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COMPARISONS OF “HIV” VIRAL LOAD MEASUREMENTS WITH ACTUAL COUNTS OF VIRIONS: CASE REPORTS AND REVIEW OF THE LITERATURE
Andrew Maniotis, PhD., 1
1. Partnership For Cures (Cures Within Reach), Chicago, Ill.
Abstract
Viral load tests have been under increasing suspicion of detecting false-positive signals. The HIV-1 viral load measurement indicates the number of copies of “HIV-1” RNA per milliliter of plasma. Here we present a case in which we calibrated an “HIV-1” viral load measurement of 204, 000 / mL as determined by PCR tests, against the actual number of “HIV-1” viral-like particles in the peripheral blood of the same patient. No HIV-1, hepatitis C, or any other kind of viral-like particles, bacteria, fungi, Lyme or syphilis spirochetes or their round bodies, or mycoplasma were observed at any magnification in or around any red or white blood cells, or in plasma. Confirmatory ultrastructural testing following PCR analysis of blood from the same patient could help eliminate false-positive viral load testing. Changes in PCR diagnostic protocols for viral load measurements are warranted, especially in cases where disease syndromes are present that are known to confound various molecular tests. Electron microscope verification also may generate significant financial savings, and better patient care, by rapidly and inexpensively dispensing with false positive PCR readings in peripheral blood and in a variety of infectious processes.
Keywords: PCR, electron microscopy, viral load, “HIV/AIDS.
This project was in part supported by a gift from The Office of Medical and Scientific Justice. No conflicts of interests of the author is reported.
Introduction
In only the past several years, “HIV/AIDS” diagnoses have encountered a series of increasing challenges regarding the specificity of any of the protein or nucleic acid tests. To address these challenges, we compared viral load readings to actual particle counts of virus-like particles of the same blood draw's sera. The first patient, whose case history is dominantly featured here, had battled “HIV” diagnoses that ruined his professional boxing career for 23 years, and he never consumed ARV's until recently. In January of 2012 he became paralyzed and unable to speak following a tetanus vaccine, and was diagnosed by neuologists as being a victim of traumatic brain injury and Guillain-Barre Syndrome. By July, 2012, he had his blood analyzed with a direct Coombs test, and he tested positive for severe anemia by various criteria. This diagnosis suggested that his red blood cells were being destroyed by self-directed antibodies. Hypergammaglobulinemia also was noted, and, could, in principle, confound protein and nucleic acid tests that measure antibodies or gene fragments as evidence of infection with an exogenous pathogen. Direct electron microscopic observation of the anemic blood failed to detect one (1) “HIV-virus-like particle” in the same peripheral blood samples in which PCR testing detected a rising and then falling count between 252,000--9,090,000 in the absense of ARV's, and a VL reading of 14,000 “HIV-1” RNA copies/ mL, following a short-course of ARV administration given by accident in an ICU. During the 15 months of this patient's profound illness, these fluctuations and these divergent results of different tests increased and decreased, while all his CBC counts improved, and his lymphocytes and RBC's steadily increased, in the absense of ARV's. The upward limit of Quest and LabCorp HIV tests using PCR is 10,000,000 copies HIV mRNA/mL, and the lowest limit of “viral-like particle detection for HIV is now reported using fluoresnt techniques and EM is < 5 particles/mL.
In this study we then wanted to analyze a number of individuals' ”HIV-positive” plasma samples (and in some cases, lymphocytic cells), by employing electron microscopy of fresh and carefully fixed serum (plasma), while simultaneously testing that same blood draw with PCR (using standard reference lab quantitative PCR VL testing), to determine the “viral load” of that sample in the absence of ARV's. As absence of evidence isn't evidence of absence, and despite our repeatedly negative findings using EM even after using different fixation protocols on a series of different patient plasma samples, we reasoned that it should be relatively straightforward to demonstrate adequate size and titration controls for any putative virus presence using nano particles, and/ or, develop a methodology for similarly equiped and experienced “HIV” diagnostic testing labs to repeat the protocol, and then provide us with a positive EM example showing the presence of at least 1-3 “HIV” viral-like particles. Alternatively, we considered that “HIV” PCR and protein readings for years have likely represented extreme oxidation states in ill persons, hypergammaglobulinemia, or more than 100 other known reasons to register positive on either nucleic acid or protein-based “HIV” tests [1-11, 13-59, 61-120]. Finally, these electron microscopy results suggest that even extremely high PCR viral load measurements do not indicate the presence of any exogenous “HIV-viral-like” particles, or viremia in vivo [69-71].
