STEP PERSPECTIVE, Volume 7, No. 2 -Summer/1995; A Publication of the Seattle Treatment Exchange Project e-mail: step@eskimo.com
Jeffrey T. Schouten, MD

Additionally, these studies have shown that the amount of HIV produced every day is much greater than previously thought, from 100-700 million new viruses per day. Equally surprising are measurements which show that the immune system is producing over a billion CD4 cells per day. The half-life of HIV is only 2 days (50% of the virus produced today is gone in two days). Therefore 98% of the viral load in the body is produced by cells infected within the last week. Also, most of the new virus produced is the result of newly infected cells. Thus, over a period of years there is a massive immune response to the production of large numbers of new viruses. Eventually, the immune system is unable to produce enough CD4 cells to overcome the viral production and the immune system weakens. This is a very dynamic process, not one characterized by a latent period, nor one characterized by a weak immune response.

Quantitative HIV Blood Tests

These new findings have been made possible by the rapid advances the last two years in techniques to accurately quantitate (measure) the number of HIV present in peripheral lymphocytes (and other mononuclear cells, or monocytes) and in the plasma (the cell-free portion of the blood). There are two techniques now readily available to measure HIV levels in the blood; they are the branched DNA test (bDNA) and the quantitative polymerase chain reaction (Q PCR)., Both tests measure HIV RNA. HIV carries its genetic information on two strands of RNA (ribonucleic acid). When HIV enters a cell it transfers its genetic information into the host cell as DNA (deoxyribonucleic acid), using an enzyme called reverse transcriptase. (The drugs AZT, ddC, ddI, d4T, and 3TC all interfere with this enzyme). The genetic information inserted into the host DNA may now be used to produce new virus by copying the information from the DNA back to viral RNA. [The Q PCR and bDNA assays measure specifically the viral RNA and not the messenger RNA (mRNA) which is produced to direct the synthesis of proteins used to build the new viruses, which include two strands of viral RNA.] Both the bDNA and Q PCR measure the amount of HIV RNA present in the plasma or in lymphocytes (and other monocytes). Studies have shown that the plasma HIV RNA is an accurate reflection of intracellular viral production.

Neither of the two methods for measuring HIV are approved by the FDA, although both companies making the tests expect to have FDA approval within a year. Once the FDA has the data which shows the consistency and reliability of these tests, they should be approved. People may obtain an HIV RNA test (Quantitative HIV test) through their physician. However, very few health insurers will pay for the test at this time because it is not yet FDA approved. Generally, Medicare and most state Medicaid programs have refused payment. Some private insurers are reimbursing for these tests. Chiron Corp. makes the bDNA test and the cost is $195. Roche Biomedical Laboratories makes the Q PCR test and the cost is $214. The Q PCR is a more sensitive test but may involve more time to perform in the lab, while the bDNA is not as sensitive, it does not measure viral levels below 10,000/ml, although soon this test may detect levels above 5,000/ml. Both tests have been standardized now so that they are available through many local laboratories. (See phone numbers at the end of this article for more information about getting a test.) Viral HIV levels below 10,000 (104)/ml are considered low, while values above 100,000 (105)/ml are considered high. Changes in HIV RNA levels over time are significant if the change is by a factor of at least 5 (or even 10) or more.

For example, an increase from 3.9 x 105 (390,000/ml) to 6.3 x 105 (630,000/ml) would not be considered significant, while an increase to 4.1 x 106 (4,100,000/m;) would be a significant change. Likewise, a decrease to 2.8 x 104 (28,000/ml) would represent a significant decrease.

Prognostic Value of HIV RNA

HIV RNA has been shown in several studies to be the best predictor of prognosis (time to progression to AIDS). It provides more information than any other marker, including CD4 cell counts. As early as 1989, David Ho measured viral blood levels and found that levels of HIV were much higher than previous estimates. He used a technique requiring viral tissue culture in the lab for three weeks to measure the amount of infectious viral particles in the blood. Persons with symptomatic HIV infection and AIDS had much higher levels of HIV in the plasma and lymphocytes (and other monocytes) than did persons with asymptomatic HIV infection. However this study did not evaluate whether HIV levels could predict which persons would progress to AIDS more rapidly. Many studies were done looking at other surrogate markers to predict prognosis including B2-microglobulin, antibodies to HIV, neopterin, interleukin, TNF, p24 (a viral surface antigen or marker), synctium-inducing (SI) phenotype (an aggressive variant of HIV), and others. However, none of these markers have proven to be sensitive enough to be of much use in clinical practice. Most of these markers are elevated (positive) only in late-stage disease, when CD4 counts are already very low, and are not of much help to predict how quickly CD4 counts will decrease.

