AIDS Treatment News #186 - November 5, 1993
John S. James

Accurate blood tests for HIV activity, and for other markers of disease severity or progression, are critically important for developing new drugs. Reliable tests might shorten the time required to show which drugs are good candidates from years to months, allowing many more potential treatments to be tested. And better viral tests could also improve medical care with the drugs we already have, by showing when a course of treatment is working for an individual and when it is not, so that the physician will have rational guidance on when to add or to switch therapies.

But what is happening today in the development of viral markers is poorly understood in the AIDS community. People do know that the tests we have now -- T-helper count as a measure of immune function, and p24 antigen as a measure of viral activity -- have serious drawbacks when used to measure antiviral drug effects. But many do not know how much progress is being made in developing better tests, both for drug trials and for clinical care. These tests are not yet commercially available to physicians, but some of them may become available in the not-distant future.

Scientists, physicians, and activists have sometimes drawn the wrong conclusions from the atmosphere of gloom which followed the Berlin conference -- suggesting, for example, that we cannot rely on laboratory tests in trials of antiviral drugs, and therefore must use only "clinical endpoints" -- death or major disease progression -- for trials of antivirals. Such a policy would add years to the time required to test potential drugs. And when a drug fails to work, clinical endpoints do not tell why.

In fact, researchers are now learning much more than was known before about HIV pathogenesis, about how to measure it, about why past trials have often failed to provide the information physicians need, and about how to run better trials in the future. To focus on the new developments in antiviral testing, which are part of this expanding knowledge, we interviewed Mark B. Feinberg, M.D., Ph.D., a scientist at the Gladstone Institute of Virology and Immunology, Director of the Virology Research Laboratory at San Francisco General Hospital, and Assistant Professor of Medicine, Microbiology and Immunology at the University of California, San Francisco. (The Gladstone Institute, a major new research center, is associated with San Francisco General Hospital but largely funded by a private charitable trust.)

Glossary: Viral Tests

This section explains some of the viral tests discussed below, which will be unfamiliar to many of our readers.

* PCR (polymerase chain reaction). This is a test which detects very small amounts of a specific kind of DNA -- the molecule which contains the genetic code of most living things. In nature, DNA is able to reproduce itself inside living cells; this is necessary for life to be able to reproduce. PCR creates conditions in the test tube so that a particular sequence of DNA (if it is present in the sample being tested) can be reproduced; each strand will become two strands. The process is repeated, and the two strands become four, then eight, then 16, etc. After 20 or more cycles of this process, each molecule of the sought-after sequence of DNA will have produced a million or more copies -- enough to be easily detected by standard methods, even when the starting concentration was far too low to be detected directly. (The inventor of PCR, Kary B. Mullis, Ph.D., received a Nobel prize for the discovery last month.)

[Note: since HIV is a retrovirus, it does not contain DNA; instead its genetic information is in the form of RNA. A PCR test for HIV usually looks for the RNA of the virus. Before the PCR can be run, the RNA is transcribed into DNA, using an enzyme (reverse transcriptase) which HIV itself uses for this purpose.]

* Quantitative PCR. Originally, PCR could only tell if a particular sequence of DNA was present in a sample or not -- it could not tell how much was there. It is difficult to accurately tell how much, because the successive steps of DNA reproduction are not 100 percent efficient, and errors accumulate throughout the process.

Today, a number of methods of quantitative PCR are being developed. But it is not clear how accurate some of the methods are. Errors of several hundred percent have been common.

* QC-PCR (quantitative competitive PCR). This is a method of quantitative PCR which was developed by Genelabs Inc. of Redwood City, California, and published in Science on March 19, 1993. In QC-PCR, a known, tiny amount of a control sequence, similar to the DNA sequence being tested but able to be distinguished, is added to the sample before the PCR process begins. During the successive doublings, both the target sequence (the one being looked for) and the control sequence, are multiplied similarly. Finally, the quantity of the target sequence in the original sample is calculated from its ratio with the control sequence. This is more accurate than measuring the total amount of the target sequence, since the inevitable errors in the doubling process affect both the target sequence and the control sequence equally.

Unfortunately, QC-PCR is labor intensive and difficult to do. Therefore it will not be available for routine medical use in the foreseeable future. But it can be used in clinical trials, and can also serve as a "gold standard" by which other tests can be measured.

* Branched DNA assay (bDNA). This test, being developed by Chiron Corporation, is much easier to run than QC-PCR and gives similar results, except that it will not work at all when the level of virus is very low.

