Abstract
Background:
The capacity of HIV Antigen/Antibody (Ag/Ab) immunoassays (IA) to detect HIV-1 p24 antigen has resulted in improved detection of HIV-1 infections in comparison to Ab-only screening assays. Since its introduction in the US, studies have shown that the Determine HIV-1/2 Ag/Ab Combo assay (Determine Ag/Ab) detects HIV infection earlier than laboratory-based IgM/IgG-sensitive IAs, but its sensitivity for HIV-1 p24 Ag detection is reduced compared to laboratory-based Ag/Ab assays. However, further evaluation is needed to assess its capacity to detect acute HIV-1 infection.
Objective:
To assess the performance of Determine Ag/Ab in serum from acute HIV-1 infections. Study design: Select serum specimens that screened reactive on a laboratory-based Ag/Ab IA or IgM/IgG Ab-only IA, with a negative or indeterminate supplemental antibody test and detectable HIV-1 RNA were retrospectively tested with Determine Ag/Ab. Results were compared with those of the primary screening immunoassay to evaluate concordance within this set of algorithm-defined acute infections.
Results:
Of 159 algorithm-defined acute HIV-1 specimens, Determine Ag/Ab was reactive for 105 resulting in 66.0% concordance. Of 125 that were initially detected by a laboratory-based Ag/Ab IA, 81 (64.8%) were reactive by Determine Ag/Ab. A total of 34 acute specimens were initially detected by a laboratory-based IgM/ IgG Ab-only IA and 24 (70.6%) of those were reactive by Determine Ag/Ab.
Conclusions:
Due to their enhanced sensitivity, laboratory-based Ag/Ab IAs continue to be preferred over the Determine Ag/Ab as the screening method used by laboratories conducting HIV diagnostic testing on serum and plasma specimens.
Keywords: HIV, acute infection, Determine, algorithm, performance
Background
The Centers for Disease Control and Prevention (CDC) published updated guidelines in 2014 for laboratory diagnosis of HIV in the United States, which recommend use of an HIV-1/2 antigen/antibody (Ag/Ab) combination immunoassay (IA) as the primary screening test in the algorithm [1]. The capacity of Ag/Ab IAs to detect HIV-1 p24 antigen has resulted in a reduction of the diagnostic window period and improved detection of HIV-1 infections in comparison to Ab-only screening assays [2–7]. Identifying individuals with acute HIV-1 infection allows for early treatment, which can reduce transmission, improves health outcomes for infected individuals, and contributes to earlier partner services [8,9]. Of the seven HIV Ag/Ab assays approved by the Food and Drug Administration (FDA) for use in the U.S., six are restricted to laboratory use (laboratory-based). The Determine HIV-1/2 Ag/Ab Combo assay (Determine Ag/Ab) is a single-use rapid test and is CLIA waived for fingerstick whole blood allowing its use in non-laboratory settings. Determine Ag/Ab is also approved for laboratory use with serum, plasma and venous whole blood. Determine Ag/Ab differentiates HIV-1/2 Ab from HIV-1 p24 Ag reactivity. Seroconversion panel studies have shown that the FDA-approved Determine Ag/Ab detects HIV infection in plasma earlier than laboratory-based IgM/IgG sensitive IAs, but its sensitivity is reduced compared to laboratory-based Ag/Ab IAs for both plasma and simulated whole blood [10–12]. However, further evaluation is needed to assess the capacity of Determine Ag/Ab to detect acute HIV-1 infection in serum.
Objective
To assess performance of Determine Ag/Ab in serum specimens identified as acute infections.