Studies conducted at the University of Washington in 1989 by Robert Coombs showed that plasma viremia (virus detected in the plasma) was predictive of a more marked decline in CD4 cell counts and correlated with progression to AIDS. The test used was a viral tissue culture, which is not as sensitive as the newer Q PCR and bDNA, and measured infectious virions, not the total number of HIV virions. This was one of the first studies, though, to show the prognostic value of the presence of HIV in the plasma.

A more recent study has convincingly shown that "plasma HIV-1 RNA is a strong, CD4+ T-cell independent predictor of a rapid progression to AIDS after HIV-1 seroconversion." The researchers compared plasma HIV-1 RNA with p24 antigen, B2-microglobulin, and CD4 cell counts to predict progression to AIDS in a group of 62 men who were part of the Multicenter AIDS Cohort Study (MACS). The authors did not correlate HIV-1 RNA levels with synctium-inducing phenotype. HIV RNA was measured using the bDNA assay (Chiron Corp.), which the authors stated had the "advantage of large sample capacity, speed, reproducibility, and a format similar to an enzyme-linked immunosorbent assay (ELISA)." The HIV RNA test was found to be the "earliest and most powerful predictor of outcome after seroconversion." This study added proof to the concept that the course of HIV infection closely follows blood levels of HIV. Even though the Q PCR can detect lower levels of HIV RNA, the authors found that the bDNA gave values accurate enough to predict the risk of progression to AIDS. The bDNA test was found to be rapid and reproducible. The HIV RNA gave more information than did the CD4 cell counts. For persons with CD4 cell counts above 500 cells/mm3, only 6% developed AIDS within 2 years of infection if there was no detectable HIV RNA in the plasma (<10,000 (104 )genome equivalents/ml), compared to 45% if the HIV RNA was detectable (>10,000 (104 ) genome equivalents/ml). The researchers also found that plasma HIV RNA levels were higher in persons with more advanced disease. This is a significant study because it is the largest study to date which correlates plasma HIV RNA levels soon after infection with later progression to AIDS.

The long-term non-progressor HIV-1 infection studies all found that persons with HIV infection and no deterioration of the immune systems after 10 years had very low to non-detectable levels of plasma HIV. Another study compared HIV RNA levels and syctium-inducing (SI) phenotype in order to predict prognosis. HIV-1 RNA copies were found to be high and stable from the time of initial infection in all persons who progressed to AIDS. Non-progressors were found to have a significant decline in the level of HIV RNA after the initial rise seen after infection. HIV RNA was found to be of greater value in predicting prognosis in persons with the non-SI phenotype. HIV RNA was measured by the Q PCR method in this study. The authors concluded that "a decline in HIV-1 RNA copy numbers during the symptom-free period of infection is strongly associated with delayed onset of AIDS." High values for this study, as in most others, were considered to be above 100,000 (1 x 105)/ml, while low values were considered to be below 10,000 (1 x 104)/ml.

The prognostic value of HIV RNA has been confirmed in other studies as well. David Baltimore and colleagues found that HIV RNA measured in lymphocytes and other mononuclear cells strongly correlated with the future course of disease, and could "have significant clinical utility as a prognostic indicator and as a means to guiding and monitoring antiviral therapies." In this study, HIV RNA provided better prognostic information than did CD4 cell counts. Saag and colleagues measured plasma HIV levels with the Q PCR assay and found that viral levels significantly decreased by as much as 235-fold with antiviral therapy or following resolution of the initial infection. HIV RNA was significantly correlated with stage of disease and CD4 cell counts.

HIV RNA As A Measure of Response to Therapy

HIV RNA provides the quickest, simplest, least expensive and most accurate indicator of response to treatment for HIV. Tests for resistance to antiretroviral drugs (like AZT) are available only through research labs, are expensive and do not directly indicate the effect of a drug on the amount of HIV in the body. Several studies have documented the rapid decrease in HIV RNA levels following antiretroviral therapy. Changes are seen in a few days. This rapid change contributed to the recent understanding of the rapid production and turnover of both HIV and CD4 lymphocytes. In 1991, Saag reported that of 8 patients who had HIV present in the blood (by tissue culture method) prior to AZT, five became tissue culture negative following AZT therapy (follow-up range 15-40 months). There was an average decrease in plasma HIV of over 100 (102 or 2 logs). David Ho reported that treatment with ABT-538, a protease inhibitor, resulted in large decreases in plasma HIV-1 levels within a few days. This study again confirmed that "replication of HIV-1 in vivo is continuous and highly productive, driving the rapid turnover of CD4 lymphocytes." Another study has found that almost complete replacement by drug-resistant strains of HIV can occur as quickly as 14 days.

