Analysis of Flegg's references 2-4 23 April 2003
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David Rasnick,
Chief Science Officer, Boveran, inc.
San Ramon, CA 94583

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Re: Analysis of Flegg's references 2-4

Dear Editor,

In keeping with all references posted so far, none of Peter Flegg's references was designed to prove the sexual transmission of HIV. All authors have assumed from the start that HIV is sexually transmitted. To get around the awkward problem of having to say just when, where and by whom it was shown that HIV and AIDS were sexually transmitted, Flegg says that, "More direct evidence accrued for [the contribution of early sexual transmission] with the finding within the Swiss HIV cohort Study, which included 191 patients with PHI [primary HIV infection]. Through case contact tracing and nucleic acid sequence analysis it was demonstrated that onward transmission to another partner had occurred in 17 of these cases during the time of PHI [Flegg's reference (2)]" His reference 2 is Yerly, S. et al. HIV drug resistance and molecular epidemiology in patients with primary HIV infection. Abstract 754, 8th Conference on Retroviruses and Opportunistic Infections. Chicago 2001.

Since this is only a citation to a conference abstract I will not discuss it because there are no data or details to analyze.

Flegg went on to say that, "A detailed analysis of 5 heterosexual couples where transmission occurred clearly demonstrated the dynamics of early HIV transmission [Flegg's ref (3), reference (1) below]". However, Pilcher et al.'s "detailed analysis" amounts to a short letter to the editor of only five paragraphs, which was published in 2001. Their letter does not claim to offer proof that HIV is sexually transmitted because the authors are compelled to assume that from the start since it had been declared to be true back in the 1980s. They report on only "5 cases drawn from 4 university hospital clinics, in whom sexual transmission was suspected to have occurred between an individual with documented PHI [primary HIV infection]. We defined PHI as p24 antigen positivity, RNA and/or DNA positivity, enzyme-linked immunosorbent assay negativity, or 2 or fewer bands on Western blot within 30 days." In short, these 5 HIV-negative cases (as the authors' criteria specify) were considered to be infected with HIV not on the basis of being antibody positive to HIV but to being positive for p24 antigen and by the so-called viral load test. Furthermore, these 5 couples were assumed to have gotten infected with HIV through sex. Before considering p24, let's first examine the PCR viral load's ability to detect infectious HIV since, after all, it is only infectious virus that can be transmitted.

Pilcher et al. do not provide experimental details in their short letter. Therefore, we do not know how they measured HIV RNA or DNA. Since (to the best of my knowledge) the Roche viral load test is the only one approved by the FDA, I will assume that they used the Roche test. Recall that on March 7, 2003, I posted a letter titled "An Abuse of surrogate markers for AIDS". I quoted from the third paragraph of the insert that comes with Roche's viral load test, which says: "AMPLICOR HIV-1 MONITOR Test is not intended to be used as a screening test for HIV or as a diagnostic test to confirm the presence of HIV infection" (Roche Diagnostic Systems AMPLICOR HIV-1 MONITOR Test package insert, PMA No. BP950005/4). Yet, Pilcher et al. offered not one word of justification for using RNA and DNA measurements to label someone as being infected with HIV.

Also in the March 7, 2003, letter I listed numerous references to false positives resulting from the viral load test. I will restate only one of the quotes here: "The availability of sensitive assays for plasma HIV viral load and the trend toward earlier and more aggressive treatment of HIV infection has led to the inappropriate use of these assays as primary tools for the diagnosis of acute HIV infection. ...Physicians should exercise caution when using the plasma viral load assays to detect primary HIV infection... Plasma viral load tests for HIV-1 were neither developed nor evaluated for the diagnosis of HIV infection..." (2).

Flegg can read the other quotes from my March 7 letter that throw into serious doubt the use of PCR viral load as a measure of infectious HIV or anything else for that matter.

Since the viral load test is not a reliable measure of infectious HIV, the only thing left to Pilcher et al. as a measure of infectious HIV was the presence of p24 antigen in the blood of their five cases. It is surprising that Pilcher et al. even tried to use p24 antigen to indicate the presence of infectious HIV since it was discredited for that purpose a decade ago (3). Kaplan et al. (4) and Kageyama et al. (5) have demonstrated that simply measuring the amount of p24 antigen present does not necessarily indicate the level of infectious HIV even in cell culture, where HIV does not have to contend with the immune system.

