Clinicians and laboratorians have praised CDC’s proposed alternative HIV testing algorithm, which calls for initial screening with a fourth-generation immunoassay. However, many labs have not yet adopted fourth-generation tests. In this issue of Strategies, researchers explain how a recent study demonstrates the benefit of the new algorithm as the lab community navigates a transition to the latest assays.

The current Centers for Disease Control and Prevention (CDC)-recommended testing algorithm, in place since 1989, calls for screening with an enzyme immunoassay (EIA), followed by validation of repeatedly reactive specimens using Western blot. However, a major weakness in this strategy is that Western blot does not detect infections in the window period of about 40 days when measurable levels of antibodies are not yet present.

CDC proposed a new algorithm to improve detection of acute infections in 2010. This alternative algorithm calls for screening with a fourth-generation immunoassay that detects p24 antigen, which increases between infection and seroconversion. Positives are followed by an HIV-1/HIV-2 differentiation immunoassay, while a nucleic acid test (NAT) is used to resolve the status of an initial positive immunoassay result followed by a negative differentiation test.

While many clinicians applauded CDC’s proposal, labs were not ready to implement the alternative algorithm right away. The Food and Drug Administration (FDA) only approved the first fourth-generation test in 2010, Abbott’s ARCHITECT HIV Ag/Ab Combo Assay.

Recently, CDC’s Bernard Branson, MD, a leader in developing the alternative CDC algorithm, employed the second FDA-approved fourth-generation HIV assay, Bio-Rad’s GS HIV Combo Ag/Ab enzyme immunoassay, and compared the results of the current and alternative testing algorithms (AIDS 2013;27:731-737). The study found that the alternative algorithm clearly improved early infection sensitivity and identified HIV-2 infections.

“Most of the previous validations we had performed with the new algorithm were with Abbott’s chemiluminescent microparticle assay. Of course, we want the new algorithm to be generic, so it was important to show similarly good performance with the second FDA-approved assay,” Branson said. “This study demonstrates that the algorithm will do what we’d hoped—pick up infections earlier. People with acute infections are at the most infectious stage, and if they are misclassified as negative or indeterminate by Western blot, I think it could delay their diagnosis.” Branson is 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.

To examine the performance of the new algorithm in a low HIV prevalence population, Branson, along with researchers from CDC and Quest Diagnostics, obtained 10,014 specimens from life insurance applicants. They also obtained 493 known Western blot-positive, 20 Western blot-indeterminate, and 230 specimens from HIV-1 seroconverters. The researchers tested the samples with both the Bio-Rad GS third-generation and fourth- generation assays, as well as an HIV-1/HIV-2 differentiation immunoassay, NAT, and Western blot. With these results in hand, the researchers compared the current and new HIV testing algorithms.

Among the low-risk insurance specimens, 13, or 0.13%, were immunoassay repeatedly reactive. Two of these were HIV-positive, confirmed by Western blot and the HIV-1/HIV-2 differentiation assay. Both algorithms led to the same result: HIV-positive. However, two repeatedly reactive third-generation specimens and nine repeatedly reactive fourth-generation specimens were false-positive. From the second set of specimens—493 known Western blot positives—all were repeatedly reactive by both third- and fourth-generation immunoassays. However, the alternative algorithm, using an HIV-1/HIV-2 differentiation assay as the second step, detected two HIV-2 positive specimens that would have gone unnoticed had testing stopped with a Western blot.

Significantly, the study found that the alternative algorithm correctly classified as positive 102 seroconverter specimens with the third-generation immunoassay and 130 with the fourth-generation immunoassay. This compared to only 56 using the Western blot under the current algorithm beginning with either generation of immunoassay.

The study reinforces the benefit of the new algorithm, Branson emphasized. “Especially with new evidence now that initiating treatment during the acute phase has clinical benefit, I think that this study really demonstrates how the new algorithm is superior to what we’re currently using,” he said.

According to Eugene Martin, PhD, a professor of pathology and laboratory medicine at the Robert Wood Johnson Medical School in New Brunswick, N.J., earlier detection using fourth-generation assays will be a major step forward in public health. “These assays allow labs to identify people who are at the highest risk of infecting others,” he said. “Fourth generation HIV tests will allow clinical focus to shift to earlier and earlier stages of the epidemic and represents a paradigm shift from a focus on simply identifying those infected, to identifying those are infected and moving into rapid therapeutic intervention to quickly reduce the HIV viral load.” Martin was not associated with the CDC study.

Laboratories will play a crucial role, not only in implementing new tests and a new algorithm, but in educating physicians and public, according to Martin. In all likelihood all labs will not adopt fourth-generation tests at the same time. “This could create some complications and confusion,” Martin said. “Labs don’t just automatically remove an older test from their menu when a new, more sensitive and specific test comes onto the market. We will be dealing with different labs using different generations of immunoassays at the same time as we all evolve toward the new algorithm.”

Not everyone will understand the difference between the new and old tests, either. “We’ve already had situations in NJ where individuals have had fourth-generation testing and then wanted to confirm the result by going to a public health facility that was running a rapid test, a less sensitive, earlier generation test,” Martin said. “So you have a discordance there that has to be carefully explained to the patient, and can be very confusing. Sometimes the infectious disease physicians haven’t even known when a fourth-generation test was moved into use at their facility. It’s really crucial that we talk to one another and make sure everyone is on the same page.”

In addition to the two FDA-approved fourth-generation immunoassays, other assays and instruments in the pipeline are expected to advance the field of HIV testing soon, Branson noted. In March, the Bio-Rad Multispot HIV-1/HIV-2 differentiation assay received a new indication from FDA for use in the new CDC algorithm as the second test in the algorithm. Until now, labs have had to perform their own verification of the assay to use it as such. In addition, Ortho-Clinical Diagnostics and Siemens are reportedly working on fourth-generation immunoassays.

CDC and FDA are also encouraging manufacturers to pursue diagnostic indications for quantitative viral load assays. Currently, the Gen-Probe Aptima HIV-1 RNA assay is the only FDA approved NAT for diagnostic use. In the meantime, CDC and the Association of Public Health Laboratories have set up a referral system so that labs not performing the Aptima assay can send specimens to one of two reference labs with a turnaround time of less than 3 days. Other assays submitted for FDA approval or under development include a fourth-generation rapid point-of-care test and a new HIV-1/HIV-2 differentiation assay.

As new assays become available, CDC will move forward with evaluating them in the context of the new algorithm. “I think the new assays have the potential to change the landscape in terms of detecting early infection, as well as dramatically reducing turnaround time,” Branson said. “I think we will see a transition where, instead of the Western blot, the only sendout test will be the NAT, and labs can perform the fourth-generation immunoassays and the rapid HIV-1/HIV-2 differentiation assays with dramatically reduced turnaround time compared to Western blot.”