A study which evaluated HIV RNA levels and resistance to Zidovudine (AZT) produced results which showed that changes in serum HIV RNA levels did not directly follow development of AZT resistance. After decreasing with initial AZT therapy, HIV RNA levels increased within weeks, while AZT resistance took longer to develop. Thus, in this study of 11 patients, apparently other factors occurred which resulted in HIV RNA levels rising even before AZT resistance developed. Drug absorption, metabolism and activation are some of the other factors that may explain these results.

HIV RNA As An Indication for Therapy

Plasma HIV RNA levels clearly predict better than any other markers the risk of progression to AIDS and loss of CD4 cells. Even in persons with CD4 cell counts >500, many people have high levels of HIV (>100,000 or 105). While the Concorde Study and ACTG 019 have not shown a survival benefit from early therapy, it is possible that the subset of persons with high levels of HIV RNA may well benefit from early therapy. The major limitation at this time is the emergence of drug resistance, and the studies of HIV RNA have shown how quickly drug-resistant viruses emerge. Long-term non-progressors all have very low plasma HIV levels, and this should be the goal of any therapy.

HIV RNA As A Possible Indicator For Risk of Maternal-Fetal Transmission of HIV

There is data that the risk of transmission of HIV from mother to child is correlated with the amount of HIV in the mother's blood. Some of this information was presented by Dr. Barbara Weiser at the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy on October 5, 1994. These results remain to be verified. Measuring a mother's blood viral level may explain how AZT reduces the risk of maternal-fetal transmission of HIV in some women. During the study which evaluated the use of AZT in pregnancy, HIV RNA levels were obtained although the results are not yet available.

Unanswered Questions

Most current HIV therapy trials measure HIV RNA levels throughout the course of the trial. Ongoing trials should help determine if lowering HIV RNA levels results in better survival and delayed onset of AIDS. Clearly, high HIV RNA levels correlate with faster progression, whether lowering those levels will slow progression remains to be demonstrated. The new understanding of the large numbers of HIV and CD4 cells produced daily would imply that the immunotherapy will not be as effective as antiviral therapy. However, more research and many trials are needed to better understand the role of the immune system in response to HIV infection and therapy. Long-term non-progressors have an effective immune response which keeps the blood levels of HIV very low. One potential danger is using HIV RNA levels to evaluate new drugs is that effective drugs may be abandoned too soon. Drugs which interfere with viral assembly may result in the replication of defective viruses, it is possible that even though HIV RNA levels do not decrease, the person receiving the drug is benefiting from it.

Current Uses For HIV RNA

HIV RNA may be useful for individuals with intermediate CD4 cell counts who are undecided about initiating antiviral therapy. High levels of HIV RNA are associated with a more rapid progression to AIDS. Although there is no proof that lowering viral levels will change the course of the infection, it seems very likely that the decline in the immune system will be slowed if viral levels are kept low. Also, HIV RNA very accurately tracks the effectiveness of antiviral therapy. It seems reasonable not to continue a drug (or add a new drug), if current therapy is not decreasing HIV RNA levels. Again, no clinical study has been done individualizing therapy based on HIV RNA results. This therapeutic strategy is limited by drug resistance and the limited number of drugs available. Preliminary data from the protease inhibitor studies have shown greater decreases in HIV RNA than with the nucleoside analogs (AZT , ddI, ddC, etc.).

Both the Q PCR and the bDNA tests have been shown to be stable from day to day and the results are reproducible for sample to sample. Relative changes over time are of greater value than one single value. The results are the most direct indicator available of HIV levels in the blood. Although CD4 cell counts are useful in determining risks of opportunistic infections, they are only an indirect indication of HIV levels. It is hoped that, more insurers will begin to reimburse patients for the costs of these new useful tests and the cost will probably decrease because there are several companies competing for this service. Soon, monitoring of HIV RNA will become a routine part of assessing drug effectiveness in clinical practice.

