Detection of Recent HIV-1 Infection Using a New Limiting-Antigen Avidity Assay: Potential for HIV-1 Incidence Estimates and Avidity Maturation Studies

Background: Accurate and reliable laboratory methods are needed for estimation of HIV-1 incidence to identify the highrisk populations and target and monitor prevention efforts. We previously described a single-well limiting-antigen avidity enzyme immunoassay (LAg-Avidity EIA) to detect recent HIV-1 infection. Methods: We describe here further optimization and characterization of LAg-Avidity EIA, comparing it to the BED assay and a two-well avidity-index (AI) EIA. Specimen sets included longitudinal sera (n = 393), collected from 89 seroconverting individuals from 4 cohorts representing 4 HIV-1 subtypes, and sera from AIDS patients (n = 488) with or without TB coinfections from 3 different cohorts. Ninety seven HIV-1 positive specimens were purchased commercially. The BED assay, LAg-Avidity EIA, AI-EIA and HIV serology were performed, as needed. Results: Monitoring quality control specimens indicated high reproducibility of the LAg-Avidity EIA with coefficient of variation of,10 % in the dynamic range. The LAg-Avidity EIA has an overall mean duration of recency (v) of 141 days (95% CI 119–160) at normalized optical density (ODn) cutoff of 1.0, with similar v in different HIV-1 subtypes and populations (132 to 143 days). Antibody avidity kinetics were similar among individuals and subtypes by both the LAg-Avidity EIA and AI-EIA compared to the HIV-IgG levels measured by the BED assay. The false recent rate among individuals with AIDS was 0.2% with the LAg-Avidity EIA, compared to 2.9 % with the BED assay. Western blot profiles of specimens with increasing avidit

The Effect of Sample Handling on Cross Sectional HIV Incidence Testing Results

To determine if mishandling prior to testing would make a sample from a chronically infected subject appear recently infected when tested by cross-sectional HIV incidence assays.Serum samples from 31 subjects with chronic HIV infection were tested. Samples were subjected to different handling conditions, including incubation at 4 °C, 25 °C and 37 °C, for 1, 3, 7 or 15 days prior to testing. Samples were also subjected to 1,3, 7 and 15 freeze-thaw cycles prior to testing. Samples were tested using the BED capture enzyme immuno assay (BED-CEIA), Vironostika-less sensitive (V-LS), and an avidity assay using the Genetic Systems HIV-1/HIV-2 plus O EIA (avidity assay).Compared to the sample that was not subjected to any mishandling conditions, for the BED-CEIA, V-LS and avidity assay, there was no significant change in test results for samples incubated at 4 °C or 25 °C prior to testing. No impact on test results occurred after 15 freeze-thaw cycles. A decrease in assay results was observed when samples were held for 3 days or longer at 37 °C prior to testing.Samples can be subjected up to 15 freeze-thaw cycles without affecting the results the BED-CEIA, Vironostika-LS, or avidity assays. Storing samples at 4 °C or 25 °C for up to fifteen days prior to testing had no impact on test results. However, storing samples at 37°C for three or more days did affect results obtained with these assays

A Comprehensive Evaluation of the Proficiency Testing Program for the HIV-1 BED Incidence Assay▿

The HIV-1 BED incidence assay was developed at the Centers for Disease Control and Prevention and since 2005 has been available as a commercial kit for use in HIV-1 incidence surveillance. A BED-specific proficiency testing (PT) program was initiated in 2006 that included a panel of eight coded specimens (six unique and two duplicates) to participating laboratories. The number of participating laboratories increased from 12 to 38 from 2006 to 2009. Overall, 96.1% of the laboratories reported results, and 95.4% of those reporting achieved a 100% score. The observed mean normalized optical density (OD-n) values of all participants correlated well with the expected OD-n values for all specimens (R2 = 0.98) used for seven PT rounds. BED testing demonstrated high reproducibility among all laboratories, with an agreement of 99.3% (574/578) between initial and confirmatory classification and regression statistics of R2 = 0.96, slope = 1.022, and intercept = 0.0066. Reproducibility among duplicate specimens was very high during each PT round, with mean deviation of 1.8%. Analysis of controls and calibrator specimen for all 343 runs showed a coefficient of variation of ca. 20% for raw ODs in the dynamic range, which was reduced to <10% when the OD was normalized (OD-n). Most laboratories that failed the PT assessment had transcriptional errors, kit reagent problems, or specimen handling errors. Thus, the BED-specific PT program enabled us to track performance of different laboratories conducting the BED assay while identifying areas for improvements. This program will also serve as a template for future PT programs for new incidence assays as they become available