Study design
The Association of Public Health Laboratories (APHL) in conjunction with CDC initiated the HIV Nucleic Acid Test Demonstration Project (NAT Demo) in August 2012 to provide access to HIV-1 RNA nucleic acid testing (NAT). The NAT Demo allows PHLs that have specimens with discordant HIV screening and supplemental antibody test results and lack access to HIV-1 RNA testing in-house to resolve the discordance using the recommended algorithm [13]. Specimens submitted to the NAT Demo reference laboratories must be 1) from a US PHL, 2) repeatedly reactive on an FDA-approved laboratory-based Ag/ Ab IA or IgM/IgG-sensitive Ab-only screening IA, and 3) negative or indeterminate on a FDA-approved supplemental Ab assay. Diagnostic testing was conducted using one of the following laboratory-based screening assays: Architect HIV Ag/Ab Combo (Abbott Diagnostics, Chicago, IL), GS HIV Combo Ag/Ab EIA (Bio-Rad Laboratories, Hercules, CA), GS HIV-1/HIV-2 Plus O EIA (Bio-Rad Laboratories), Advia Centaur HIV 1/O/2 Enhanced (Siemens Healthcare Diagnostics, Tarrytown, NY) or VITROS Anti-HIV 1 + 2 assay (Ortho-Clinical Diagnostics, Raritan, NJ). Reactive specimens were tested using one of the following supplemental Ab tests: Multispot HIV-1/HIV-2 Rapid Test (Bio-Rad Laboratories), Geenius HIV-1/2 Supplemental Assay (Bio-Rad Laboratories) or GS HIV-1 Western Blot (Bio-Rad Laboratories). All NAT Demo referral specimens that met the manufacturer’s collection and storage requirements were tested with the APTIMA HIV-1 RNA Qualitative Assay (Hologic, San Diego, CA) at either the New York State Department of Health’s Wadsworth Center Laboratory (WCL) or the Florida Bureau of Public Health Laboratories (FBPHL). Specimens that screened reactive, had a negative or indeterminate supplemental antibody test and detectable HIV-1 RNA were classified as laboratory algorithm-defined acute HIV-1 infections. Data were not collected regarding whether persons previously knew they were HIV-infected or had received antiretroviral treatment. Sample eligibility criteria were the following: at least 60 μl available, three or fewer freeze-thaws and classified as laboratory algorithm-defined acute HIV-1 based on NAT Demo data. A total of 159 serum specimens submitted between January 2013 and December 2016 met these criteria and were included. Specimens were deidentified and tested in singlet with the FDA-approved Alere Determine HIV-1/2 Ag/ Ab Combo test (Alere Inc., Waltham, MA) according to the manufacturer’s instructions. Results of Determine Ag/Ab and the originating laboratory’s primary screening immunoassay that identified these laboratory algorithm-defined acute infections were evaluated for concordance. The project was approved as research not involving identifiable human subjects by the Division of HIV/AIDS Prevention at CDC.
Results
Of 159 acute HIV-1 specimens, Determine Ag/Ab was reactive for 105 resulting in 66.0% concordance (Table 1). Determine Ag/Ab reactivity based on HIV-1 antibody detection (Table 1, Ag+/Ab+ and Ag−/Ab+) was 54.7% (87/159) and an additional 11.3% (18/159) were reactive for p24 Ag only.
Table 1.
Determine Ag/Ab results for serum specimens classified as acute HIV-1 infections.
| Lab screening assay Acute specimens N | Determine Ag/Ab Results N (%) | |||||
|---|---|---|---|---|---|---|
| Reactive | Analyte-specific reactive results |
NR | ||||
| Ag+/Ab− | Ag+/Ab+ | Ag−/Ab+ | ||||
|
| ||||||
| Ag/Ab IA | 125 | 81 (64.8) | 12 (9.6) | 5 (4.0) | 64 (51.2) | 44 (35.2) |
| Ab-only IA | 34 | 24 (70.6) | 6 (17.6) | 2 (5.9) | 16 (47.1) | 10 (29.4) |
| Total | 159 | 105 (66.0) | 18 (11.3) | 7 (4.4) | 80 (50.3) | 54 (34.0) |
Ag, HIV-1 p24 antigen; Ab, HIV-1/2 antibody; +, Reactive; −, Nonreactive (NR).