Further Information for Laboratories Performing HIV RNA Tests:

Roche Biomedical Laboratories- Human Immunodeficiency Virus (HIV-1) RNA, Quantitative (Q PCR). 800-872-5727. Cost - $214.

Chiron Corp.- Branched DNA HIV RNA Assay. 800-553-5445 (Nichols Lab, for the nearest local laboratory). Chiron also provides a reimbursement service to assist physicians in insurance claims. 800-775-7533 (M-F, 9-5 Pacific Time). Cost - $195.

Jeffrey Schouten is a general surgeon and co-chair of STEP's Scientific Review Committee.

References

1. Emberton J, Zupancic M, Ribas JL, et al. Massive Covert Infection of Helper T Lymphocytes and Macrophages by HIV During the Incubation Period of AIDS Nature 1993;362:359-62.

2. Pantaleo G, Graziosi C, Demarest JF, et al. HIV Infection is Active and Progressive in Lymphoid Tissue During the Clinically Latent Stage of Disease. Nature 1993;362:359-62.

3. Saag M. AIDS Update: Issues on the Cutting Edge Conference. Fred Hutchinson Cancer Research Center, Seattle, Washington. May 17,1995.

4. Pachl C, Todd JA, Hern DG, et al. Rapid and Precise Quantification of HIV-1 RNA in Plasma Using a Branched DNA Signal Amplification Assay. J AIDS 1995;8:446-454.

5. Mulder J, McKinney, Christopherson C, et al. Rapid and Simple PCR Assay for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma: Application to Acute Retroviral Infection. Journal of Clinical Microbiology 1994;32:292-300.

6. Ho D, Moudgh T, Alam M. Quantitation of Human Immunodeficiency Virus Type 1 in the Blood of Infected Persons. N Engl J Med 1989;321:1621-5.

7. Tsoukas CM, Bernard NF. Markers Predicting Progression of Human Immunodeficiency Virus-Related Disease. Clinical Microbiology Reviews 1994;7:14-28.

8. Coombs, RW, Collier AC, Allain JP, et al. Plasma Viremia in Human Immunodeficiency Virus Infection. N Engl J Med. 1989;321:1626-31.

9. Mellors JW, Kingsley LA, Rinaldo CR, et al. Quantitation of HIV-1 RNA in Plasma Predicts Outcome after Seroconversion. Ann Int Med 1995;122:573-79.

10. Schouten JT. Viral and Immunologic Studies in 30 Long-term Survivors With Non-progressive HIV-1 Infection. STEP Perspective 1995;7(1):7.

11. Jurriaans S, VanGemen B, Weverling GJ, et al. The Natural History of HIV-1 Infection: Virus Load and Virus Phenotype Independent Determinants of Clinical Course?. Virology 1994;204:223-33.

12. Saksela K, Stevens C, Rubinstein P, Baltimore D. Human immunodeficiency virus type 1 mRNA expression in peripheral blood cells predicts disease progression independently of the numbers of CD4+ lymphocytes. Proc Natl Acad Sci 1994;91:1104-8.

13. Piatak MJ, Saag MS, Yang LC, et al. High Levels of HIV-1 in Plasma During All Stages of Infection Determined by Competitive PCR. Science 1993;259:1749-54.

14. Saag MS, Crain MJ, Decker WD, et al. High-Level Viremia in Adults and Children Infected with Human Immunodeficiency Virus: Relation to Disease Stage and CD4+ Lymphocyte Levels. Journal of Infectious Diseases 1991;164:72-80.

15. Ho DD, Neumann AU, Perelson AS, et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature 1995;373:123-6.

16. Wei X, Ghosh SH, Taylor ME, et al. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 1995;373:117-22.

17. Loveday C, Kaye S, Tenant-Flowers M, et al. HIV-1 RNA serum-load and resistant viral genotypes during early Zidovudine therapy. Lancet 1995;345:820-4.

18. Burroughs Wellcome Co. News Release, October 5, 1994.

These articles were provided by the Seattle Treatment Education Project - Copyright (c) 1997 - Seattle Treatment Education Project. Noncommercial reproduction encouraged. Distributed by AEGIS - http://www.aegis.com
950601
STEP7201

Copyright © 1995 - Seattle Treatment Education Project (STEP) - All rights reserved. Noncommercial reproduction is encouraged. STEP is published four times a year by the Seattle Treatment Education Project, 127 Broadway East, 3rd Floor, Seattle, WA 98102.    Email: step100@aol.com  STEP web page