What’s the Best Testing Strategy for HIV Infection?
Proposed Algorithm Incorporates Latest Technologies, Testing Priorities
By Genna Rollins
Knowledge about all aspects of human immunodeficiency virus (HIV) epidemiology, diagnosis, treatment, and prevention has advanced significantly since scientists first isolated the virus in 1984. But in a field characterized by rapid breakthroughs, one thing has remained unchanged for more than 2 decades: the diagnostic testing algorithm recommended by the Centers for Disease Control and Prevention (CDC). Since 1989, the diagnostic standard for HIV in the U.S. has involved initial screening with an enzyme immunoassay (EIA), followed by validation of repeatedly reactive specimens using a more specific test such as Western blot or immunofluorescence assay. This strategy has served its purpose well, but now public health officials are poised to overhaul the current algorithm completely to reflect the latest diagnostic technologies and challenges.
“Western blot testing and the current algorithm were initiated at a time when all we had were antibody tests, so we were stuck trying to make a diagnosis from a single test,” explained Bernard Branson, MD, associate director of laboratory diagnostics in the division of HIV AIDS prevention in CDC’s National Center for Viral Hepatitis, HIV, STD and TB Prevention. “Now, we have these other alternatives that weren’t available in the past, and labs are using them. So people recognize that the Western blot is a bit tarnished for several reasons.” Branson presented a proposed algorithm at the 2010 CDC-American Public Health Laboratories (APHL) HIV Diagnostics Conference, which took place last March. The algorithm came from ongoing collaboration between the two organizations through the APHL/CDC HIV Steering Committee.
Over the years, laboratorians and clinicians have watched the Western blot assay slowly being left in the dust by new technologies and an increasingly nuanced understanding of HIV, making now an ideal time to introduce a new algorithm, according to Eugene Martin, PhD, professor of pathology and laboratory medicine at the UMDNJ-Robert W. Johnson Medical School in New Brunswick, N.J. “The proposed algorithm is setting the stage for a major shift that’s absolutely appropriate and in some respects is late in coming,” he said. “It gives us a tremendous opportunity to shorten the cycle time to find out whether a person is infected, and if so, to do something about it.”
In the past 2 decades, HIV diagnostics have advanced from first-generation EIAs, which detected antibody bound to solid-phase viral lysate, to third-generation EIAs, which detect antibody using an antigen-antibody-antigen sandwich method. Earlier this year, the Food and Drug Administration (FDA) approved the first fourth-generation test for use in the U.S., Abbott’s HIV Ag/Ab Combo assay, which detects both HIV-1 and HIV-2 antibodies and the HIV p24 antigen. Bio-Rad has submitted a premarket approval application for a similar assay. Ortho Clinical Diagnostics also is developing a fourth generation assay and plans to submit for FDA approval, according to a company spokesperson.
While the FDA requires sensitivity and specificity of HIV EIAs to exceed 98%, different antigen targets and assay formats have made it possible to detect a broader range of HIV subtypes, groups, and antibody isotypes from saliva, whole blood, serum, or plasma specimens. The introduction of rapid HIV tests, first approved by the FDA in 2002, has sped-up turnaround time for screening results and enhanced the role of point-of-care contact in HIV testing and counseling. Diagnostic manufacturers also have developed nucleic acid amplification tests (NAAT), making it possible to identify an HIV infection before seroconversion. While all these advances are significant in their own right, the bottom line is that a positive HIV diagnosis is now possible up to 26 days prior to positive results by Western blot, the recommended confirmation test (J Clin Microbiol 2008;46:1588–95). This has real implications for public health officials focused on stopping the spread of HIV infection and getting newly infected patients into therapy as soon as possible.
“There’s been a growing body of literature recognizing the importance of that window period between the time of infection and the time the infection is detected,” said Peter Leone, MD, professor of medicine and medical director of the North Carolina HIV/STD prevention and control branch at the University of North Carolina in Chapel Hill. “It’s important at the individual level in that we’re missing people very early on when there’s a chance to impact long-term their infection by starting them on therapy and linking them to care. And it’s important from the public health perspective to decrease transmissions within networks.” Leone and his colleagues have published on their experience in detecting acute HIV infection using NAAT (N Engl J Med 2005;352:1873–83).
