Introduction
Effective and timely surveillance of the HIV epidemic is critical for the prevention of virus transmission and a prerequisite for achieving high rates of early antiretroviral treatment. However, approximately 17 million of the 37 million people living with HIV are unaware of their HIV status [
1,
2]. Boosting and expediting the identification of infected individuals is therefore a major challenge for the global health sector in the fight against HIV/AIDS [
2].
Patients suffering from acute HIV infection are particularly difficult to diagnose [
3]. These individuals typically suffer from rather non-specific symptoms, yet have viral loads that are about 20 times higher than in the untreated chronic phase of infection, which also correlates with a high risk of subsequent transmissions [
4,
5]. Besides that, patients with primary HIV infection will likely directly benefit from early antiretroviral therapy by improving the overall clinical outcome [
6], preserving the diversity of their immune response [
7,
8], possibly reducing the size of the latent HIV reservoir [
9,
10], and in some cases even achieving functional cure [
11]. Consequently, there is a demand for diagnostic tests that are applicable for monitoring large groups of individuals and that are capable of rapidly identifying acute HIV infections with high sensitivity and specificity.
Today, the recommended laboratory-based screening tests are fourth-generation antigen/antibody immunoassays (4G-EIAs) [
12,
13]. Because of their ability to simultaneously detect anti-HIV-1/2 antibodies (IgM, IgG) and the major structural HIV-1 protein, HIV-1 p24CA, with high sensitivity, 4G-EIAs are the current gold standard to screen for chronic as well as acute HIV infections. The efficiency of diagnostic tests to identify early acute virus infections is largely determined by their ability to detect HIV-1 p24CA. The limit of detection for HIV-1 p24CA in 4G-EIAs is below 20 pg/mL and thus comparable to those of standard p24CA-EIAs [
14,
15]. 4G-EIAs generally score positive 4–7 days after the HIV-1 RNA is detectable by nucleic acid amplification in plasma [
16,
17]. However, 4G-EIAs require well-equipped laboratories with trained personnel, total costs in resource-poor settings can be high or even unattainable and turn-around times may be too slow for effective patient management [
18].
Consequently, over the last years, rapid diagnostic tests (RDTs) for HIV screening have been widely implemented. To perform a RDT typically takes less than 30 min even for inexperienced users and tests accept different patient samples, including serum, whole blood and/or other body fluids (urine, oral swaps). Besides their utility in developing countries, rapid tests are increasingly being used also as a consumer test for self-testing in Europe and the US [
19,
20].
A concern for the widespread use of RDTs has been their diagnostic performance compared to laboratory-based assays [
21]. One of the most frequently employed fourth-generation RDTs is the Determine™ HIV-1/2 Ag/Ab Combo (Alere) that works with either serum or whole blood samples. Several studies indicated a poor sensitivity for the detection of HIV-1 p24CA for the early version of this RDT [
22‐
28], and therefore, Alere developed and released a modified version of this test. The sensitivity of this modified RDT to detect HIV-1/2 antibodies was shown to be high with low rates of false positive results [
29‐
31]. However, its reported sensitivity for the detection of HIV p24CA antigen, the critical determinant for identifying acute HIV-1 infection, varied considerably: some studies found relatively high rates of p24CA detection (> 85%) in samples from infected individuals prior to seroconversion [
29,
31,
32]. In contrast, other studies reported considerably lower p24CA detection rates (< 30%) in similar patient cohorts [
30,
33]. Not surprisingly, the detection rates of the HIV antigen were particularly poor in samples from patients infected with the closely related HIV-2 [
30].
The aim of this study was to evaluate the performance of the new compared to the old version of the Determine™ HIV-1/2 Combo to detect p24CA as well as anti-HIV antibodies in serum samples from patients with acute (prior to or post-seroconversion) or chronic HIV-1 and HIV-2 infection. Importantly, we compared the results obtained by these RDTs to those from multiple HIV detection assays including nucleic acid amplification (PCR), 4G-EIA and p24CA-EIA. Moreover, we evaluated the capability of the new RDT to detect p24CA from several clade B HIV-1 transmitted/founder (T/F) viruses that had been cloned using viral consensus sequences isolated from patients briefly after infection [
34,
35]. The performance of the new RDT to detect capsid antigen was also tested for HIV-2 strains and HIV-2 primary isolates. Furthermore, we assessed the analytical sensitivity of the RDTs for p24CA using a well-characterized protein standard [
36].