Results: Ultrastructural analysis (electron microscopy-EM) for various pathogens in the peripheral blood including “HIV”
The first patient's PCR viral load tested plasma indicated 204,000 “HIV”VL/mL---approximately one twentieth of what had been obtained in December, 2011, 6 months before, which was 4,100,000 copies/mL. In fact, this patient's viral load was nearly the same as was measured in the summer of 2010, which was 252,000 copies/mL in the absence of any ARV administrations. The comparison and EM survey of this blood draw is shown in Figures 1,2. No viruses of any kind, no curvularia or yeast or fungi, no bacteria, no mycoplasma, no Lyme disease spirochetes or round-bodies, no syphilis spirochetes or round bodies, no diphtheria or diphtheroids, and no pseudomonas were identified. The only pathology shown in these high resolution micrographs of whole blood was consistent with anemia, as evidenced by the retention of organelles in immature red blood cells known as reticulocytes, and, the patient's plasma was negative for any virus. Nine other patients' VL readings/EM particle counts are presented in TABLE 1.
Figure 1. Representative samples of 60 randomly chosen EM grid fields of a hemolytic anemia patient's red blood
cells (A,B,C) harboring an “HIV-1” viral load of 204,000/microliter. Note that organelles are observed freqently in the red blood cells (A) because the red blood cells are immature, and/or exhibit drug injury. No “HIV” viral particles were observed in any of 60 EM fields that were analyed, or within any intercellular spaces (A-C).
Figure 2. TEM's of various white blood cells from the 204,000 PCR-measured viral load containing sample obtained from the peripheral blood of this hemolytic anemia/ Guillain-Barre patient (A, B, C, D, E) . No viral-like particles of any kind can be seen, nor can any obvious abnormalities in any of the white blood cells. Also note there are no viral-like particles in the intercellular regions between the cells shown in A-E. TEM micrograph of virus-like particles pubished in Nature Protocols, 2007 claimed to be “HIV-1” grown in Petri dishes (not from peripheral blood or any in vivo context) (F). In this micrograph from a Nature Protocols published study, cultured peripheral blood lymphocytes were said to be “productively infected” with “HIV, “ and then processed with agar and en-bloc stained with uranyl acetate and photographed. Budding and mature virus like particles are clearly visualized between the two cells. According to the paper's authors, the magnification = 7,800 X*. (From Nature Protocols 2, 2439 -2450, 2007, from Figure 12: “Lymphocytes replicating HIV. Processing tissue and cells for transmission electron microscopy in diagnostic pathology and research.” Lesley Graham & Jan Marc Orenstein. Published online: 4 October 2007 doi:10.1038/nprot.2007.304. The official report provided by by the Electron Microscopy Diagnostic Laboratories at Massachusetts General Hospital described their findings following the surveys represented of 60 EM grids in Figures 1 and 2 in the following way: “No viral inclusions, fungi, or bacterial forms are identified in the sample.” An independent hematologist also was consulted who determined that the immature red blood cells and non-excluded organelles were not necessarily characteristic of immature red blood cells, but also the possibility of drug or tetanus vaccine injury (or Job's disease) was suggested..
Demonstration that In Vitro made “HIV” can be spiked into human plasma and easily photographed using an electron microscope, and that precise bead titration can indicate how many particles should be visualized/EM field, given an initial “known” concentration (VL reading)
We reasoned that it might be instructive to demontrate that we could in fact find and photograph what “experts” condider “real” “HIV” of the type most identified in America. The following “HIV-1” clone pNL4-3 is the first so-called “non-Gallo” clone from 1984, and its spiked concentration is supposedly 500,000,000 virions/mL in the photographs shown below (Figure 3). However, according to our calculations, there should be at least 300-3,000 virions/field at this “neat” concentration. Moreover, the particles do not resemble “HIV” in these negatively stained preparations, as they are said to appear in TEM electron micrographs of tissue culture-grown “HIV.”
