Andrew F. Longosz

Performance of a limiting-antigen avidity enzyme immunoassay for cross-sectional estimation of HIV incidence in the United States

Background A limiting antigen avidity enzyme immunoassay (HIV-1 LAg-Avidity assay) was recently developed for cross-sectional HIV incidence estimation. We evaluated the performance of the LAg-Avidity assay alone and in multi-assay algorithms (MAAs) that included other biomarkers. Methods and Findings Performance of testing algorithms was evaluated using 2,282 samples from individuals in the United States collected 1 month to >8 years after HIV seroconversion. The capacity of selected testing algorithms to accurately estimate incidence was evaluated in three longitudinal cohorts. When used in a single-assay format, the LAg-Avidity assay classified some individuals infected >5 years as assay positive and failed to provide reliable incidence estimates in cohorts that included individuals with long-term infections. We evaluated >500,000 testing algorithms, that included the LAg-Avidity assay alone and MAAs with other biomarkers (BED capture immunoassay [BED-CEIA], BioRad-Avidity assay, HIV viral load, CD4 cell count), varying the assays and assay cutoffs. We identified an optimized 2-assay MAA that included the LAg-Avidity and BioRad-Avidity assays, and an optimized 4-assay MAA that included those assays, as well as HIV viral load and CD4 cell count. The two optimized MAAs classified all 845 samples from individuals infected >5 years as MAA negative and estimated incidence within a year of sample collection. These two MAAs produced incidence estimates that were consistent with those from longitudinal follow-up of cohorts. A comparison of the laboratory assay costs of the MAAs was also performed, and we found that the costs associated with the optimal two assay MAA were substantially less than with the four assay MAA. Conclusions The LAg-Avidity assay did not perform well in a single-assay format, regardless of the assay cutoff. MAAs that include the LAg-Avidity and BioRad-Avidity assays, with or without viral load and CD4 cell count, provide accurate incidence estimates.

Incorrect identification of recent HIV infection in adults in the United States using a limiting-antigen avidity assay.

Objectives:To evaluate factors associated with misclassification by the limiting-antigen avidity (LAg-avidity) assay among individuals with long-standing HIV infection. Design:Samples were obtained from the Multicenter AIDS Cohort Study and AIDS Linked to the IntraVenous Experience cohort (1089 samples from 667 individuals, 595 samples collected 2–4 years and 494 samples collected 4–8 years after HIV seroconversion). Paired samples from both time points were available for 422 (63.3%) of the 667 individuals. Methods:Samples were considered to be misclassified if the LAg-avidity assay result was 1.5 or less normalized optical density (OD-n) units. Results:Overall, 4.8% (52/1089) of the samples were misclassified, including 1.8% [16/884, 95% confidence interval (CI) 1.09–3.06%] of samples from individuals with viral loads above 400 copies/ml and 1.4% (10/705) of samples from individuals with viral loads above 400 copies/ml and CD4+ cell counts above 200 cells/&mgr;l (95% CI 0.68–2.60%). Age, race, sex, and mode of HIV acquisition were not associated with misclassification. In an adjusted analysis, viral load below 400 copies/ml [adjusted odds ratio (aOR) 3.72, 95% CI 1.61–8.57], CD4+ cell count below 50 cells/&mgr;l (aOR 5.41, 95% CI 1.86–15.74), and low LAg-avidity result (⩽1.5 OD-n) from the earlier time point (aOR 5.60, 95% CI 1.55–20.25) were significantly associated with misclassification. Conclusions:The manufacturer of the LAg-avidity assay recommends excluding individuals from incidence surveys who are receiving antiretroviral therapy, are elite suppressors, or have AIDS (CD4+ cell count <200 cells/&mgr;l). The results of this study indicate that those exclusions do not remove all sources of assay misclassification among individuals with long-standing HIV infection.

A comparison of two measures of HIV diversity in multi-assay algorithms for HIV incidence estimation.

Background Multi-assay algorithms (MAAs) can be used to estimate HIV incidence in cross-sectional surveys. We compared the performance of two MAAs that use HIV diversity as one of four biomarkers for analysis of HIV incidence. Methods Both MAAs included two serologic assays (LAg-Avidity assay and BioRad-Avidity assay), HIV viral load, and an HIV diversity assay. HIV diversity was quantified using either a high resolution melting (HRM) diversity assay that does not require HIV sequencing (HRM score for a 239 base pair env region) or sequence ambiguity (the percentage of ambiguous bases in a 1,302 base pair pol region). Samples were classified as MAA positive (likely from individuals with recent HIV infection) if they met the criteria for all of the assays in the MAA. The following performance characteristics were assessed: (1) the proportion of samples classified as MAA positive as a function of duration of infection, (2) the mean window period, (3) the shadow (the time period before sample collection that is being assessed by the MAA), and (4) the accuracy of cross-sectional incidence estimates for three cohort studies. Results The proportion of samples classified as MAA positive as a function of duration of infection was nearly identical for the two MAAs. The mean window period was 141 days for the HRM-based MAA and 131 days for the sequence ambiguity-based MAA. The shadows for both MAAs were <1 year. Both MAAs provided cross-sectional HIV incidence estimates that were very similar to longitudinal incidence estimates based on HIV seroconversion. Conclusions MAAs that include the LAg-Avidity assay, the BioRad-Avidity assay, HIV viral load, and HIV diversity can provide accurate HIV incidence estimates. Sequence ambiguity measures obtained using a commercially-available HIV genotyping system can be used as an alternative to HRM scores in MAAs for cross-sectional HIV incidence estimation.