Flegg says that the biological evidence linking higher rates of sexual transmission of HIV with high levels of HIV can be found in Rakai, Uganda, as reported by Quinn et al. (6). As is typical of much of AIDS research, Quinn et al. piggybacked their study onto one that was designed to address a different question from the one they were interested in. Quinn et al. retrospectively selected 415 discordant HIV-seropositive couples from a 1994-1998 Wawer et al. study of 15,127 individuals from Rakai, Uganda (7). The intention of the Wawer et al. study had been to reduce HIV incidence by mass treatment of STDs with conventional antibiotics. More about this study below.

As Quinn et al. acknowledged, their respective "study differs from other investigations that selectively identified and followed HIV-1 discordant couples." The authors are probably referring to Padian et al. (8) and similar studies but they don't tell us. As is customary in AIDS research, the presence of HIV infection was presumptively determined by testing for antibodies to HIV and or using the Roche Amplicor viral load test. Again, as is typical of AIDS publications, there was no discussion of the appropriateness of using these tests to diagnose HIV infection (both of which are specifically not approved for detecting HIV infection) or even of the limitations of their accuracy under the specific conditions prevailing in sub- Saharan Africa. Because Quinn et al. were looking only for and thinking only of sexual transmission of HIV, they automatically assumed that the appearance of antibodies to HIV in their discordant couples (after retrospective analysis) had to be from sexual transmission.

The lack of consideration of the appropriateness and accuracy of the HIV antibody and viral load testing may explain the otherwise inexplicable results reported by Quinn et al. The authors reported that "22 percent of the HIV-1-negative partners seroconverted during the course of the study, for an overall incidence of 11.8 per 100 person-years."

But surprisingly, "There were no significant differences in the rate of acquisition of HIV-1 infection according to either the presence or absence of symptoms of sexually transmitted diseases or the presence or absence of syphilis, gonorrhea, Chlamydia, trichomonas, and bacterial vaginosis." There was also "no significant difference in risk of infection among HIV-1 negative partners according to the level of formal education, history of travel outside the district within the previous year, the number of sexual partners within the past year (one vs. two or more), or condom use or nonuse."

These results were completely contrary to the expectations outlined in the first paragraph of the introduction: "A wide variety of behavioral and biologic risk factors are associated with the risk of transmission, including the frequency and types of sexual contact, the use or nonuse of condoms, immunological status,... and sexually transmitted diseases."

The discordance between results and expectations only gets worse when the entire Wawer et al. study population is taken into consideration. The rationale behind the Wawer et al. study (7) was that reducing STDs (which was assumed to be a co-factor in the transmission of HIV) should reduce the transmission of HIV. However, the result of the study was paradoxical. While the investigators were very successful in significantly reducing STDs, their intervention had "no [effect] on incidence of HIV-1 infection..." (7).

The results of Wawer et al. were no fluke. An even more recent study by Kamali et al. "showed that in a "community-randomised trial in rural southwestern Uganda, behavioural and STI interventions were associated with an increase in condom use with the last causal partner--a proxy measure for consistent condom use in high-risk encounters, and substantial reductions in incidence of active syphilis and prevalence of gonorrhoea. There was also evidence...of reduced incidence of HSV2--a proxy measure of unprotected sexual contact, and increased recognition of symptoms of STIs... . These effects were not translated into any measurable reduction in HIV-1 incidence over a median follow-up of 3-6 years" (9).

In South Africa, pregnant women were tested for syphilis and antibodies to HIV in order to see how the two diseases were correlated by geographical location and over time. But, there was no correlation (10). On the contrary, KwaZulu-Natal, which is leading when it comes to HIV, has the lowest rate of syphilis in all provinces. Western Cape, on the other hand, had the highest rate of syphilis in 2000 but the lowest HIV prevalence. Northern Cape had the highest rate of syphilis in 2001 but the third lowest prevalence of HIV antibodies in that year. Paradoxically, then, there is an inverse geographical correlation between syphilis and HIV although both are said to be transmitted by heterosexual intercourse. An even more extraordinary result is the divergence over time between an increasing prevalence of antibodies to HIV and a declining rate of syphilis (10). This is also difficult to understand given the assumption that both are sexually transmitted.