Figure 3. The electron microscope images show the images of “neat” HIV-1 in ammonium acetate buffer and negatively stained for electron microscope analysis for particle detection. Notice the empty particles (blue arrows) outnumber the filled particles (red arrows). These“HIV-1” particles had been previously fixed with glutaraldehyde (zoom in on the arrow-designated particles...these are high resolution photos). Photo provided by Gregory Hendricks, UMass Medical School Core Electron Microscope facility, University of Massachusetts, Worcester, September, 2012. The “HIV-1” clone pNL4-3 sample of 500,000,000 “virons” of “neat” “HIV-1”/mL was kindly provided by Dr. Heinrich Gottlinger, University of Massachusetts Medical School, Worcester, Massachusetts.
Demonstration as to how known concentrations of 10 nM gold beads were titrated according to viral load-determined “virus” concentrations
For 8 samples, we precisely titrated 10 nM gold beads into human plasma, and fixed them identically to 10 mL plasma samples that were centrifuged to concentrate putative viral-like particles according to the centrifugation speeds used in“HIV” genotyping protocols, that concentrate virus when initial viral load readings are 1000/mL or less (Figure 4). This titration was used as a measure of determining how many viral-like particles should be visualized per EM grid, given the pre-determined viral load reading as determined by PCR. The following sample belonged to a patient harboring a VL reading of 4,000,000 mRNA/mL.
Figure 4. Demontration of size and number dilutions/titrations using 10 nM gold nanoparticles obtained from . Although these nano-gold particles are 10 times smaller than "HIV," their number and size can be easily documented when spiked into human plasma, even at concentrations beginning at 1000 particles/mL. Calculations suggest that the starting concentration of these gold nanoparticles was approximately 10,000/mL, a value 100,000 times less than the supposed VL of the patient's plasma. Table 1.
Viral load numbers compared to particle counts in the sera of 10 individuals: Patient identifiers have been removed and replaced with viral load readings rather than names.
VIRAL LOAD NUMBER OF VIRAL-LIKE PARTICLES OTHER PATIENT INFO
|252, 000, 4,100,906, 204,000,1,930,000, |0 |Professional athlete, Guillain-Barre Syndrome |
|9,090,000, 14,000* | |following tetanuus vaccination |
|49, 000 |0 |Bell's Palsy, “HIV+ Scotland |
|2,000, 000 |0 |Life threatening C. difficille infection, |
| | |“HIV+woman” |
|4, 000, 500 |0 |Hemophilia |
|5, 700, 000 |0 |African Immigrant, “HIV+” |
|700, 500 |0 |“HIV-positive” woman |
|3, 090, 500 |0 |“HIV+ “Gold-coast” African |
|2, 120, 000 |0 |“HIV+” African American female |
|3, 000, 200 |0 |“HIV+ American male |
|7, 050 000 |0 |Young mother naive to ARV's |
Discussion and review of the literature
This is the first time that a direct comparison of “”HIV-positive” blood has been specifically studied using electron microscopy of the same seraum patient whose viral load was measured in June, 2010 to be 252,000 copies/mL on a LabCorp “HIV-1 mRNA/mL” test, 4,100,906 + “HIV” mRNA/mL on a Cobra Ampliprep Roche PCR test in December 2011, 204,000 copies/mL 6 months later on aLabCorp PCR “HIV” viral load test (Figures 1,2 ), 1,930,000 on another Cobra Roche PCR viral load test in August 10, 2012, on August 28, 2012, 9,090,000 copies/mL, and December 2012 14,000 copies/mL. These fluctuations and differing results increased and decreased, while his CBC counts steadily improved, and his lymphocytes steadily increased, in the absense of ARV's. The electron microscopy results, as well as the ANA (anti-nuclear antigen) negative results of this patient suggest that even extremely high PCR viral load measurements do not indicate the presence of any “HIV-viral-like” particles or viremia in vivo. The confirmation of these results have important implications. To date, “viral-like particles” only have been documented by AIDS researchers to occur in Petri dishes or in test tubes after certain oxidizing chemicals are added such as PHA, IL2, interferon antibodies, and the like. And these in vitro studies demonstrate, rather than infectious or pathogenic “HIV” harboring surface projections or any other pathogenic viral parts, that most, if not all of these viral load readings are spurious chemical reactions that probably represent endogenous “junk” nucleic acid sequences or HERVs that are synthesized by oxidized cells or caused by various local or systemic pathologies. Yet many front line AIDS clinicians and virologists also believe these ill-defined nucleic acid sequences correlate with pathology and demise of their patients, despite the fact that other