Sometime toward the end of the 1990s or early 2000s, it occurred to Berry Bennett, MPH, that the recommended HIV test practice that guided his public health laboratory was lagging behind available HIV test technology.
This was a period during which the Food and Drug Administration (FDA) had been approving third-generation HIV immunoassays that were more sensitive and could detect antibodies earlier than older assays. Yet the Centers for Disease Control and Prevention’s (CDC) HIV test algorithm and guidelines, first issued in 1989 and updated only once, in 1992, still required labs to perform confirmatory tests using Western blot or immunofluorescent antibody (IFA).
Both technologies had arrived within 10 years of French researchers unmasking HIV types 1 and 2 in the mid-1980s. The Western blot had grown more popular than IFA; however, both types of tests had their limitations. They were not as sensitive as newer immunoassays, were more vulnerable to subjective interpretations, and could not differentiate between an HIV-1 and HIV-2 infection.
Bennett, who heads the Retrovirology Unit of the Florida Bureau of Public Health Laboratories (FBPHL) in Jacksonville, noticed that more specimens were producing repeatedly reactive results on initial screening but negative or indeterminate results on the Western blot. A fair number of those specimens came from people with high-risk lifestyles, such as men having unprotected sex with men, or IV drug abusers.
“Most public health laboratories (PHLs) received minimum information on an individual’s clinical symptoms and behavioral risk factors when they received a blood specimen to test,” Bennett says. “However, PHLs experiencing unconfirmed HIV diagnostic results with that algorithm often became aware of clinical manifestations that caused them to suspect that this person potentially could be in an acute or early HIV infection stage.”
If a laboratory issued a negative or indeterminate report, it would be up to the public health providers, “the last gatekeepers,” as Bennett describes them, to “advise the person he or she should come back to get another blood draw in two to three weeks, knowing that it may have been too early [in his or her infection] for the algorithm to confirm the diagnosis.” The variability of individual immune responses also played a part, considering that the body starts producing antibodies between two and 12 weeks of infection. “So that was basically our only option to avoid false negative diagnostic results.”
The problem of missing early acute-stage HIV infections—and losing touch with patients—led the CDC to embark on a six-year quest to change its recommended HIV test protocol, one that does not use either Western blot or IFA as confirmatory tests. The changes, embodied by a new algorithm and set of guidelines published in June 2014, may save many lives over the next few decades, and be a significant advance both from the clinical and public health perspectives.
Three goals
S. Michele Owen, PhD, Acting Associate Director for Laboratory Diagnostics in the CDC’s Division of HIV/AIDS Prevention, says the process of overhauling the algorithm began in earnest in 2008-2009, with the publication of a status report by the Association of Public Health Laboratories. The report was based on the finding of a group that analyzed various algorithms that labs were using and identified the pros and cons of different schemes.
At the CDC’s HIV Diagnostics Conference of 2010, attendees heard data on various algorithm configurations, but it wasn’t until the Diagnostic Conference of 2012 that “the real guidelines writing process really started,” says Dr. Owen.1 Early on, the writing group identified three objectives: to diagnose people earlier in infection; to better and more accurately diagnose HIV-1 and HIV-2; and to get results back to people sooner.
“Diagnosing HIV earlier has public health benefits because very early in infection, people, particularly those who are antigen positive but not yet antibody positive, have more virus floating around and so have a greater ability to transmit to other people,” she says, pointing to a large study by M. Cohen et al.2 “Also, more and more evidence is suggesting that the sooner an individual starts therapy the better the preservation of their immune system. And so it probably increases life span.”
Referring to the second objective, she says that HIV-2 infections are very rare and usually involve people living in or connected to Africa, but they must be identified or risk being treated with ineffective medicine.
“Some of the reverse transcriptase inhibitors used to treat HIV-1 certainly are effective against HIV-2,” she says. “However, other classes of drugs, like the protease inhibitors, do not work for HIV-2.”
And getting results back on the same day instead of a week later is important because a significant number of people who come in for screening leave their specimen but fail to return to obtain results. At the 2010 conference, Bernard Branson, MD, then Associate Director of HIV Laboratory Diagnostics, said that at the time about 20 percent of those tested in the United States never obtained Western blot confirmatory results.
Evolution of HIV testing
There are two ways to detect HIV. One way is to look for the markers that the human immune response to the infection generates, usually antibodies. These can be detected by enzyme immunoassay (EIA), immunofluorescent (IFA), or Western blot. Another way is to detect the genetic material of the virus itself, which can be done with a polymerase chain reaction test (PCR) or a nucleic acid amplification test (NAT).