Of 125 acute specimens initially detected with a laboratory-based Ag/Ab assay, 81 (64.8%) were reactive by Determine Ag/Ab and 44 (35.2%) were nonreactive. A total of 34 acute specimens were initially detected by a laboratory-based IgM/IgG Ab-only IA and 24 (70.6%) of those were reactive by Determine Ag/Ab, with 18 of 24 indicating antibody reactivity (Ag+/Ab+ plus Ag−/Ab+) and 6 indicating HIV-1 p24 antigen detection only. Ten of 34 (29.4%) specimens initially detected by an Ab-only IA were not detected by Determine Ag/Ab (Table 1).
Discussion
In this study, performance of Determine Ag/Ab was assessed retrospectively on serum specimens from individuals with laboratory algorithm-defined acute HIV-1 infection. Determine Ag/Ab failed to detect HIV in 35.2% (44/125) of HIV-1 positive specimens that screened reactive with a laboratory-based Ag/Ab assay. Previously published data on plasma indicate that the median time to reactivity for Determine Ag/Ab is 2.3–5.5 days earlier than for laboratory-based HIV IgM/IgG Ab-only IAs [12]. However, data from our study indicate that about 30% of acute HIV-1 infections that were detected by a laboratory-based IgM/IgG Ab-only IA would not have been detected if the serum had been screened using Determine Ag/Ab. Of the 24 Determine Ag/ Ab-reactive specimens initially detected by an Ab-only IA, 6 were nonreactive for antibodies, but had detectable p24 antigen by Determine Ag/Ab. For these 6 specimens with discordant antibody results, it is possible that aspects of Determine Ag/Ab’s design which differ from instrument-based, antibody-only IAs (e.g. lateral flow format, limited IgM detection, lower specimen testing volume and different antigenic components) could have contributed to this discordance. The presence of multiple envelope, and in some cases p24 core, epitopes in the IgM/ IgG IAs may have allowed antibodies to be detected earlier during seroconversion than the single gp41 protein in Determine Ag/Ab. These data suggest that there may be a window within the seroconversion period in which Ab-only IAs can detect HIV-1 antibodies that are not reliably detected by Determine Ag/Ab.
Although this study provides data on the ability of Determine Ag/Ab to detect HIV-1 in serum collected from individuals with laboratory algorithm-defined acute HIV-1 infection, we do not have data to evaluate concordance between Determine Ag/Ab and laboratory-based IAs in specimens that screened nonreactive with a laboratory-based IA. Determine Ag/Ab may have detected acute HIV-1 infections that were missed by laboratory-based screening IAs, especially when laboratory-based IgM/IgG Ab-only screening IAs were used [10,12]. Our data indicate that the specimen storage conditions were appropriate for maintaining antigen detection, however we cannot exclude the possibility that p24 Ag may have degraded in some specimens during the period after initial screening. Despite the ability of Determine Ag/ Ab to distinguish between p24 Ag and HIV-1/2 Ab reactivity, data from this study indicate that some acute HIV-1 infections will be missed using Determine Ag/Ab. Due to their enhanced sensitivity, laboratory-based Ag/Ab IAs continue to be the preferred screening method for laboratories conducting HIV diagnostic testing on serum and plasma. However, some laboratories may choose to use the Determine Ag/Ab rapid test rather than a laboratory-based Ag/Ab IA for practical reasons including low testing volume, need for quick turnaround and instrumentation costs [14]. Therefore, when reporting negative Determine Ag/Ab results, laboratories should consider informing providers of the assay’s reduced sensitivity relative to other FDA-approved HIV Ag/Ab assays and the potential for false negative results during acute HIV-1 infection.
Acknowledgements
This project was 100% funded with federal funds from a federal program of $115,163. This publication was supported by Cooperative Agreement #5NU60OE000103 funded by the Centers for Disease Control and Prevention. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of CDC or the Department of Health and Human Services. No competing interests are declared. Ethical approval was not required. The project was approved as research not involving identifiable human subjects by the Division of HIV/AIDS Prevention at the Centers for Disease Control and Prevention.
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