Laboratory assays for determining recent HIV-1 infection are of great public health importance for aiding in the estimation of HIV incidence. Concerns have been raised about the potential for misclassification with serology-based assays due to fluctuations in the antibody response, particularly following progression to AIDS. We characterized longitudinal antibody responses to HIV using a cohort of men who have sex with men (MSM) sampled for up to 17 years, in which 57% of the 65 study subjects included in the current analyses progressed to AIDS during the study period. Envelope-specific total IgG antibody levels, avidity, and p24-specific IgG3 levels were evaluated using a multiplexed Bio-Plex assay. For the majority of the analytes, no significant difference in IgG reactivity was observed between AIDS and non-AIDS specimens. Although a slight decline in gp120 reactivity was noted with decreasing CD4+ T cell count, the drop in assay values was relatively minimal and would likely not lead to an increase in the misclassification rate of the assay. A peak in HIV-1 p24 IgG3 levels was observed during early infection, as confirmed by testing 1,216 specimens from 342 recent seroconverters with the Bio-Plex assay. As expected, IgG3 reactivity declined with disease progression and decreasing CD4+ T cell count in the MSM cohort; however, 37% of the study subjects exhibited relatively high IgG3 levels late in the course of infection.

Laboratory-based HIV tests for recent infection (TRIs), which primarily measure a specific serological biomarker(s) that distinguishes recent from long-term HIV infection, have facilitated the estimation of population-based incidence. Dried blood spots (DBS) on filter paper are an attractive sample source for HIV surveillance, given the simplified and cost-effective methods of specimen collection, storage, and shipment. Here, we evaluated the use of DBS in conjunction with an in-house multiplex TRI, the HIV-1-specific Bio-Plex assay, which measures direct antibody binding and avidity to multiple HIV-1 analytes. The assay performance was comparable between matched plasma and DBS samples from HIV-1 infected individuals obtained from diverse sources. The coefficients of variation, comparing the median antibody reactivity for each analyte between plasma and DBS, ranged from 2.78% to 9.40% and the correlation coefficients between the two sample types ranged from 0.89 to 0.97, depending on the analyte. The correlation in antibody reactivity between laboratory and site-prepared DBS for each analyte ranged from 0.87 to 0.98 and from 0.90 to 0.97 between site-prepared DBS and plasma. The correlation in assay measures between plasma and DBS indicate that the sample types can be used interchangeably with the Bio-Plex format, without negatively impacting the misclassification rate of the assay.

BACKGROUND: Accurate and reliable laboratory-based assays are needed for estimating HIV-1 incidence from cross-sectional samples. We recently described the development of a customized, HIV-1-specific Bio-Plex assay that allows for the measurement of HIV-specific antibody levels and avidity to multiple analytes for improved HIV-1 incidence estimates. METHODS: To assess intra- and inter-laboratory assay performance, prototype multiplex kits were developed and evaluated by three distinct laboratories. Longitudinal seroconversion specimens were tested in parallel by each laboratory and kit performance was compared to that of an in-house assay. Additionally, the ability of the kit to distinguish recent from long-term HIV-1 infection, as compared to the in-house assay, was determined by comparing the reactivity of known recent (infected <6 months) and long-term (infected >12 months) drug naive specimens. RESULTS: Although the range of reactivity for each analyte varied between the prototype kit and in-house assay, a measurable distinction in reactivity between recent and long-term specimens was observed with both assays in all three laboratories. Additionally, kit performance was consistent between all three laboratories. The intra-assay coefficient of variation (CV), between sample replicates for all laboratories, ranged from 0.5% to 6.1%. The inter-laboratory CVs ranged from 8.5% to 21.3% for gp160-avidity index (a) and gp120-normalized mean fluorescent intensity (MFI) value (n), respectively. CONCLUSION: We demonstrate the feasibility of producing a multiplex kit for measuring HIV antibody levels and avidity, with the potential for improved incidence estimates based on multi-analyte algorithms. The availability of a commercial kit will facilitate the transfer of technology among diverse laboratories for widespread assay use.

BACKGROUND: FDA-approved HIV Antigen/Antibody combo (4th generation) immunoassays (IAs) can identify HIV-1 infections before the Western blot (WB) becomes positive. In the US, increased detection of acute HIV infections has been facilitated by using 4th generation IAs, but there is no FDA-approved 4th generation rapid test (RT). The Alere Determine HIV-1/2 Ag/Ab Combo (Determine Combo) RT detects and distinguishes HIV p24 Antigen (Ag) from Antibody (Ab) to HIV-1+HIV-2 and thus has the potential to improve diagnosis of acute HIV infection. OBJECTIVE: To evaluate the ability of Determine Combo RT to detect acute/early HIV-1 infections and HIV-2 antibody in well-characterized plasma specimens. STUDY DESIGN: In HIV-1 seroconverters from the US, Determine Combo reactivity was evaluated by performing the 50% cumulative frequency analysis and by comparing with 3rd and 4th generation IAs' reactivity. HIV-2 plasma specimens from Ivory Coast were tested with Determine Combo. RESULTS: The 50% cumulative frequency analysis in 17 seroconverters placed Determine Combo (Ag+/Ab-, Ag+Ab+, Ag-/Ab+) and Ab-component reactivity at 15.5 and 7 days before WB positivity, respectively. In 26 seroconverters, Determine Combo was reactive in 99.0% and 92.5% of 3rd and 4th generation IAs-reactive specimens, respectively. All HIV-2 plasma specimens were Ab-reactive/Ag-non-reactive by Determine Combo. CONCLUSIONS: Based on previous results with the same seroconversion panels, combined Ag/Ab reactivity of the Determine Combo appears between FDA-approved 4th and 3rd generation laboratory IAs. These data indicate that this RT could detect HIV-1 infection earlier than other RTs and it performs well in HIV-2 specimens.