In 1985, Abbott Laboratories received FDA approval to market the first antibody-based screening assay, an HIV-1 EIA, and by the end of the decade the company also had licensed an HIV-1 antigen assay. The first molecular test was not approved until 1999, so for 14 years, antibody tests, and some antigen tests, basically made up the HIV test market. (Current HIV antibody tests can detect antibodies as early as three weeks after exposure, which is earlier than the first generation antibody tests. Second generation tests can detect IgG antibodies, third generation can detect both IgM and IgG antibodies, and fourth generation can detect both HIV antibodies and the p24 antigen.)
From the beginning, CDC test protocol called for an initial assay that, if repeatedly positive, was to be followed by a confirmatory test. In fact, two years before Cambridge Biotech licensed the first Western blot kit for HIV-1 in the U.S. in 1991, the CDC had published an algorithm recommending both a first-line assay and confirmatory Western blot or IFA.3
Screening assays typically use synthetic antigenic peptides and recombinants (antigens produced by recombinant DNA), while Western blot is made from the inactivated virus itself. As a result of its reliability and superior specificity, Western blot rose in popularity. However, the test was not easy to manufacture, and involved many steps and significant training to perform. It also took two to four days to produce results in the form of protein bands that were not always easy to interpret. More significantly, it sometimes confused the much rarer HIV-2 for HIV-1 and was vulnerable to missing early cases.
After Western blot came on the scene, the next six or seven years were an intense period of research and competition. By 1999, there were five major Western blot products on the market.
Antigen technology breakthrough
A new test using its own synthetic antigens, which could detect both HIV-1 and HIV-2 more quickly and more accurately, was developed by Bio-Rad. The test, whose design resembled an ICON pregnancy test, was marketed in Europe and eventually received FDA clearance in 2004 as the HIV-1/HIV-2 Multispot Rapid Test. It was at the 2010 HIV Diagnostics Conference in Orlando, FL, that the CDC’s Branson suggested to an audience that it consider using Multispot as a way to improve “follow-up” on higher-risk people infected with HIV. Branson said, “The people with the highest incidence are not coming back. They don’t know they’re infected. We need something better and faster.”
Bio-Rad applied to the FDA to change the package insert of the Multispot assay to include a second intended use as a supplemental test. Approval was granted in 2013.
With the addition of Multispot as a supplemental test, the CDC was able to publish its new algorithm. Now HIV testing begins with a combination assay that detects HIV-1 and HIV-2 antibodies and the HIV-1 p24 antigen. Specimens reactive on this initial assay undergo supplemental testing with the differentiation assay. Specimens reactive on the initial immunoassay and nonreactive or indeterminate on the antibody differentiation assay are subject to HIV-1 nucleic acid testing for resolution.
Brisk implementation
Dr. Owen says that the new algorithm is rapidly being implemented: Both Quest Diagnostics and LabCorp have adopted it, as have 50 to 75 percent of state public health labs. The earliest adopters, in 2012, were the public health labs in Massachusetts and Florida.
Not surprisingly, Bennett of FBPHL was part of the group that promulgated the new algorithm and supporting guidelines. Since adopting it, his lab has tested more than 300,000 blood specimens with the new algorithm.
Just in 2014, FBPHL tested 108,000 specimens, had about 2,100 positives (repeatedly reactive to the fourth-generation test), and found 18 “algorithm-defined acute HIV-1 infections,” or about 0.9 percent of the confirmed cases. In other words, they were repeatedly reactive to the initial fourth-generation immunoassay, non-reactive to the confirmatory (differentiation) test, and positive for HIV-1 RNA on the NAT. The lab’s 2014 data has not yet been published but similar data from 2012 was published in the Journal of Clinical Virology.4
“So if we can have an algorithm that detects established infections, which it does very well, as well as acute infections, then we can really concentrate on these people in the early stage,” he says. “We get them into care for their own benefit very early and prevent downstream infections, which may be transmitted without their knowledge.”
References
- Summaries of 2005, 2007, 2020 and 2012 HIV Diagnostic Conferences, and extensive data on the test algorithm development, can be found at http://www.hivtestingconference.org. Accessed February 25, 2015.
- Myron Cohen, MD, an HIV researcher at the University of North Carolina, Chapel Hill and lead investigator of HPTN 052, headed a study that proved treating HIV also prevents transmission of HIV. See Cohen MS, Chen YQ, Mccauley M, et al., “Prevention of HIV-1 infection with early antiretroviral therapy. NEJM. 2011; 365:493-505.
- Starr D. Blood: An Epic History of Medicine and Commerce. (2nd Ed., page 300). 2012. New York, N.Y.: Perennial/HarperCollins Publishers.
- Performance of the new HIV-1/2 diagnostic algorithm in Florida’s public health testing population: a review of the first five months of utilization. J Clin Virol. 2013. Dec;58 Suppl 1:e29-33.doi: 10.1016/j.jcv.2013.08.
