof et al. (90) there are arrows pointing to five  = =93HIV=94=20 particles devoid of surface projections whose dimensions are 121 X = 145;=20 121 X 169; 121 X 145, 121 X 145 and 133 X 145 nM=20 respectively.  
In Bess et al. (91) there = are a total=20 of six =93HIV particles=94 also devoid of surface projections = whose dimensions=20 are 160 X 240; 200 X 240; 280 X 280; 208 X 250; 167 X 250 and 250 = X 292=20 and nM respectively. =93Actually, the authors maintain these are = HIV=20 particles, but without any proof. In the material of that density = band=20 there ought to be many viral particles, on the contrary they point = only=20 some, among many impurities.  
They bear only the vaguest = resemblance=20 to retroviral particles=94. =93The point is that any genuine = retroviral=20 particle contains a fixed amount of RNA and protein. No more and = no less.=20 This means that if we consider diameters of particles pointed in = the two=20 studies, they are  1.14 times to 1.96 times larger than the = estimated=20 maximum for a normal HIV particle. Translating this in volumes, = and=20 comparing them to a particle with a diameter of 120nM,  the=20 Franco/German particles have 50% more volume than a retroviral = particle=20 and the US particles have 750% more volume=94, that is to say = incompatible=20 with the same definition of a retrovirus! Even if the authors do = not admit=20 it, the data of the two cited papers contribute to show that where = should=20 be present pure HIV, of HIV there is no trace.  =

CONCLUSION =20

None of the tests = examined=20 seems to be sufficiently sure to detect an HIV infection. = Paradoxically=20 not even the =93direct virus detection=94 is able to do answer to = the same=20 question. As a matter of facts there is an incompatible = discordance among=20 the results obtained with different methods. These discordances = are by=20 themselves logical incompatibilities that invalidate them. = Besides, the=20 existence of an entity that one can reasonably name =93HIV = virus=94 is not=20 proved, as far as its proteins, its genome, its physical particle = are=20 concerned. The true meaning of the tests is better understood if = its=20 origin is examined. To show the existence of a new virus, both = Montagnier=20 group in 1983 and the group of Gallo in 1984 separated supernatant = of=20 cultures they considered infected in density gradients. They = erroneously=20 considered that the material that banded at 1,16 mg/ml was = constituted by=20 =93pure HIV particles=94. The proteins which were present in this = band=20 and  were reactive with AIDS patients sera (but not only with = their=20 sera) were considered HIV proteins. In the same manner, a = particular=20 fraction of RNA which they found in the same band was considered = the=20 genome of the new virus. By then, some of these proteins are used = to=20 get  the reagent for some kind of tests or as immunogens to = produce=20 in laboratory animals the antibodies used for other kind of tests = ( =20 similar procedure was followed to obtain RNA=20 segments).  
At this point the explanation of Gallo experiments in = 1984 appears=20 clearer: he used a mixture of proteins (believed to be viral but = in fact=20 cellular) to immunize the rabbits and obtain a serum which = obviously=20 reacted with the same proteins that were in the culture from which = the=20 initial mixture came (95).  
In other words Gallo = believed to show=20 an hypothesis (the existence of the virus) with other hypothesis = shown to=20 be false (that is to say that inverse transcription activity, the = presence=20 of virus-like particles, the antibodies obtained in the way just = mentioned=20 were all virus specific).  
In this way were born the = tests which=20 to-day are still used and perfectioned to put so engaging a = diagnosis. An=20 important prove of the correctness of this interpretation of the = facts=20 stays in the results that they obtained on mice and monkeys in = 1991=20 (96).  
In those studies, with the scope to evaluate a candidate = vaccine,=20 some laboratory animals were injected with cellular components = from=20 cultures which were =93not infected by HIV=94: the results were = similar to=20 those obtained with =93the real vaccine=94 (i.e. with virus from = =93infected=20 cultures=94).  
No significant difference was found, so that the comment = -=20 perfectly suited  and still up to date of John Maddox - was: = =93AIDS=20 research turned upside down=94.   =
That is to say that it = wasn't necessary=20 at all to hypothesize the presence of a virus to obtain those=20 phenomena. =20

Fabio Franchi   =
Specialista in=20 Igiene,  
Medicina=20 Preventiva  
e in = Malattie=20 Infettive 
Trieste
 
 
Fig.=20 1
 
Fig.=20 2
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig.=20 3
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig.=20 4
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig.=20 5
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