leading clinicians and AIDS researchers have published how viral load or viremia measured by PCR has nothing to do with declining T-cell numbers, or patient morbidity or wellness [82,120,121,122]. It is known that the freezing of biological material can create ice-crystals that could in principle disrupt “HIV” structural and infectious integrity, as the CDC, blood banks, Factor XIII and IX preparative industries first discovered in their quest to to protect the blood supply, and to establish the pathogenic requirements and/or disruptive conditions that would destroy the virulence of various blood borne pathogens including “HIV.” Therefore, ten blood samples processed for EM in this study were not dry-ice frozen and thawed, in order to avoid disruption of “HIV,” avoiding this potential pitfall regarding how these physical changes were thought to destroy structure and pathogenicity of “HIV” early in the AIDS era. The samples were fixed in gluteraldehye shortly after the blood draw, and thus were shipped to diagnostic EM labs in a protein cross-linked state, that is impervious to degradation. Therefore, using the precautionary principle to best capture at least one non-damaged HIV particle in samples containing between 102,000 putative “HIV” particles in the first sample (Roche), or what LabCorp PCR suggested were first approximately 800,000 viral particles in 2010, 102,000 particles or 4.5 million particles in 2012, depending if you believe LabCorp or Roche's viral load tests, we reasoned that one would not want to freeze and thaw a sample, to insure that the postulated gp120/160 projections would not be missing or destroyed after freezing and thawing. However, the EM data in this study, strongly reinforced by the patient's medical history, suggest that PCR “HIV” viral load measurements in vivo may be detecting something other than specific viral nucleic acid sequences of any virus, or any viremia at all. Let us first review possible caveats to this simple PCR-EM test for “HIV” from the same peripheral blood of this patient, who was said to harbor extremely high viral loads over many years in the complete absence of ARV's. First, let us assume that the “HIV” virus in the first patient is hiding in the lymph nodes or brain and is not found in peripheral blood. Then, what do LabCorp or Roche's peripheral blood viral load measurements mean? In this patient, they supposedly detected 252,000 (2010), 4,100,000 (2011), 204,000 (June 2012), 1,900,000 (early August) 2012, and then 9,090,000 “HIV-1” mRNA molecules on August 28, 2012, suggesting that in any of these samples, one should see 1/2 as many “HIV” virions as indicated by the viral load measurement, since two genomes are said to be present in every “HIV” virus particle. The blood was drawn from the patient's arm, and not from a lymph node, even close to a lymph node, or from a brain biopsy. Regarding the sample we first analyzed, the PCR viral load test indicated that there are 204,000 mRNA copies of the “HIV” genome in that patient's peripheral blood (sent in a test tube tested for viral load). Electron microscopic analysis couldn't detect even 1 of the anticipated 102,000 putative virions in that same blood (2 copies/204,000 mRNA PCR-detected copies). Therefore, the argument that “HIV” is hiding in the lymph nodes or brain does not address this discrepency, and is not logical. It is more likely that Roche's test and LabCorps test detect something very different than viral nucleic acid fragments or entities. Second, let us assume that the samples are not fixed well enough to preserve the anticipated 102,000 -4.5 million HIV virions at the ultrastructural level (using EM), but the PCR accurately detects the the real number of genomic molecules of “HIV.” This assumption does not apply to most if not any of the samples, as they were fixed in gluteraldehyde minutes after the blood draw. Another way of stating this is that, in the first sample, somehow PCR is capable of seeing 204,000 genomes representing 102,000 virions/mL, 9,090,000 genomes representing 4,500,000 virions.mL, 1,900,000 genomes representing approximately 800,000 virions/mL, but EM, that could visualize ribosomes and mitochondrial cristae in photographs of cells presented in the photographs of this anemia patient, did not detect a single “HIV” virus particle either among his cells, or in his plasma. This caveat would suggest that at least part of the the 204,000 mRNA genomes of the lowest PCR measurement taken within a month of EM testing and which theoretically exist as 2 genomes/virion, can survive intact, while every component that surrounds the two supposed delicate RNA “HIV” genomic threads, including the proteins of the rigid supportive