HIV Guidelines: Some Evolve; Some Don't. What's Up With That? Part Two

We learned of Dr. Bob's unexpected passing just days before this blog entry was scheduled to post.

Read part one of this two-part article.

Gentle Readers, welcome to Part Two. Let's talk about detecting very recent HIV infections and emerging diagnostic guidelines. As we have learned more about the natural history of untreated HIV infection and the body's immune response to the virus, we've developed a better understanding of measurable events occurring during recent HIV infection. For instance, the HIV viral load spike that occurs shortly after acquiring the virus correlates in time with a corresponding spike in HIV p24 antigen level. As the body's immune response kicks into gear several weeks after infection, the immune system begins making specific anti-HIV antibodies. The first to appear is anti-p24 antibody. As that antibody increases, the p24 antigen and HIV viral load decline.

I realize this sounds confusing, and that's because it is confusing. The basic point is that in early HIV infection we've learned when to measure what and what to measure, so we can make the diagnosis of HIV infection sooner than waiting for seroconversion (developing detectable levels of anti-HIV antibodies in the blood).

Earlier detection of HIV would have benefits for the positively charged individual, because it would allow for early intervention with antiretrovirals to preserve immune function and limit immune activation/inflammation. It would also improve public health by decreasing the number of new infections. Studies have shown that those who know their status are more likely to practice safer sex techniques to prevent further spread of the virus. Plus, HIVers on effective combination antiretroviral therapy are significantly less infectious.

Perhaps this would be an appropriate time to discuss the various types of HIV-diagnostic tests and some of their limitations.

The first- and second-generation ELISA/EIA (enzyme immunoassays) HIV-diagnostic tests are indirect measurements. For all you science geeks (and premed or med students reading this blog) I'll briefly describe how these tests are performed. Those who are not science geeks may want to skip to the next section.

Serum or plasma from the individual being tested is added to plastic cells that have been coated with viral antigens (first-generation assays) or recombinant HIV proteins and/or synthetic peptides (second-generation assays). If the person whose blood is being tested is HIV infected, specific anti-HIV antibodies (termed anti-HIV-IgG antibodies) would bind to the antigen in the wells. Then an enzyme that links to IgG antibody is added. This in turn binds to the anti-HIV-IgG. A color reagent is then added, and a change in color would indicate the presence of anti-HIV-IgG in the blood being tested. Really, that's how it's done. I'm not making this stuff up!

In third-generation ELISA/EIA tests, both anti-HIV-IgG antibody and anti-HIV-IgM antibody are detected. Third-generation assays detect HIV antibodies earlier than first or second generation assays, because IgM antibodies are the first class of antibodies to be produced by the body's immune system in response to an invader (such as HIV or other germs).

Fourth-generation ELISA/EIA tests assay for p24 antigen as well as anti-HIV antibodies of the IgG and IgM classes. Because the viral antigen p24 becomes present in the blood before anti-HIV antibodies, tests assaying for p24 antigen can detect HIV infection earlier than tests that assay only for anti-HIV antibodies (first, second and third generation tests).

Rapid HIV tests using oral fluid or blood can detect anti-HIV-IgG and anti-HIV-IgM antibodies, but not p24 antigen. Last year the Food and Drug Administration (FDA) approved an automated rapid test with enhanced sensitivity and specificity.

The Western Blot test is much more complex than ELISA/EIA testing. It involves an analytical technique that detects not only the presence of anti-HIV antibodies, but also specific types of anti-HIV antibodies directed against the core of the virus (p17, p24, p55), polymerase enzymes (p31, p51, p66) and the viral envelope (gp41, gp120, g1p60). Western Blot (WB) testing is indicated only as confirmatory testing for preliminarily positive ELISA/EIA tests, due to the high rate of false positives seen with WB testing (~2%). A negative WB shows no bands (no anti-HIV antibodies detected). A positive WB shows reactivity to gp120/160 plus either gp41 or p24. An indeterminate WB reveals any band pattern that does not meet the criteria for a positive test.

Newer diagnostic testing techniques can detect HIV infection during the acute infection stage earlier than Western Blot testing. NAAT (nucleic acid amplification testing), also called PCR (polymerase chain reaction), tests directly for a piece of HIV's genetic material (RNA). The concept here is that NAAT assays for the actual virus while ELISA/EIA and WB tests measure antibodies to the virus. Because it takes a period of time for the body's immune system to manufacture specific anti-HIV antibodies, the "window period" period is longer for antibody testing than for NAAT/PCR testing.

Last year the FDA approved an improved nucleic acid-based test (Aptima-1) that can be used both to detect (diagnose) acute HIV infection and/or to confirm positive preliminary ELISA/EIA or rapid tests. It's worth noting the use of Aptima-1 NAAT testing as a confirmatory test remains outside of the current guidelines.

After acquiring HIV, acute retroviral syndrome (ARS) symptoms generally manifest within two to three weeks. HIV p24 levels (measurable with fourth generation ELISA/EIA tests) typically peak just after the onset of ARS symptoms at 2.5 to 3.5 weeks after infection. HIV RNA levels (measurable by NAAT/PCR testing) begin to increase at 1.5 to 2 weeks, peaking around three to six weeks following infection. Anti-HIV antibodies generally become detectable in the blood with third generation ELISA/EIAs as early as three to four weeks, while second generation tests usually detect positive results beginning around 4.5 to 5 weeks after infection. Of particular note is that Western Blot testing doesn't begin detecting positive results until around five weeks.