In this test, copies of a DNA probe are attached to the wall of a small laboratory vessel; then the sample is put in. The probe binds to a certain part of HIV RNA, if it is found in the sample, holding the RNA in the vessel. Then another DNA probe is put in; one end of this attaches to another part of the HIV RNA. The other end of this second probe has many branches, and each branch ends with a "reporter" chemical which, under certain conditions, will produce light, which can be detected by laboratory equipment. Each molecule of HIV RNA can attach to one of these branching structures and hold onto a number of the light sources, not just one. In this way, very small amounts of the target RNA can be detected, without the need for PCR.

* P24 antigen test. This blood test has been used in medical practice for several years. But recent data is showing that it does not work well to predict clinical outcome. The reason seems to be that the test is often inaccurate; people can be p24 negative and still have large amounts of infectious virus in their blood.

A new version of this test, called the acid-dissociated (or ICD) p24 antigen test, offers some improvement. But it appears to have most of the problems of the original p24 test.

* Viral cultures. In this test, virus is grown from blood cells or blood plasma. To estimate how much virus is present, the sample is successively diluted until no virus is found.

Quantitative viral-culture tests have been used in research for several years. But they are labor intensive and require special facilities, so they have seldom been used in clinical care. Also, they may be less accurate than the newer tests. The Interview

JJ: What is your main work at the Gladstone Institute?

MF: Our Virology Lab (at San Francisco General Hospital) is responsible for doing virologic characterization of volunteers in drug trials. But our main work at Gladstone is guided by our own interests in better understanding the natural history of HIV disease. We need to understand the natural history to determine whether any intervention is beneficial or not. If you don't know what correlates with disease progression, you're in the dark.

Over the past couple of years, a better understanding of HIV disease has emerged. There is more viral replication going on, even during the asymptomatic phase, than people had suspected. Also, clinical deterioration is presaged in many ways by evidence of increased viral replication -- as if the immune system of the infected person is losing its ability to contain viral growth. What may be going on is that as the virus replicates more, it can inflict more damage on the immune system, which consequently further weakens its ability to hold the virus in check.

We, and others, are trying to find ways of accurately monitoring these processes. We certainly need a better baseline description of what's going on in people with HIV. We need to nail down the association between increased viral replication and disease progression. We also need accurate, practical markers for drug trials. The tests that have been available, and the ones that are commonly followed in drug trials, are not very good.

The ideal would be a test that is accurate, that correlates with the fundamental biology of the disease, that predicts future disease progression and the effects of treatments, and that is doable on a large scale. Then clinicians in practice could monitor their patients, and get insight directly into what is going on, instead of relying on surrogate markers, which have obviously been incompletely helpful.

JJ: You would not describe a good viral test as a surrogate marker?

MF: Right, it's a direct marker. Surrogate markers are by definition things other than what you want to measure. Surrogate markers give you a clue about what's going on. But they are only meaningful if they accurately reflect the underlying process. Certainly CD4 counts (T-helper counts) do to a certain extent, but what controls the level of CD4 cell is not entirely understood. Part of it could depend on virus replication, part of it could be disruption of the architecture of the immune system. There are many variables, many unknowns, which may affect CD4 counts. People used the CD4 test because it was available, and it seemed like it would be a meaningful marker. Early on it showed some response to therapy. In retrospect, in more detailed analysis, it has not turned out to be that good.

Clearly CD4 is useful for staging people, for knowing when to begin prophylaxis for opportunistic infections. Here it is a meaningful marker. And falling CD4 counts clearly correlate with development of immunosuppressive disease.

JJ: Concerning the p24 antigen test, are its problems caused by the fact that much of the virus is complexed with antibody, so it isn't detected -- even with the newer acid dissociated (ICD) p24 antigen test?

MF: Yes. We have long had the sense that was true. But you get a much clearer picture when you have other ways of looking for the virus that's there.

There are a number of proteins, or other parts of the HIV virus, that one could conceivably measure, if we had tests to do so. P24 was the first test that was developed. For many purposes it's useful. For example, as a laboratory measure for monitoring virus replication in tissue culture, it is very good, because there are no antibodies to HIV in those tissue culture flasks. The test is very sensitive, very straightforward to do, and it is accurate in that context. But in the body, antibodies complicate the detection of p24.

Another kind of entity you can look for, when testing for HIV, is the RNA genome. HIV, being a retrovirus, has in the virus particle two copies of its RNA, which can transmit the genetic information of the virus to another cell. There are a number of ways now -- two primary ways -- to look at the amount of HIV RNA in the plasma of an infected person. One uses PCR, the other uses branched DNA (bDNA) technology.

When you do those tests and compare their results with those of the p24 antigen tests, you can find people who have very high levels of HIV RNA in their plasma and no detectable p24. So you have to conclude the p24 is falsely negative. In some of those people, you can culture infectious virus from their plasma, when their p24 antigen test is negative.