viral matrices, capsids, and the rest of the virus, are degraded, leaving nothing for the electron microscopist to detect. To be clear on this point, in effect, the result would have to mean that LabCorp and Roche PCR technology can detect “HIV” genomic fragments after freezing and thawing them, and can detect the two delicate mRNA genomes/virion that the PCR amplifies (which wouldn't be necessary to amplify in the first place with a viral load of 204,000, 252,000, 2,900,000, or 4,100,000, or 9,090,000 mRNA copies/mL). Yet, as shown in Figures 1, and 2 especially in the higher magnification electron micrographs, we see preservation of delicate cellular structures such as bi-membrane structures, ribosomes, mitochondrial christae, and the inner and outer mitochondrial membranes, and thus this interpretation is unlikeley. These criticisms are also applicable to the samples fixed shortly after blood draw, and then concentrated in a centrifuge with gluteraldehyde.
The single digit nanometer-sized organelle features (ribosomes, mitochondrial double membranes measuring 7 nM) shown in the sample of patient 1, can also serve as an internal control for magnification(s) of each micrograph if and when cells are present, as the inner and outer mitochondrial membranes are known to each be approximately 7nM in diameter, whereas Petri dish generated “HIV” viral-like particles are assumed to be approximately 120 nM. This means the data show that we were able to detect single-digit nanometer structures of the blood cells that are perfectly preserved and that are 17 times smaller than “HIV,” or gold nano beads measuring 10 nM (1/10th the size of “HIV”) but not “HIV” virus particles in any peripheral plasma specimen. We are at a loss, therefore, to explain how at far lower titres than 204,000 mRNA, one can find “HIV” viral-like particles easily in vitro, as many groups have reported previously since the beginning of the AIDS era, and as is shown in Figure 2F from Nature Protocols. This paradox invites a larger number of electron microscope analyses of human peripheral blood samples, each of which accompanied by a high PCR viral load reading, to determine in a small population of tested individuals, if any virus-like particles ever can be detected. A third caveat is that the preparation protocols demanded by Quest Diagnostics, LabCorp, and Roche that were rigidly followed in the extraction, sample preparation, and handling steps of this patient's blood are not standardized, and the results of these tests are contradictory and differ by hundreds of thousands and even millions copies/mL, but B-DNA and RT PCR-obtained readings are known to vary by factors of 10 to 100 fold differences in VL reading, which is why SOP's suggest to begin and continue with the same kind of VL testing in patients first diagnosed and who subsequently begin ARV therapy. The various VL protocols also do not hold sample times constant, as room temperature exposure times are different, plasma versus cells plus plasma are transported in some cases, and differential exposure to oxygen occurs, because of the necessity of transferring plasma to screw cap tubes for transport when on dry ice, versus 0 degrees centrigrade transport. EM fixation can avoid all of these variables by maintaining whole blood or plasma at room temperature, or at 0 degrees C, avoiding ice-crystal formation, and with fixation with gluteraldyhyde, immediately upon blood draw or arrival at the EM laboratory, crosslinking all the proteins, cells, or viruses, as was done with these patient samples. Historically, there have been other kinds of AIDS described: ideopathic CD4 cell lymphocytopenia, for instance, where the patient shows T-cell deficits yet no “HIV” antibodies can be discovered. This is not the case in this patient, although there are some 4,000 or more ideopathic CD4 cell lymphocytopenia cases described in the literature. These syndromes may owe their etiology instead in many cases, to complex autoimmune dysfunction, which are syndromes in many cases known to produce “HIV-1-specific” antibodies, such as lupus, or as in the state of multiparous pregnancy (which is not a medical disorder), severe atrophy of the thymus in children, flu vaccination, hepatitis B vaccination, tetanus vaccination, rheumatoid arthritis, malaria, 50% of dog sera test “HIV-1” positive, and approximately 80 other syndromes/situations that have been published in the peer-reviewed literature. In this case, we seem to have repeated progression to an AIDS-like syndrome and recovery from it, without any evidence of “HIV-viral like particles” being present. None of the patient's intimate contacts or children have tested “HIV” positive for 26 years.