Head-to-head comparisons of newer techniques, such as NAAT/PCR, with standard WB testing reveal positive results are obtainable days to weeks earlier with NAAT/PCR. A particular problem with WB testing is indeterminate test results. Anti-p24 antibody is the most commonly detected antibody in the setting of false-positive ELISA/EIAs and indeterminate WB results. That rapid HIV testing techniques do not assay for p24 antibodies explains why rapid tests have a lower incidence of false-positive results than WB tests. Decreasing the rate of false-positive test results is always desirable and particularly important in clinical settings where it would lead to avoidance of unnecessary exposure to antiretroviral therapy (for instance in testing women in labor who have not had previous HIV testing).

The Centers for Disease Control and Prevention (CDC) workgroups are now evaluating new HIV-testing algorithms for both laboratory and point of care settings. Combining rapid tests with NAAT-based confirmatory testing is under consideration. I would predict ultimately Western Blot testing will be phased out completely. That's right folks; you heard it here first!

Some may wonder if detection of acute HIV infection is really essential to public health. Actually it is. As mentioned above, the acute phase of HIV infection is associated with high viral loads and enhanced infectiousness. In epidemiological studies up to 50% of new HIV transmission is attributed to those HIVers who are very recently infected. Following current guidelines for standard HIV testing would miss much of the acute-stage infection.

Acute-phase-HIV-infection diagnosis is difficult, because many recently infected HIVers may not have major ARS symptoms or they have symptoms that are missed or attributed to another ailment. It is estimated that 92% of those acquiring HIV will have some ARS symptoms, but the diagnosis of HIV based on symptoms is missed 80% of the time.

Further complicating HIV clinical diagnosis is that symptoms are notoriously unreliable in predicting who is and is not HIV infected, because most ARS symptoms are generic and seen in many common ailments, such as "flu."

Options for improving HIV-diagnostic testing during acute HIV infection include enhanced screening techniques, such as p24 antigen (fourth generation tests, already in widespread use) and pooled HIV NAAT testing (this is available in some locations through specific programs). Pooled NAAT testing simultaneously screens 100 samples divided into 10 "pools" of 10 samples each. Any pool detecting HIV RNA then individually samples each patient's blood in the pool. A positive NAAT/PCR is indicative of acute HIV infection in someone with a negative rapid test. This two-stage method in essence detects HIV virus in the blood before seroconversion (development of antibodies).

Clinical studies have convincingly demonstrated that pooled NAAT/RNA/PCR screening has the ability to detect a significant number of HIV-positive samples missed by routine HIV-antibody testing during early HIV infection. Similarly there are clinical studies demonstrating that the use of fourth generation ELISA/EIAs can detect HIV infection earlier than routine antibody-only screening. (An Australian study showed fourth generation tests identified 93.2% of infected HIVers compared to 63.2% in those who were simultaneously tested with third generation antibody-only ELISA/EIA tests.)

It is estimated that 20% of HIV-positive Americans have no idea they are infected with the virus. It is also estimated that those positively charged individuals who are unaware of their HIV status are responsible for 54% of new HIV infections. Knowing one's serostatus has been shown to be associated with increased adherence to safer sexual practices. The current concept of "test-and-treat" would pay significant dividends by dramatically reducing HIV incidence. This model would require widespread testing and immediate institution of antiretroviral therapy for those found to be "virally enhanced." Early diagnosis and treatment would in essence lower the "community viral load," thereby decreasing the risk for new infections.

It is now widely agreed that testing is a critical first step in our effort to reduce the rate of new HIV infections, which have remained remarkably stable at approximately 56,000 per year in the U.S over recent years. This strategy is reflected in the CDC's 2006 revised guidelines for HIV screening in all health care settings. Patients are screened unless they "opt out" and specifically decline HIV testing. Unfortunately many locales are not complying with these recommendations, due to a variety of reasons, including cost.

The science is now clear. We know how to significantly impact the new-infection rate. All that's required is improved implementation. In addition, we have new tools available (NAAT/PCR, p24 antigen, etc.) that should be incorporated into a new set of guidelines for HIV screening of acute HIV infection. The FDA has made the first step in this process by approving Aptima-HIV-1 for HIV-diagnostic testing. Watch for additional and hopefully speedier evolution of other HIV testing guidelines.

Gentle Readers, I realize this blog was rather complex and perhaps a bit dry. Sorry about that, but I felt it was important to discuss this topic and have been meaning to do so for quite some time. I can now take it off my "to do" list. The basic take-home messages regarding routine HIV-antibody testing currently in use and the newer NAAT/PCR/RNA tests are:

  1. ELISA/EIA and WB test for antibodies while NAAT/PCR/RNA assays for a piece of the actual HIV virus. The FDA has approved Aptima-HIV-1, a NAAT/PCR/RNA test, for HIV-1 diagnostic testing.
  2. The window period for ELISA/EIA remains three to six months while the window period for NAAT/PCR/RNA is approximately two weeks. Both ELISA/EIA and NAAT/PCR/RNA tests require confirmatory testing.
  3. HIV-diagnostic testing can be confusing to those who do not understand the complexities of HIV immunopathogenesis or the limitations of individual HIV-diagnostic tests. However, HIV-diagnostic testing really isn't rocket science. HIV-knowledgeable physicians understand the subtleties and limitations of HIV-diagnostic testing. Even with its limitations and complications, HIV-diagnostic testing is remarkably accurate when conducted and interpreted appropriately.
  4. Watch for evolution of the testing guidelines as our testing technologies and knowledge of HIV continue to improve. Stay tuned to We promise to keep you updated on updates!

Get in touch with the Robert James Frascino AIDS Foundation.