Performance Characteristics of the Immunoglobulin G-Capture BED-Enzyme Immunoassay, an Assay To Detect Recent Human Immunodeficiency Virus Type 1 Seroconversion

Recently, we developed an immunoglobulin G (IgG)-capture BED-enzyme immunoassay (BED-CEIA) to identify recent human immunodeficiency virus (HIV) type 1 (HIV-1) seroconversion for use in incidence estimates. We have established an algorithm for its use; developed quality control reagents to monitor the assay; and evaluated its performance for interrun, intrarun, and operator variability. Analysis of 144 individual plates, which involved multiple plate lots and several operators over more than a year, indicated that the coefficients of variation (CVs) were between 10 and 15% for raw optical density (OD) values in the dynamic range between 0.5 and 2.0 OD units; the CVs decreased to 5 to 10% when the OD was normalized (OD-n; OD-n = specimen OD/calibrator OD). The intrarun CVs were generally in the range of 5 to 10% for specimens with ODs <0.5 and less than 5% for specimens with ODs >0.5. The level of concordance between multiple plate lots (n = 6) and multiple operators (n = 7) was quite high (R(2) > 0.9). Comparison of the results of the initial and the confirmatory tests with specimens with OD-n values ≤1.5 demonstrated a high degree of correlation (R(2) = 0.92); 566 (92%) of 615 of specimens tested in the two modes retained the same classification (recent or long-term infection). The values for those specimens with changed classifications (n = 49) were close to the cutoff (OD-n = 1.0), as expected. The twofold difference in the HIV IgG contents between the controls and the calibrator reagents was exploited to monitor individual plate runs by using a control plot, which was incorporated into the spreadsheet for data entry and run monitoring. This information provides baseline data for the successful transfer of BED-CEIA to other laboratories and the use of BED-CEIA for the detection of recent HIV seroconversion and the calculation of incidence estimates worldwide

Performance evaluation of the Asante Rapid Recency Assay for verification of HIV diagnosis and detection of recent HIV-1 infections: Implications for epidemic control.