In 2004, the Red Cross reported in The New England Journal of Medicine that even after repeated testing using different test kits, low-risk populations, such as blood donors (or military recruits) will typically yield 12 (PCR-positive) or 2 (ELISA positive) out of 37,000,000 samples, leaving potentially 10 out of 12 false positives, depending on which test kit you believe accurately detects "HIV's" molecular signatures [101]. While it has been pointed out that of the 2 of the 12 in this study who initially tested positive on ELISA seroconverted in subsequent months to the molecular signature of "HIV" detected on PCR, and thus may warrant an investigation as to what the meaning of these molecular markers represent among persons who exhibit clinical symptoms. And it still is apparent to date that none of these tests have been validated against the isolation of pure "HIV" itself in vivo. They have been validated instead against other test kits that were assumed to detect "HIV." Acording to Mark Pandori, PhD. Chief microbiologist at San Francisco Department of Public Health Laboratory:
“Many samples on WESTERN blots have spurious bands that are difficult to interpret. Or because WB's are complex to administer and may react as positive if a person is infected with another virus (http/testing.htm). Therefore, many reference labs and physicians are questioning the WB as a gold standard. The latest APHL/CDC (an “HIV” testing regulatory body of the government) proposed algorithm omits WB's altogether.”
Moreover, it has been recently proposed that viral load does not correlate with T-cell numbers, and the rate of progression (when an individual will exhibit symptoms of AIDS) can only be predicted in 4%-6% of HIV-positives studied (out of 2,800)[82]. In fact, as early as 1995, the Nobel Laureate and developer of The Polymerase Chain Reaction used to detect viral load against his warnings that PCR cannot detect viral load, Dr. Kary Mullis, published a new hypothesis attempting to explain how immune collapse need not be due to any particular virus, but by what he imagined as an immunological chain reaction:
“If previously latent virus with a distinct epitope would provoke a new immune response, every immune response would be perpetually generating new immunogens. The immune system so infected would be perpetually generating new immunogens. As the frequency of infection increased such an immune chain reaction would be progressively more debilitating for the stability and effectiveness of immune function” [60].
This hypothesis was important, because it predicted that a vaccine against any specific virus would be ineffective against AIDS, and to date there have been more than 100 failed and abuptly halted “HIV” vaccine trials. Currently there are not less than 224 “HIV” vaccines that have failed or are failing in the “HIV” vaccine pipeline, indicating perhaps, that the true antigens associated with Acquired Immune Deficiency Syndrome still have not been isolated (please see Kary Mullis Notes following the Reference List).
Finally, in a recent issue of AIDS Clinical Care, front-line AIDS clinicians published statements that should concern clinicians and scientists studying “HIV.” “AIDS” is unexpectedly progressing despite treatments with “life saving anti-retrovirals” and in many cases without “HIV” being detected [121, 122]. In other words, these patients were taking a double or triple AIDS-drug cocktail regimen, no “viral load” could be detected, yet their T-cells were plunging from relatively normal levels to the worrisome and CDC-defined level reached when doctors suggest that drug therapy should begin, at around 200-300 CD4+ cells/mm. Drug “resistance” was “checked” and found not to be an issue.
With the recruitment of a few of the many electron microscope diagnostic labs situated in many of the outstanding medical institutions in this country and throughout the world to employ this calibration method described here, together with the cooperation of physicians and PCR viral load testing reference labs, this PCR/EM comparison makes it now within our means to insure that we can now avoid the discrimination inherent in false positive molecular nucleic acid or protein tests that have not been validated in vivo at the ultrastructural level, to detect or eliminate the presence of the very microbial culprits said to cause a disease. The use of spiked tissue culture dish-generated “HIV” viral-like particles and serial dilution of gold nanoparticles together can both insure that enough target virus should be detectable in a given VL reading, (especially when a low to moderate pre-centrifugation step is used to concentrate any potential virus-like particles before PCR is attempted, which is consistent with current state-of-the-art genotyping protocols of NUGENE used to concentrate “virus,” and now many other ones).