Part of the problem is that a p24 antigen result is not only a function of how much virus is there. It is also a function of how much antibody is there, and both the viral and antibody levels could be changing, either together or independently, in ways that we don't understand. This will not necessarily be the same at each stage of the disease, or for each individual. Probably the majority of people with AIDS will not have detectable p24.

With the new methods, you can measure HIV RNA in the vast majority of people.

Differences Between Tests

MF: We have tried to compare a number of different viral markers head-on -- plasma culture, p24 antigen, QC-PCR, and bDNA -- to see how they relate to each other, and try to figure out which is the most meaningful one to follow. To answer those questions, you need to do all the tests and compare them directly, studying patients at different stages in the infection, and doing all these assays on the same specimens. This tells you how good the assays are, but it also can tell you important things about the pathogenesis of the disease.

JJ: Are you doing these tests as part of the trials run by the ACTG (AIDS Clinical Trials Group, of the U.S. National Institute of Allergy and Infectious Diseases)?

MF: We are doing this work independently, because we think it's important. There is a frustration with the fact that clinical researchers have not arrived at a consensus about what they think is the best quantitative parameter to follow. People are searching for better tools.

JJ: Where do the samples come from?

MF: Mostly they come from people participating in various trials where extra blood samples are available, or from people being cared for in the clinics here.

JJ: Is there a way somebody could sign up if they are interested in donating blood and learning their results?

MF: Unfortunately we don't have a mechanism for that now. Hopefully there will be more opportunities in the future, as we organize trials that are based on viral quantitation as a guide to therapy. We're working hard to make this available.

If we treat people based on their direct virologic measures, could we do better than we've done by treating them based on surrogate markers? My feeling is that the answer will be yes. But it's essential that we answer that question as rigorously and correctly as we can. If we falsely assume that something is logical and therefore would be a good marker, and it turns out not to be, a few years from now we're basically in the same situation we are now with CD4 counts. My belief is that measuring the viral parameters directly will be more useful, more accurate, and a better way to monitor the activity of drugs.

JJ: Beyond being sure that the new tests measure the virus accurately, people also want to see clinical confirmation. Do the tests help predict which patients will do well in the future, and which patients are benefiting by their drug therapy? Of course we don't want to wait many years to get that information from people who are being tested now. Can we speed this process of validating the new viral markers by testing samples frozen years ago, so that the future clinical course of the patient is already known?

MF: An advantage of both QC-PCR and bDNA is that they can be done on frozen specimens, provided they were frozen in an appropriate way. We may be able to learn a lot from trials that have already been done.

Other research groups are finding that measuring viral burden quantitatively is in fact predictive of disease progression. It's not yet proven that intervening to decrease viral burden, if that is possible, would slow down progression. That's exactly the kind of information one needs. For a number of ACTG trials, there are lots of stored specimens that might be used.

There is already a fair amount of evidence that increasing viral replication is an indicator that disease progression is accelerating. What we don't yet know is whether, if you can slow down the virus, you can slow down progression. It sounds logical, but it does need to be demonstrated.

We do not know whether AZT alone will give a convincing demonstration. And we do not always have the best samples -- for example, where you know how long somebody has been on the drug, and whether they had resistant viruses.

JJ: After the Concorde results last summer (which raised serious doubts about using T-helper counts as a measure of antiviral efficacy in trials), some people have suggested that we need to return to clinical endpoints for trials, instead of using blood tests to determine how well a drug is working.

MF: My feeling about the reaction to the Concorde results is that people were understandably disappointed, but they sometimes took the wrong lesson from it. I don't think it's correct to conclude that it does not make sense to intervene earlier in the course of the disease. Everything I know as a basic scientist studying HIV pathogenesis tells me that the time to intervene is earlier, if you had effective drugs.

What I take from the Concorde results is that, if a drug or a test did not work, we need to know why. The results do not condemn early intervention; they may condemn the drug or how it was used. But nothing about that trial has swayed my belief that intervening early would be beneficial if we had good drugs.

The Concorde study left some people with the notion that we don't have a good marker for antiviral drug activity, and we need to follow clinical endpoints. That basically puts us back to where were seven years ago. We can do better today, and we must do better. Following clinical endpoints will be essential to correlate with other markers, like direct virologic markers. But clinical outcomes take time.

And a clinical endpoint does not tell you why a drug did or did not work. It's 1993, and we have to learn to understand these things. Then it will be important to have a consensus on what's best to look for in trials.

My feeling is that this movement toward saying, "We need clinical endpoints," is like people throwing their hands up in frustration, some in despair and some in confusion about what do to. Many of us who work in this area do have a fairly clear idea about what might be done.

JJ: In which patients are QC-PCR and branched DNA appropriate? My understanding is that the branched DNA test is much easier to do, and feasible for widespread use, but it won't work if the viral level is very low, because of background noise in the test.