Finding the Right Testing Strategy
CDC’s 2006 guidelines recommending routine, voluntary HIV screening for all individuals age 13–64 in healthcare settings expanded the scope of testing, but providers still have not made huge inroads with a critical sub-group. CDC data indicate that about 21% of the population with HIV is unaware of it. Yet these individuals are responsible for more than half of new HIV infections, reflecting the fact that acute infection is more communicable than long-standing illness. “As we’ve learned more about HIV, acute HIV, and expanded testing, particularly to high-risk groups, the importance of that acute period has actually increased. But we’re looking at a widening gap of people either being missed by antibody testing or having discrepant results, yet they’re infected,” Leone added.
Although there’s considerable focus on identifying acute infection, public health officials also place priority on catching more people earlier in the course of infection who are past the acute infection stage. A sizable minority of patients continue to be diagnosed late in the disease process, about 1 year before developing AIDS. To the extent that the proposed algorithm would help identify them sooner, for example, by getting definitive results more quickly for anyone who is tested, it would be welcomed, according to Branson.
APHL and CDC have proposed several alternative algorithms over the years, but none quite hit the mark in terms of testing strategy priorities, so they were never advanced to the point of formal endorsement. For instance, two preliminary algorithms called for either HIV-1 EIA only or HIV-1 and HIV-2 EIAs followed by NAAT for repeatedly reactive results. “There are two difficulties with that. One is, somewhere between three and seven percent of cases confirmed as antibody-positive tested negative for RNA, so RNA alone was not sufficient to confirm HIV infection,” Branson explained. “Secondly, if you were to immediately perform an RNA test, you wouldn’t be able to tell if the person had acute or long-standing HIV infection, because both of those people would be RNA positive.”
In recent years, public health officials also have become concerned about identifying HIV-2 infection, which is growing in prevalence in the U.S. and has a different treatment approach than HIV-1. “There’s a misperception that HIV-2 is not a problem in certain areas of the country,” noted Barbara Werner, PhD, an infectious diseases consultant to the Massachusetts Department of Public Health. “It certainly is less of an issue outside some of the eastern cities like New York that are ports of entry from around the world. However, people from HIV-2 endemic areas are settling in other parts of the country, and if you don’t look, you’re not going to find it.” Werner represented APHL during an AACC webcast titled “An Update on HIV Testing: New Tools and Optimized Algorithms,” which was held on September 16.
A Tarnished Gold Standard
Aside from being less sensitive than many screening assays, Western blot has other liabilities, not the least of which is turnaround time. “An individual can be found to be preliminarily positive with a rapid HIV test, but the current algorithm requires the specimen to be confirmed with Western blot, which is a fairly complex test that can’t be performed in some locations where rapid HIV tests are performed,” explained Martin. “The Western blot results have to be analyzed, returned to the sample collection site, and then you have the issue of getting back to that patient. We’ve discovered in New Jersey, as many as 25 percent of patients don’t come back to find out their results, so it’s oftentimes a process that gets blunted on the way to a diagnosis.”
The Western blot assay also presents technical challenges that most clinical chemists would be happy to forego, according to Mark Pandori, PhD, chief microbiologist for the San Francisco Department of Public Health Laboratory. “From a laboratorian’s point-of-view, Western blot is somewhat problematic. It’s slow and somewhat time-intensive to run, and many samples will have spurious bands, making it often difficult to interpret,” he said.
Given these shortcomings, many, including Leone, have questioned the Western blot’s status as a gold standard. “I think it’s an impediment to making an accurate diagnosis during the critical six-week period of acute infection. As our screening tools have improved, we need more sensitive confirmatory testing too,” he said.