Antibody Maturation in Women Who Acquire HIV Infection While Using Antiretroviral Preexposure Prophylaxis

The CAPRISA 004 preexposure prophylaxis (PrEP) randomized trial demonstrated that women who used a vaginal gel containing the antiretroviral drug tenofovir (TFV) had a 39% lower risk of acquiring human immunodeficiency virus (HIV). It is not known whether topical TFV alters the antibody response to breakthrough HIV infection. In this study, antibody maturation was evaluated using 3 serologic assays: the BED capture enzyme immunoassay (CEIA), the Bio-Plex (Luminex) assay, and the Bio-Rad avidity assay. Tests were performed using serum samples collected 3, 6, 9, 12, 24, 36, 48, and >48 months after seroconversion from 95 women in the CAPRISA 004 trial (35 in the TFV gel arm and 60 in the placebo arm). For the BED CEIA and Luminex assay, linear mixed effects models were used to examine test results by study arm. Cox proportional hazard analysis was used to examine time to avidity cutoff. Anti-HIV antibody titers did not differ between study arms. Women assigned to TFV gel demonstrated slower antibody avidity maturation, as determined by the Bio-Rad (P = .04) and gp120 Bio-Plex (P = .028) assays. Women who were assigned to receive topical TFV but became infected had slower antibody avidity maturation, with potential implications for diagnosis and antibody-based incidence assays as access to antiretroviral therapy-based PrEP is increased.

Immune responses in Ugandan women infected with subtypes A and D HIV using the BED capture immunoassay and an antibody avidity assay.

Introduction:Analysis of samples from Uganda using serologic HIV incidence assays reveal that individuals with subtype D infection often have weak humoral immune responses to HIV infection. It is unclear whether this reflects a poor initial response to infection or a waning antibody response later in infection. Materials and Methods:Samples (N = 2614) were obtained from 114 women aged 18–45 years in the Ugandan Genital Shedding and Disease Progression Study cohort (2001–2009; 82 subtype A, 32 subtype D; median 23 samples/women, range 3–41 samples, median follow-up of 6.6 years). Samples were analyzed using the BED capture immunoassay (cutoff, 0.8 OD-n) and the avidity assay (cutoff, 90% Avidity Index). Antibody maturation was assessed by having the BED capture enzyme immunoassay (BED-CEIA) or avidity value exceed the assay cutoff 1 or 2 years after infection. The waning antibody response was measured by having the BED-CEIA or avidity value fall >20% below the maximum value. Results:For the BED-CEIA, 8 women with subtype A infection and 3 women with subtype D infection never progressed previously the cutoff value (median, 5.9 years follow-up after infection). Six women with subtype D infection never achieved an avidity index >90%. Subtype did not impact the proportion of women whose assay values regressed by >20% of the maximal value (for BED-CEIA: 33% for A, 41% for D, P = 0.51; for avidity: 1% for A, 6% for D, P = 0.19). Discussion:The higher frequency of misclassification of individuals with long-term subtype D infection as recently infected using serologic incidence assays reflects a weak initial antibody response to HIV infection that is sustained over time.

Inter-laboratory assessment of a prototype multiplex kit for determination of recent HIV-1 infection.

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 naïve 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.