MF: That's right. But it will pick up virus in most people, if not almost everybody, with a CD4 count of under 500. For people over 500, it may pick up 70 or 80 percent. And for those who cannot be measured, it may be good enough to know that they're below the cutoff of the test, because they may have relatively little risk of rapid disease progression (because the viral level is so low).

JJ: Whereas with the p24 antigen test, if you're below the cutoff, it doesn't mean very much?

MF: Right, you could have AIDS and have p24 below the cutoff, and still have a hundred thousand, or a few million, copies of the virus per milliliter of blood at that time.

The QC-PCR, or other quantitative PCR methods, are very important; when we have learned enough to be sure of their accuracy, these tests might serve as a gold standard for the branched DNA or other assays. But quantitative PCR assays are labor intensive, and demand high levels of skill from the people doing them. It's hard to imagine that these will become available enough that physicians would use them in routine care.

The branched DNA assay could be widely used because it is more like other clinical tests that are commonly done. Also, it has a much more rapid turnaround time than quantitative PCR.

But it is still too early to give a blanket endorsement to the bDNA assay. It still needs rigorous testing, with additional comparisons with other tests; that is what we and others groups are doing. Hopefully we will have answers to this in the relatively near future. But also, the test has to be validated in clinical practice; that means doing clinical trials using this test as a marker.

JJ: What is the range of the number of virus copies which you find in patients' blood with these tests?

MF: In people who have just recently been exposed to HIV and are undergoing the primary infection -- where the virus is widely disseminated in their body, and there is no immune response to it which has developed yet -- they can have millions of copies of RNA per milliliter of blood. You divide that by two to get the number of virus particles, since there are two copies of RNA in each virus particle. So people can have millions of virus copies per milliliter.

Once they have an immune response to the virus, the levels of viremia go down significantly. But still people will have 10,000, 20,000, or 100,000 copies of the virus per milliliter, and some people have more.

JJ: What are the limits of the tests -- the number of copies below or above which they do not work?

MF: The cutoff for the bDNA assay is presently 10,000 copies of RNA per milliliter. That is the background level that was established after screening many HIV-negative plasma samples. Researchers are working to lower that background.

The QC-PCR assay can go down to about 10 copies per millimeter. And smaller amounts can be detected by special preparation of the sample. If it is done well, there's no background. So QC-PCR is the best assay to determine whether a sample is negative.

JJ: How high can these tests go?

MF: You can cover the whole range, to several million copies per milliliter of blood.

JJ: Do you have any thoughts about how to get the new tests quickly accepted? Presumably the first step is to finish getting the data, and make a good scientific case.

MF: That's an essential first step. It is important that this work comes from a number of laboratories. Because what will matter is that you get the same result from the test no matter where you do it.

After we make sure that the assays are accurate, which I think we're pretty close to, we need to validate them in clinical trials, to show that they make a difference (in clinical outcomes). It would not help to spend money for these tests if it turns out that monitoring them does not add anything to clinical care of a patient. We must make sure that monitoring these tests allows us to treat patients better with the drugs that we have -- and also allows us to identify effective drugs more expeditiously, and to eliminate those that don't work.

JJ: How might these tests be used in clinical trials?

MF: Perhaps a valuable initial screening, which you could do fairly rapidly, without necessarily needing a lot of people, is to test a drug in HIV-infected people and see how well it decreased viral replication. If it did, then in a short period of time the drug could go on to larger studies, to look at endpoints.

Many people have participated in large trials of AZT and related drugs. These trials are expensive, and very demanding on lots of patients who participate. And at the end of the day we don't know clear answers from the studies. We need to be more selective about what we do, to develop a more productive framework to test drugs.

Later, if we prove in one trial, or in a few trials, that diminishing the viral burden correlates with a better outcome, there may be a day in the not too distant future when viral reduction might be sufficient proof of efficacy for the FDA. They don't yet know what criteria to settle on. I hope that the data about direct virologic markers will be persuasive enough that it will be easy for the FDA to decide whether or not to use them.

There are a number of research groups now working on viral tests to measure HIV disease progression; there is much work in this area. The lessons I stated above were derived not only from this laboratory, but from many other groups as well.

For me, the important point is that we are not in the dark. It's not like there is no course we can take that will give us definitive answers. Unfortunately, that is the lesson that many people derived from recent disappointments in therapeutic interventions.

What everybody has to do, from physicians who take care of patients, to the patients themselves, to the scientists, is to tackle the difficult questions and devote energy to that. There is important work that can be done today.

This should be a source of optimism. We will learn why the drugs we have today don't work as well as we hoped. We may also be able to learn how to use them better -- which would improve patient care in the short term. And we will have a better way of testing new drugs rapidly.

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