Many in the HIV field apparently share Leone’s assessment. In fact, the latest APHL/CDC proposed algorithm—the one it is taking through the validation process towards eventual adoption—omits Western blot altogether. Experts, however, are quick to point out that the assay still provides relevant clinical information. “Western blot is fine as long as you’re confirming long-standing infection, because those people have developed a full antibody response and the Western blot will be positive,” explained Werner. “It’s also useful for diagnostic dilemmas and for testing vaccine recipients, because you can see the pattern of response in people who are not infected but have the antibody because they’ve been vaccinated.” The assay also continues to have a role in monitoring patients with end-stage AIDS, according to Pandori.
Proposed HIV Testing Algorithm*
*Proposed in March 2010. CDC is seeking validation data and stakeholder feedback before it formally endorses the Algorithm.
**Nucleic Acid Amplification Test
Source: 2010 CDC APHL HIV Diagnostics Conference
Cutting Time, Indeterminate Results
The 2010 proposed algorithm calls for initial screening with a fourth-generation immunoassay (See Figure, above). Positive results would be followed by an HIV-1/HIV-2 differentiation immunoassay. Positive results from this step would prompt the start of HIV treatment, whereas a negative result would lead to an additional step of NAAT testing.
This strategy appears to meet all the priorities for an HIV diagnostic algorithm identified by the APHL-CDC HIV Steering Committee. These criteria include detecting HIV infection through a range from acute to long-standing illness, differentiating HIV-1 from HIV-2, providing timely results, and eliminating indeterminate and inconclusive results whenever possible. “The proposed algorithm is being called ‘ideal’ because it gets at all those characteristics,” explained Werner. “There’s a very small percentage of specimens that you’d have to go to RNA testing to get a result on. You could do a fourth-generation assay, then an HIV-1 HIV-2 differentiation assay and get results pretty quickly on the vast majority of people you were testing. You’d also know whether or not you were dealing with acute infection with this algorithm.”
How many labs will implement the new algorithm after it is published remains to be seen. During the AACC webinar, participants were asked, “If this HIV algorithm is recommended for use, will your laboratory implement it within 12 months?” While 40% indicated they would, 17% said they would not, and another 42% reported being uncertain or said they might. Not all labs will feel obligated to invest in a fourth-generation assay, at least initially, according to Werner. “Some labs already are using the traditional algorithm with a supplemental NAAT option for Western blot indeterminate or inconclusive results, and I’m sure some will want to continue doing that for a while,” she said. Pandori suggested that the proposed algorithm likely will have the proviso that labs implement the best available assay, and some will be able to achieve this standard without necessarily implementing a fourth-generation assay. “If you’re not in a high-prevalence area, there’s probably not going to be much difference between running a third- and fourth-generation test. There may not be a lot of acute infection out there for you to detect in your community,” he explained.
Branson emphasized that before CDC recommends the new algorithm, the agency plans to collect ample validation data from a variety of users in multiple different settings and he encouraged labs to submit data to him. “The so-called gold standard algorithm was developed in the 1980s based on a lot of data that were submitted by labs, and that’s why I see the process for the new algorithm being similar,” he said. “When different labs in different parts of the country come together in order to assure that it works in many different circumstances that we encounter, then we’ll have the evidence needed to support such a change.” Branson hesitated to predict when the new algorithm will be issued, given that CDC not only will collect validation data but also feedback from stakeholders before making any formal recommendations.
He cautioned, however, that even though the proposed algorithm will tighten the diagnostic timeline considerably and address key testing priorities, it will not be a total solution in every clinical situation. “For the acute HIV infection period, it may be too early for any test to detect HIV,” he observed. “That’s not just a limitation of Western blot but also of any other technologies. In many of those situations, the only solution is the test of time.”
For Further Information
- Copies of AACC’s Webinar titled “An Update on HIV Testing: New Tools and Optimized Algorithms,” which took place on September 16, are available through AACC Customer Service at www.aacc.org or (800) 892-1400 or (202) 857-0717, Select Option 2.