Evaluation of postpartum HIV superinfection and mother-to-child transmission

This brief shares the findings of a study conducted to assess the quality of integrated services under the National Rural Health Mission (NRHM) in Jharkhand India. The NRHM is mandated by the Indian Ministry of Health and Family Welfare to integrated family planning into immunization services. FHI 360 and CARE India conducted a cross-sectional descriptive study to assess the quality of integrated services provided under the National Rural Health Mission (NRHM) in Lohardaga district of Jharkhand. The NRHM as mandated by the India Ministry of Health and Family Welfare seeks to integrate family planning into immunization services nationwide. The assessment covered service delivery at two primary health centers and 15 village health and nutrition days using semistructured interviews with immunization service providers service managers and women who brought their babies to receive immunization services. The study also looked at what services were being provided at the 17 delivery locations and the potential for improved integration. (excerpt)Objective:This study examined HIV superinfection in HIV-infected women postpartum, and its association with mother-to-child transmission (MTCT). Design:Plasma samples were obtained from HIV-infected women who transmitted HIV to their infants after 6 weeks of age (transmitters, n = 91) and HIV-infected women who did not transmit HIV to their infants (nontransmitters, n = 91). These women were originally enrolled in a randomized trial for prevention of MTCT of HIV in Malawi (Post-Exposure Prophylaxis of Infants trial in Malawi). Methods:Two HIV genomic regions (p24 and gp41) were analyzed by next-generation sequencing for HIV superinfection. HIV superinfection was established if the follow-up sample contained a new, phylogenetically distinct viral population. HIV superinfection and transmission risk were examined by multiple logistic regression, adjusted for Post-Exposure Prophylaxis of Infants study arm, baseline viral load, baseline CD4+ cell count, time to resumption of sex, and breastfeeding duration. Results:Transmitters had lower baseline CD4+ cell counts (P = 0.001) and higher viral loads (P < 0.0001) compared with nontransmitters. There were five cases of superinfection among transmitters (rate of superinfection = 4.7/100 person-years) compared with five cases among the nontransmitters (rate of superinfection = 4.4/100 person-years; P = 0.78). HIV superinfection was not associated with increased risk of postnatal MTCT of HIV after controlling for maternal age, baseline viral load, and CD4+ cell count (adjusted odds ratio = 2.32, P = 0.30). Longer breastfeeding duration was independently associated with a lower risk of HIV superinfection after controlling for study arm and baseline viral load (P = 0.05). Conclusion:There was a significant level of HIV superinfection in women postpartum, but this was not associated with an increased risk of MTCT via breastfeeding.

Characterization of the Neutralizing Antibody Response in a Case of Genetically Linked HIV Superinfection.

This report describes the identification of a genetically confirmed linked heterosexual human immunodeficiency virus (HIV) superinfection (HIV-SI) in a woman with chronic HIV infection who acquired a second strain of the virus from her husband. Serum neutralizing antibody (NAb) responses against their homologous and heterologous viruses, including the superinfecting strain, in the woman and her husband were examined before and after onset of HIV-SI. The woman displayed a moderately potent and broad anti-HIV NAb response prior to superinfection but did not possess NAb activity against the superinfecting strain. This case highlights the unique potential of linked HIV-SI studies to examine natural protection from HIV infection.

Evolution of HIV-1 within untreated individuals and at the population scale in Uganda

HIV-1 undergoes multiple rounds of error-prone replication between transmission events, resulting in diverse viral populations within and among individuals. In addition, the virus experiences different selective pressures at multiple levels: during the course of infection, at transmission, and among individuals. Disentangling how these evolutionary forces shape the evolution of the virus at the population scale is important for understanding pathogenesis, how drug- and immune-escape variants are likely to spread in populations, and the development of preventive vaccines. To address this, we deep-sequenced two regions of the HIV-1 genome (p24 and gp41) from 34 longitudinally-sampled untreated individuals from Rakai District in Uganda, infected with subtypes A, D, and inter-subtype recombinants. This dataset substantially increases the availability of HIV-1 sequence data that spans multiple years of untreated infection, in particular for different geographical regions and viral subtypes. In line with previous studies, we estimated an approximately five-fold faster rate of evolution at the within-host compared to the population scale for both synonymous and nonsynonymous substitutions, and for all subtypes. We determined the extent to which this mismatch in evolutionary rates can be explained by the evolution of the virus towards population-level consensus, or the transmission of viruses similar to those that establish infection within individuals. Our findings indicate that both processes are likely to be important.

The Impact of Viral Suppression and Viral Breakthrough on Limited-Antigen Avidity Assay Results in Individuals with Clade B HIV Infection.

We analyzed the impact of HIV viral load on the performance of a limiting antigen avidity enzyme immunoassay (LAg-Avidity assay) and determined if this assay could be used to identify viral breakthrough. Three groups of samples were tested: (1) 18 individuals (30 samples) previously identified as elite suppressors; (2) 18 individuals (72 samples) who were continually suppressed on antiretroviral treatment (ART) with 1 sample before and 2-6 samples (one/year) after ART initiation; and (3) 20 individuals (179 samples) on ART who had evidence of viral breakthrough (>400 copies/ml) with subsequent viral suppression. Elite suppressors had the lowest LAg-Avidity assay values. Among those who were continually suppressed on ART, 83% (15/18) had LAg-Avidity assay values that decreased over time. Although the LAg-Avidity assay on a single sample cannot identify when a viral breakthrough occurs, paired longitudinal samples could identify viral breakthrough (sensitivity: 65%, specificity: 84%).