The data from Thailand show that these paradoxical results are not peculiar to Africa. Even though Thailand is said to be severely hit by a heterosexually transmitted HIV-epidemic, we find yet again the same scenario presented by South Africa and Uganda. Bangkok has the highest rate of STDs but low HIV prevalence. Conversely, the so called Golden Triangle of northern Thailand has the highest rate of HIV but the second lowest STD morbidity of all regions. And, even within the different provinces of the Northern Region there is a negative correlation between HIV and syphilis (11). The conclusion from these observations is obvious: HIV cannot be heterosexually transmitted.

About the only thing Quinn et al. can find that correlates with the rate of appearance of antibodies to HIV in discordant couples are the numbers produced by the viral load test. In addition to the problems discussed above and in my March 7, 2003, letter, the viral load test in the Quinn et al. study was not performed during the study but actually one year after its completion. Furthermore, the authors did not perform the obvious control of performing the viral load test on the HIV-negative partners. This, at the very least, would provide a negative control, which is certainly called for given Flegg's reference to Pilcher et al. who had HIV antibody negative but viral load positive individuals (1).

AIDS researchers chronically assume too much.

David Rasnick

References

1. Pilcher, C. D., Eron, J. J., Jr., Vemazza, P. L., Battegay, M., Harr, T., Yerly, S., Vom, S., and Perrin, L. (2001) Sexual transmission during the incubation period of primary HIV infection, Jama 286, 1713-1714

2. Rich, J. D., and et al. (1999) Misdiagnosis of HIV infection by HIV-1 plasma viral load testing: a case study, Annals of Internal Medicine 130, 37-39

3. Papadopulos-Eleopulos, E., Turner, V. F., and Papadimitriou, J. M. (1993) Is a positive Western blot proof of HIV infection?, Biotechnology 11, 696-707

4. Kaplan, A. H., Zack, J. A., Knigge, M., Paul, D. A., Kempf, D. J., Norbeck, D. W., and Swanstrom, R. (1993) Partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles, J Virol 67, 4050-4055

5. Kageyama, S., Hoekzema, D. T., Murakawa, Y., Kojima, E., Shirasaka, T., Kempf, D. J., Norbeck, D. W., Erickson, J., and Mitsuya, H. (1994) A C2 symmetry-based HIV protease inhibitor, A77003, irreversibly inhibits infectivity of HIV-1 in vitro, AIDS Res Hum Retroviruses 10, 735-743

6. Quinn, T. C., Wawer, M. J., Sewankambo, N., Serwadda, D., Li, C., Wabwire-Mangen, F., Meehan, M. O., Lutalo, T., and Gray, R. H. (2000) Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group, N Engl J Med 342, 921-929

7. Wawer, M. J., Sewankambo, N. K., Serwadda, D., Quinn, T. C., Paxton, L. A., Kiwanuka, N., Wabwire-Mangen, F., Li, C., Lutalo, T., Nalugoda, F., Gaydos, C. A., Moulton, L. H., Meehan, M. O., Ahmed, S., and Gray, R. H. (1999) Control of sexually transmitted diseases for AIDS prevention in Uganda: a randomised community trial. Rakai Project Study Group, Lancet 353, 525-535

8. Padian, N. S., Shiboski, S. C., Glass, S. O., and Vittinghoff, E. (1997) Heterosexual transmission of human immunodeficiency virus (HIV) in northern California: results from a ten-year study, Am J Epidemiol 146, 350-357

9. Kamali, A., Quigley, M., Nakiyingi, J., Kinsman, J., Kengeya- Kayondo, J., Gopal, R., Ojwiya, A., Hughes, P., Carpenter, L. M., and Whitworth, J. (2003) Syndromic management of sexually- transmitted infections and behaviour change interventions on transmission of HIV-1 in rural Uganda: a community randomised trial, Lancet 361, 645-652

10. Makubalo, L. E., Netshidzivhani, P. M., Mulumba, R., Levin, J., du Plessis, H., Ratsaka, M., Mahlasela, L., Mudzanani, L., Johnson, C., and Shikweni, F. (2001) Summary Report:NATIONAL HIV AND SYPHILIS SERO-PREVALENCE SURVEY IN SOUTH AFRICA, Pretoria, South Africa, Directorate: Health Systems Research, Research Coordination and Epidemiology, http:// 196.36.153.56/doh/index.html

11. Chitwarakorn, A. e. a. (1998) Sexually Transmitted Diseases in Asia and the Pacific, Ministry of Public Health, AIDS Division, HIV/ AIDS Situation in Thailand, Region 10, Chiang Mai, Thailand

Competing interests:   None declared