- Copies of the April 2009 status report of HIV Testing Algorithms are online at the American Public Health Laboratories website.
- Copies of abstracts and presentations from the 2010 CDC-American Public Health Laboratories HIV Diagnostics Conference are available online.
What Clinical Labs Can Do
Before the new algorithm goes into effect, considerable education will be required about both the limitations of Western blot and advantages of the new strategy. Several experts, including Martin, cited the loyalty of clinicians in continuing to rely on the Western blot even after the introduction of more sensitive tests. “We’ve had problems with providers being unwilling to see a new patient until they’ve had Western blot. So we’ve experienced the realization that there’s an educational process that needs to go on,” he said.
Leone concurred that provider education will need to go hand-in-hand with implementation of the new algorithm. “Even when HIV testing is thought of and testing obtained, the problem is, doctors are still not certain which test to order. They’re also reluctant to order RNA testing because they don’t view it as a diagnostic test,” he explained. Use of the proposed algorithm and a fourth-generation assay may make providers more comfortable with ordering an HIV test and more confident in making a diagnosis, he added.
Leone also encouraged labs on the fence about implementing a fourth-generation assay and/or the proposed algorithm, to consider the cost of not detecting HIV infection early on. “They need to think beyond the lab and look at institutional costs,” he said. “Missing HIV costs the system money because these folks come in later with advanced HIV or transmit to other people.”
Although APHL has been working closely with CDC to update proposed testing algorithms, Pandori emphasized the need for clinical labs to keep up with the latest technologies and testing strategies. “In a sense, these recommendations are more important at the clinical lab level than at the public health level. Clinical labs are really important in terms of cutting those transmission networks because more people are getting missed in clinical settings than in public health,” he observed. For that reason, he urged lab directors to implement the proposed algorithm. “Hesitancy to change will do harm in this case. It’s not like just moving from culture to PCR for influenza. The ability to detect recent HIV infection is essential in doing anything about stopping transmission of the disease.”
Two Decades of HIV Testing
Here’s a look back at major HIV testing advances during the past two decades.
The first combination test to detect HIV-1 and HIV-2 antibodies was licensed.
FDA licensed SUDS HIV-1, a ten minute diagnostic test kit which can be used by health professionals to detect the presence of HIV-1.
FDA published an interim rule establishing a requirement for certain infectious disease testing, donor screening, and record keeping to help prevent the transmission of HIV and hepatitis B and C through human tissue used in transplantation.
FDA approved the first non blood-based collection kit utilizing oral fluid for use in the detection of the antibody to HIV-1.
FDA cleared for marketing the first blood test to measure latex antibodies in the blood. The test can be used to help identify people who are allergic to latex and as such assist people in the use of barrier products to prevent HIV transmission.
FDA recommended that blood establishments should implement donor screening for HIV-1 antigen using licensed test kits.
FDA approved the first antigen test kit, Coulter HIV-1 p24 Antigen Assay, to screen blood donors for HIV-1. The agency also approved the first HIV test system that can be used at home and can be purchased over-the-counter (OTC). The Confide HIV Testing System (Direct Access Diagnostics), is comprised of three integrated components: an OTC home blood collection kit, HIV-antibody testing at a certified lab, and a test result center that provides test results, counseling and referral anonymously.
FDA approved Amplicor HIV-1 Monitor Test, an in vitro nucleic acid amplification test for the quantitation of HIV-1 RNA in human plasma (viral load).
FDA approved the HIV-1 western blot confirmatory test for OraSure’s oral collection system. The oral collection system was originally approved in 1994 using an enzyme-linked immunosorbent assay (ELISA) method.
FDA approved the first HIV test which uses urine samples. The urine-based test detects the presence of antibodies to HIV-1 using an enzyme-linked immunosorbent assay (ELISA) method.
FDA approved Cambridge Biotech HIV-1, a Human Immunodeficiency virus Type 1 (Western Blot) test with a new indication for urine specimen testing.