Discussion
To our knowledge, this is the first study to analyze the performance of the novel and old RDT versions of the Determine™ HIV-1/2 Combo to detect acute (including T/F viruses) and chronic HIV infection in comparison with results from 4G-EIA, p24CA-EIA, immunoblotting, and quantitative nucleic acid amplification. Furthermore, we investigated the capabilities of the new RDT to detect chronic HIV-2 infection as well as p27CA from several HIV-2 strains and primary isolates.
The old version of the RDT reacted to only 25% (4/16) of the specimens from acutely infected, treatment-naïve, immunoblot-negative patients in Fiebig stages II–III. In only 18.8% (3/16) of the samples in this group, the old RDT identified the presence of HIV-1 p24CA. Even though the size of our cohort was limited, these data are in accordance with the results from other studies that reported poor HIV-1 p24CA sensitivity for the old RDT, ranging from 10.1 to 65.4% [
22,
24,
26,
28]. Two studies conducted in sub-Saharan Africa concluded that the old version of the RDT had 0% sensitivity in specimens from acutely infected patients [
44,
45]. In comparison, in our study, the novel version of the Determine™ HIV-1/2 Combo reacted to 77.3% (17/22) of the serum samples from patients in Fiebig stages II–III. This test identified the presence of p24CA in 63.6% (14/22) of the specimens from this study group. These results indicate an increased sensitivity of the new version of the RDT for detecting early HIV-1 infection prior to seroconversion. Several studies that have recently investigated the performance of the new RDT to detect infection in p24CA-positive, immunoblot-negative serum samples showed lower reactivity rates (< 30%) [
30,
33], while other groups demonstrated higher reactivity rates of more than 85% in similar specimens [
29,
31,
32].
All specimens from acutely HIV-1-infected, treatment-naïve, immunoblot-indeterminate/positive patients (Fiebig stages IV–VI) scored positive in both versions of the Determine™ HIV-1/2 Combo, confirming the high sensitivity of this type of RDT to detect HIV-1 infection in acute infection during or after seroconversion [
29‐
31]. Of note, the novel RDT failed to detect HIV-1-specific antibodies in one specimen from an HIV-1 subtype C-infected patient in Fiebig stage IV. The performance of the Alere RDT to detect HIV-1 subtype C-specific antibodies in seroconverters should be studied more extensively in the future. Of note, cross-sectional data from patients with chronic HIV-1 infection in Malawi and Swaziland indicate that the Alere RDT has high sensitivity for detecting HIV-1-specific antibodies in sera from these predominantly subtype C-infected individuals [
44,
45]. However, a comparison of RDT test results with immunoblot analyses and HIV-1 subtyping is missing in these studies and little is known about the performance of the RDT for detecting antibodies in immunoblot-indeterminate specimens from acutely HIV-1 subtype C-infected patients.
Of particular interest, the new version of the RDT showed HIV-1 antibody reactivity in 8/23 (34.8%) specimens from acutely infected, still immunoblot-negative patients (Fiebig stages I–III). These results indicate higher sensitivity for HIV-1-specific antibodies compared to the frequently used New LAV Blot I Assay and the INNO-LIA HIV I/II Score. This RDT may thus potentially be capable of detecting seroconversion in acute infection earlier than standard immunoblot assays, in line with recent reports [
26,
46]. The old version of the RDT was reactive to HIV-specific antibodies in only 1/16 (6.3%) specimens from acutely infected seronegative patients.
Specimens from chronic HIV-1-infected patients were antibody-reactive in both versions of the Alere RDT (7/7 positive). In addition, we evaluated the performance of the novel RDT to detect chronic HIV-2 infection. Also here, the test reliably detected HIV-specific antibodies (11/11). Neither the new nor the old Determine™ HIV-1/2 Combo reacted to serum samples from uninfected individuals, underlining the already described low false positive rate of this assay [
30‐
32].
Next, we wanted to determine the specificity of the new Alere RDT to detect p24CA antigen from clade B HIV-1 T/F viruses, originally created using viral consensus sequences from recently infected patients [
34]. These strains likely resemble the genotype and phenotype of the earliest stages of HIV-1 infection better than classical laboratory strains, the latter, in specific cases, were shown to have altered capsid stability and function [
47]. The capsid antigen of all T/F viruses tested (8/8) was detected by the novel RDT. However, this required p24CA concentrations ~ 10- to 20-fold higher than suggested by the test manufacturer, again raising concerns about the sensitivity of this assay component.
Due to its structural similarity to HIV-1 p24CA, HIV-2 capsid antigen can occasionally be detected by some 4G-EIAs, albeit with reduced sensitivity [
48,
49]. In the current study, the new Determine™ HIV-1/2 Combo was not reactive with any of the six HIV-2 viruses tested at high viral titers, suggesting that it would be incapable of detecting acute HIV-2 infections prior to seroconversion.