OBJECTIVES: Accurate methods of estimating HIV-1 incidence are critical for monitoring the status of the epidemic and the impact of prevention strategies. Although several laboratory-based tests have been developed strictly for this purpose, several limitations exist and improved methods or technologies are needed. We sought to further optimize a previously described bead-based, HIV-1-specific multiplex assay with the capability of measuring multiple immune responses for determining recent infection. METHODS: We refined the customized HIV-1 Bio-Plex assay by determining cutoffs and mean durations of recency (MDR), based on the reactivity to longitudinal seroconversion specimens (n = 1347) from 311 ART-naive, HIV-1-infected subjects. False-recent rates (FRRs) were calculated for various long-term cohorts, including AIDS patients, individuals on ART, and subtype C specimens. Incidence was estimated for each individual assay analyte from a simulated population with a known incidence of 1%. For improved incidence estimates, multi-analyte algorithms based on combinations of 3 to 6 analytes were evaluated and compared to the performance of each individual analyte. RESULTS: The MDR for the six analytes varied from 164.2 to 279.4 days, while the multi-analyte algorithm MDRs were less variable with a minimum and maximum value of 228.4 and 277.9 days, respectively. The FRRs for the 7 multi-analyte algorithms evaluated in this study varied from 0.3% to 3.1%, in a population of ART-naive, long-term individuals. All algorithms yielded improved incidence estimates as compared to the individual analytes, predicting an incidence of 0.95% to 1.02%. CONCLUSIONS: The HIV-specific multiplex assay described here measures several distinct immune responses in a single assay, allowing for the consideration of multi-analyte algorithms for improved HIV incidence estimates.

BACKGROUND: Blood specimens for HIV testing are frequently collected using serum collection tubes. The Aptima HIV-1 RNA Qualitative test, originally approved for diagnostic use as a supplemental test for use with plasma, is now approved for use with serum, and may be used in new laboratory-based HIV testing algorithms which detect acute and established HIV infections. OBJECTIVES: To compare the sensitivity of Aptima using serum and plasma specimens from persons with established HIV infection. STUDY DESIGN: Parallel serum and plasma specimens were collected from 325 persons with established HIV-1 infection who had positive immunoassay (IA) and Western blot (WB) results. Samples with negative Aptima results were considered false-negative and were subjected to repeat testing. Aptima sensitivity for serum and plasma was calculated relative to IA and WB, and compared using the McNemar test. RESULTS: The sensitivity of Aptima using serum (97.23%, 95% confidence interval [CI] 94.81-98.73) was similar to that using plasma (97.54%, 95% [CI] 95.21-98.93) p=1.00. Five of ten specimens initially false-negative on either serum or plasma were reactive on repeat testing. No specimens initially classified as false-negative on both matrices were reactive on both matrices on repeat testing. CONCLUSIONS: In specimens from persons with established infections, Aptima performed with similar sensitivity when used with serum or plasma. Using serum for immunoassay screening and supplemental testing may provide added convenience for laboratories.

Pre-exposure prophylaxis (PrEP) is a novel intervention strategy for the prevention HIV transmission. As several clinical trials are at various stages of completion, it is important to understand the impact of PrEP treatment on the development of the immune response to HIV, particularly in individuals that exhibit breakthrough infections despite PrEP. A model of HIV infection, using rhesus macaques and the simian/human immunodeficiency virus (SHIV), was employed to evaluate the effects of PrEP on the evolution of the humoral immune response. Time to seroconversion, neutralizing and binding antibody levels, and antibody avidity were measured in 12 rhesus macaques infected during daily or intermittent PrEP with FTC (emtricitabine) or Truvada (FTC/Tenofovir combination) and compared to 11 untreated, SHIV-infected controls. Macaques that became infected while receiving PrEP exhibited significantly lower peak virus loads during acute infection as compared to untreated animals. While the timing of seroconversion and SHIV binding and neutralizing antibody levels were not impacted by treatment, lower maturation rates of antibody avidity for anti-p27, gp120, gp160, and gp41 were observed. This study suggests that reduced virus loads associated with PrEP treatment has little impact on timing of seroconversion and neutralizing/binding antibody levels: however, maturation of antibody avidity was suppressed.