As shown in TABLE 1, other patients in recent months also have been subjected to this analysis, and we have found that even with high viral loads, they too, like the first patient discussed here, have no “HIV” virual-like particles in their peripheral blood.
In conclusion, “HIV” is most likely due to a Petri dish artifact created by Luc Montagnier's group and then Robert Gallo's group because of PHA and IL2 oxydation of both normal cord lymphocyte cells or cancer cells respectively in vitro [69-71], and thus, “HIV” virus particles have never been or will never be seen in the blood or other tissues of a human being. As of this writing, 3 other “high viral load”-bearing ARV-negative individuals have been similarly tested, and no virions have been detected in their blood either. We are currently collecting a group of 30 similar individuals whose VL readings are in the hundreds of thousands or millions, and whose same blood draw will be examined using the EM methodology described here, or others perhaps, in addition to a new optical method we have devised. Ongoing repetition of this type of comparison is warrented until one “HIV” virus particle is discovered in any human peripheral blood sample.
Materials and Methods: Preparation of Buffy Coat for Transmission Electron Microscopy (TEM).Two samples of approximately 7.0 ml of peripheral blood in purple top (EDTA) tubes were received on ice via over night carrier. The tubes were set aside to settle for one hour at room temperature in a vertical position. The tubes were then centrifuged at 1000 rpm for 10 minutes without using the centrifuge brake option. The tubes were then carefully removed and placed into a sturdy test tube rack. The result is a top layer of serum proteins over a platelet layer, a thin white cell layer and finally a red blood cell layer at the bottom. The serum was carefully removed with a plastic pipet and very carefully replaced with a modified Karnovsky’s KII fixative (2.5% glutaraldehyde, 2.0%parafomaldyhyde, 0.025% calcium chloride in a 0.1M sodium cacodylate buffer pH7.4). The fixative was allowed to rest over the platelets and white cell layer for 1.5 hours after which a solid disk of platelets and white cells with a thin layer of red blood cells forms. The disk was loosened from the test tube walls with a sharpened wooden dowel, and fixed for another hour in fresh KII fixative. The disk was then cut into eighths (pie fashion) and routinely processed for TEM in a Leica Lynx™ automatic tissue processor. Briefly, they were post-fixed in osmium tetroxide, stained En Bloc with uranyl acetate, dehydrated in graded ethanol solutions, infiltrated with propylene oxide/Epon mixtures, flat embedded (full thickness) in pure Epoxy resin, and polymerized over night at 60°C. One micron sections were cut, stained with toluidine blue, and examined by light microscopy. Representative areas were chosen for electron microscopic study and the Epoxy blocks were trimmed accordingly. Thin sections were cut with an LKB 8801 ultramicrotome and diamond knife, stained with Sato’s lead, and examined in a FEI Morgagni transmission electron microscope. Images were captured with an AMT (Advanced Microscopy Techniques, Danvers, MA) 2K, digital CCD camera.
Gluteraldehyde fixation:
Twenty-two mL of peripheral blood is drawn from an arm vein. 2 mL of thiws plasma.Two mLs of plasma is then sent to Roche. Roche's viral load tests typically test 3-10 times higher than LabCorp's tests or others. We tried to obtain the highest viral loads available from people naive of ARV's to increase the probablitity of finding one virion in 10 people's plasma samples. A low speed centrifugation (1000 RPM/G) step is performed to separate cells from the remaining 20 mLs of plasma before packaging for VL testing using this algorithm: First, the cells are removed using a low centrifugation spin (1000 G), then a centrifugation step is performed at 28,000 RPM/G for 2 hours to concentrate potential "HIV" in that plasma. This speed and time is warranted because when they do genotyping of "HIV" of a sample that contains less than 1000 VL, in order to "boost" the "virion particles" in that sample of peripheral blood to amplify it to more than 1000 VL/mL, which is the limit needed for genotyping. 18 mL of supernatant is then discarded. 1mL of concentrated plasma is then placed into two plastic tubes, and 120 nM beads is placed in one of them as a size standard, but not the other tube containing plasma only. Both 1mL tubes of concentrated "VL" plasma and concentrated VL and 120 nM beads are then fixed in gluteraldehyde (2%), then ship them to EM diagnostic labs. Most diagnostic EM labs will comply with this approach, because they only take glut-fixed blood samples from patients. Diagnostic labs then are sent the fixed 1mL, they deposit it in methacrylate or a comparable embedding plastic medium, and then thin sectioning of the plastic embedding medium is performed, and the silver sections are placed on 5 EM grids. A survey of 100 different EM grid fields is then performed and documented.