The analytic sensitivity of the Determine™ HIV-1/2 Combo to detect HIV-1 p24CA antigen in serum is stated by the company to be at least 25 pg/mL [
37]. In our experiments, both versions of the RDT assay failed to detect a well-characterized, highly purified p24CA antigen standard from a clade B virus strain at these concentrations [
36], reaching sensitivities of only ≥ 50 pg/mL (first experiment) or even ≥ 100 pg/mL (second experiment). In one case, the novel version of the Alere RDT was reactive only at p24CA levels ≥ 200 pg/mL. In contrast, the quantitative p24CA-EIA used in this study detected the p24CA standard at concentrations ≥ 6.25 pg/mL illustrating the higher sensitivity of this type of antigen assay compared to current RDTs. Furthermore, we observed a correlation between the concentrations of p24 antigen measured by the p24CA-EIA and those that were prepared for the experiments indicating the capability of this assay to faithfully estimate the concentrations of HIV-1 p24CA antigen also in patient sera (Fig.
1). In line with the reduced sensitivity of the RDT assay for recombinant capsid antigen, detection of several clade B T/F viruses also required considerably higher p24CA antigen concentrations to score positive in the novel test version (Table
6).
The p24CA-EIA was reactive in all specimens from acutely infected, still non-seroconverted patients tested in Fiebig stages II–III. In serum samples from this study group, both versions of the Determine™ HIV-1/2 Combo were negative at p24CA concentrations ≤ 315 pg/mL. Moreover, the RDTs failed to detect the structural viral antigen also in several samples with higher p24CA concentrations (Table
1).
For acutely infected, immunoblot-indeterminate/positive specimens (Fiebig stages IV–VI), the p24CA-EIA was reactive in 23/27 (85.2%) cases. The old version of the RDT did not show reactivity to p24CA in this study group, even though 4/8 samples tested with this assay had estimated p24CA concentrations of ≥ 529 pg/mL. The new RDT detected the HIV-1 capsid antigen in 10/27 (37%) of all specimens, but all antigen-reactive specimens had estimated p24CA concentrations of ≥ 485 pg/mL. Taken together, these results strongly suggest that the sensitivity of the Determine™ HIV-1/2 Combo to detect HIV-1 p24CA antigen is considerably lower, in some cases more than 20-fold, than specified by the manufacturer [
37].
The HIV-1 patient specimens evaluated in this study are mostly from subtype B, but also include the subtypes A1, C, CRF01_AE, CRF02_AG, F and URF. For future studies, it would be valuable to evaluate the performance of the RDTs with an even more diverse panel of HIV-1 specimens, especially since the old version of the RDT was shown to have impaired sensitivity to capsid antigen from non-B subtype HIV-1 [
24].
Patient specimens from all HIV-1 study groups that showed positive nucleic acid amplification (except for the one Fiebig I sample) and/or positive HIV-1 immunoblot results were also reactive in the 4G-EIA, confirming that this assay has a high sensitivity to detect HIV-1 infections in plasma samples from both early acutely infected and chronically infected patients.
In samples from acutely infected patients (prior to or post-seroconversion), a larger fraction of strong reactive test results were observed using the new RDT compared to the old version of the test (Tables
1 and
2). As a consequence, positive results may be easier to spot with the new version of this rapid test, which is especially important for untrained users and self-testers.
In our study, both versions of the Determine™ HIV-1/2 Combo proved to be reactive to HIV-1-specific antibodies from several HIV-1 subtypes. Furthermore, the new RDT was reactive to antibodies from patients with HIV-2 groups A and B infection. Thus, this assay might pose a viable alternative to screen for chronic as well as acute HIV-1 and HIV-2 infections after or during seroconversion, with several advantages to other non-RDT assays, including fast turn-around times and independence from professional laboratories with trained personnel. However, even though the novel version of the RDT seems to have an improved capacity to detect also HIV-1 p24CA antigen compared to the old one, its sensitivity is markedly lower compared to p24CA-EIAs or 4G-EIAs. Moreover, based on the lack of detection of 6 HIV-2 viruses, the Determine™ HIV-1/2 Combo is likely unable to detect acute HIV-2 infection prior to seroconversion.
Taken together, the p24CA-EIA and 4G-EIA test systems should still be considered assays of choice to reliably screen for acute HIV infection. Even though the new version of the RDT shows enhanced performance, the manufacturer still needs to improve the sensitivity and likely also clade diversity of the Determine™ HIV-1/2 Combo for p24CA, to make this RDT a viable alternative to established laboratory tests for detecting acute HIV infection in immunoblot-negative patients.
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