FDA approved a supplement to AMPLICOR HIV-1 MONITOR Test (Roche Molecular Systems). This supplemental approval extends the lower limit of quantitation from 400 HIV RNA copies/mL down to 50 copies/mL; the upper limit of quantitation, 750,000 HIV RNA copies/mL remains the same.
FDA approved TrueGene HIV-1 Genotyping Kit and Open Gene DNA
Sequencing System (Visible Genetics) to be used to identify drug resistance in HIV patients.
FDA approved NucliSens HIV-1 QT (bioMerieux), for use in prognostic assessment of HIV-1 infected patients, and for monitoring the effects of anti-retroviral therapy.
FDA approved the VERSANT HIV-1 RNA 3.0 Assay (Bayer) for direct quantitation of HIV-1 in plasma of HIV-infected individuals, as an aid in management of individuals infected with HIV-1.
FDA approved the first rapid HIV diagnostic test kit for use in the U.S, the OraQuick Rapid HIV-1 Antibody Test (OraSure Technologies) can quickly and reliably detect antibodies to HIV-1.
Under a CLIA waiver the Department of Health and Human Services (HHS) expanded availability of the rapid HIV test from the current 38,000 laboratories to more than 100,000 sites, including physician offices and HIV counseling centers.
FDA approved the OraQuick test (OraSure Technologies) in November as a moderate complexity test.
FDA approved the OraSure rapid test (OraSure Technologies) for detection of HIV-2 when used with a whole blood sample. The agency also approved the use of oral fluid samples with a rapid HIV diagnostic test kit and the OraQuick Rapid HIV-1 Antibody Test (OraSure Technologies) for testing plasma for HIV-1 and HIV-2.
FDA granted a CLIA waiver to the OraQuick ADVANCE Rapid HIV-1/2 Antibody Test (OraSure Technologies) for use with oral fluid.
The agency also issued final guidance on the “Use of Nucleic Acid Tests on Pooled and Individual Samples from Donors of Whole Blood and Blood Components (including Source Plasma and Source Leukocytes) to Adequately and Appropriately Reduce the Risk of Transmission of HIV-1 and HCV”, as well as draft guidance for industry entitled “Role of HIV Drug Resistance Testing in antiretroviral Drug Development”.
FDA approved the Multispot HIV-1/HIV-2 Rapid Test (Bio-Rad Laboratories) for the detection and differentiation of circulating antibodies associated with HIV-1 and HIV-2 in human plasma and serum, as an aid in the diagnosis of infection with HIV-1 and/or HIV-2.
FDA granted marketing approval for the Abbott RealTime HIV-1 Assay (Abbott Molecular), an in vitro reverse transcription-polymerase chain reaction (PCR) assay for the quantitation of HIV-1 in human plasma from HIV-1 infected individuals over the range of 40 to 10,000,000 copies/mL, for use in managing the treatment of HIV infection. The agency also granted marketing approval for the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (Roche Diagnostics), an automated PCR test, for quantitation of HIV-1 nucleic acid in human plasma to aid in clinical management of HIV-1 infected patients.
FDA also approved the Procleix Ultrio Assay (Gen-Probe—marketed by Chiron), a fully automated qualitative in vitro nucleic acid test to screen for HIV-1 and hepatitis C virus RNA in donated blood from donors of whole blood, blood components, or source plasma.
FDA approved the VITROS Anti-HIV 1+2 assay (Ortho Clinical Diagnostics) for diagnosis and donor screening for antibodies to HIV-1 and HIV-2 in human serum and plasma.
The agency also approved the cobas TaqScreen MPX Test (Roche), the first nucleic acid test to detect the presence of HIV-2, HIV-1 Group O, HIV-1 Group M, and Hepatitis C and B virus in donated blood plasma and human tissue.
FDA approves for use in the U.S. the first fourth-generation HIV test, the ARCHITECT HIV Ag/Ab Combo Assay (Abbott), which detects HIV-1 Group M and Group ?), HIV-2 and HIV p24 antigen in serum and plasma.