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Notes From Kary Mullis, Inventor of The Polymerase Chain Reaction and Nobel Recipient:The answer lies in THE SEQUENCE? Re: PCR question. “HIV” DNA/RNA sequences in your BLOOD is sufficient cause for PHYSICIANS to pronounce AIDS.
Dear Kary~as you mentioned~there is another independent measure not related to DNA~but its still BLOOD related- using an EM to photograph the blood to detect the virus named “HI.”
How come I can have a viral load of 9 million copies/mL using your PCR method, and not one virus show in the blood?
What sequence are they using? And how can YOU prove this?
EM is the ultimate method ~ but we need to disprove the sequence of “HIV.”
Trisha Harding Morrison, Date: Sat, May 11, 2013 5:55 pm
Dear Tommy (and Trisha),
A number of diseases are detected by the fact that using two sequences known to be contained in the DNA/RNA of some organism and running a PCR reaction, containing that DNA, a fragment of a known size predictable from the sequence of that organism will be produced. Further evidence (and much more credible) for the presence of that particular organism can be obtained by sequencing the fragment so obtained. An intermediate level of confidence can be obtained by so-called hybridization of the obtained fragment with a known sequence standard.
Most diseases can be diagnosed with some independent measure not related to DNA, but over the years DNA evidence has become a well-trusted method, and the mere presence of HIV DNA sequences in your blood is sufficient cause for most physicians to pronounce that you have AIDS and treat you for it. (You can have your prostate gland surgically removed based on higher than expected levels of prostate hormone, but I wouldn't recommend it without collaborative evidence of rampant prostate cancer.)
The issue of assigning meaning to the evidence of HIV sequences in people is in my opinion the real outstanding issue, and my opinion is that there is insufficient evidence to conclude that such sequences are dangerous enough to the person (if at all) to justify a treatment that may very well be.
I have made myself clear on this issue for years. If I were to discover something which would make me think otherwise, I would feel honor-bound to make that very public.
Sadly anybody who can read and really wants to know about the issue of AIDS can read about it forever and still not know much about it as it is a highly contested issue and the financial consequences are immense. There are a large number of such issues in the world. Welcome to Earth.
PCR detects a very small segment of the nucleic acid which is part of the virus itself. The specific fragment detected is determined by the somewhat arbitrary choice of DNA primers used which become the ends of the amplified fragment (not virus isolation). They have to be in the sequence for it to be amplified in the first place, but they can be rather a small part of the total sequence. (Two to three hundred nucleotides is usually chosen out of several thousand in the total retrovirus). When incorporated in a cell the virus exists as DNA, when it is released from the cell in its infectious form it is RNA; which can be converted easily into DNA in the cell or in vitro for purposes of amplification. There are many sequence variations among the sequences called HIV. Any one of them can get you classed as what they consider HIV positive. And due to the tiny amounts of nucleic acid detectable after many cycles of PCR amplification (after 30 cycles one copy will get you about a billion copies) the test is super sensitive. Antibodies are detected by a non-PCR test called an EIisa. Elisa testing is not so specific as PCR as there are many antibodies not specific to HIV which may cross-react in this test. If a sequence amplifies with primers designed to HIV, it is HIV by definition. In order to amplify and produce a fragment of approximately the correct size the original sequence must hybridize with the primers, and therefore it must be at least a very close match (at least at the primer sites) to what has been defined as HIV. (now called HIV-AIDS in case there are any skeptics).
Kary Mullis, Date: Tue, May 7, 2013 4:44 pm
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