We previously described development of a rapid test for recent infection (RTRI) that can diagnose HIV infection and detect HIV-1 recent infections in a single device. This technology was transferred to a commercial partner as Asante Rapid Recency Assay (ARRA). We evaluated performance of the ARRA kits in the laboratory using a well-characterized panel of specimens. The plasma specimen panel (N = 1500) included HIV-1 (N = 570), HIV-2 (N = 10), and HIV-negatives (N = 920) representing multiple subtypes and geographic locations. Reference diagnostic data were generated using the Bio-Rad HIV-1-2-O EIA/Western blot algorithm with further serotyping performed using the Multispot HIV-1/2 assay. The LAg-Avidity EIA was used to generate reference data on recent and long-term infection for HIV-1 positive specimens at a normalized optical density (ODn) cutoff of 2.0 corresponding to a mean duration of about 6 months. All specimens were tested with ARRA according to the manufacturer's recommendations. Test strips were also read for line intensities using a reader and results were correlated with visual interpretation. ARRA's positive verification line (PVL) correctly classified 575 of 580 HIV-positive and 910 of 920 negative specimens resulting in a sensitivity of 99.1% (95% CI: 98.0-99.6) and specificity of 98.9% (95% CI: 98.1-99.4), respectively. The reader-based classification was similar for PVL with sensitivity of 99.3% (576/580) and specificity of 98.8% (909/920). ARRA's long-term line (LTL) classified 109 of 565 HIV-1 specimens as recent and 456 as long-term compared to 98 as recent and 467 as long-term (LT) by LAg-Avidity EIA (cutoff ODn = 2.0), suggesting a mean duration of recent infection (MDRI) close to 6 months. Agreement of ARRA with LAg recent cases was 81.6% (80/98) and LT cases was 93.8% (438/467), with an overall agreement of 91.7% (kappa = 0.72). The reader (cutoff 2.9) classified 109/566 specimens as recent infections compared to 99 by the LAg-Avidity EIA for recency agreement of 81.8% (81/99), LT agreement of 9% (439/467) with overall agreement of 91.9% (kappa = 0.72). The agreement between visual interpretation and strip reader was 99.9% (95% CI: 99.6-99.9) for the PVL and 98.1% (95% CI: 96.6-98.9) for the LTL. ARRA performed well with HIV diagnostic sensitivity >99% and specificity >98%. Its ability to identify recent infections is comparable to the LA-Avidity EIA corresponding to an MDRI of about 6 months. This point-of-care assay has implications for real-time surveillance of new infections among newly diagnosed individuals for targeted prevention and interrupting ongoing transmission thus accelerating epidemic control

Mean Recency Period for Estimation of HIV-1 Incidence with the BED-Capture EIA and Bio-Rad Avidity in Persons Diagnosed in the United States with Subtype B

<div><p>HIV incidence estimates are used to monitor HIV-1 infection in the United States. Use of laboratory biomarkers that distinguish recent from longstanding infection to quantify HIV incidence rely on having accurate knowledge of the average time that individuals spend in a transient state of recent infection between seroconversion and reaching a specified biomarker cutoff value. This paper describes five estimation procedures from two general statistical approaches, a survival time approach and an approach that fits binomial models of the probability of being classified as recently infected, as a function of time since seroconversion. We compare these procedures for estimating the mean duration of recent infection (MDRI) for two biomarkers used by the U.S. National HIV Surveillance System for determination of HIV incidence, the Aware BED EIA HIV-1 incidence test (BED) and the avidity-based, modified Bio-Rad HIV-1/HIV-2 plus O ELISA (BRAI) assay. Collectively, 953 specimens from 220 HIV-1 subtype B seroconverters, taken from 5 cohorts, were tested with a biomarker assay. Estimates of MDRI using the non-parametric survival approach were 198.4 days (SD 13.0) for BED and 239.6 days (SD 13.9) for BRAI using cutoff values of 0.8 normalized optical density and 30%, respectively. The probability of remaining in the recent state as a function of time since seroconversion, based upon this revised statistical approach, can be applied in the calculation of annual incidence in the United States.</p></div

Identifying Risk Factors for Recent HIV Infection in Kenya Using a Recent Infection Testing Algorithm: Results from a Nationally Representative Population-Based Survey

<div><p>Introduction</p><p>A recent infection testing algorithm (RITA) that can distinguish recent from long-standing HIV infection can be applied to nationally representative population-based surveys to characterize and identify risk factors for recent infection in a country.</p><p>Materials and Methods</p><p>We applied a RITA using the Limiting Antigen Avidity Enzyme Immunoassay (LAg) on stored HIV-positive samples from the 2007 Kenya AIDS Indicator Survey. The case definition for recent infection included testing recent on LAg and having no evidence of antiretroviral therapy use. Multivariate analysis was conducted to determine factors associated with recent and long-standing infection compared to HIV-uninfected persons. All estimates were weighted to adjust for sampling probability and nonresponse.</p><p>Results</p><p>Of 1,025 HIV-antibody-positive specimens, 64 (6.2%) met the case definition for recent infection and 961 (93.8%) met the case definition for long-standing infection. Compared to HIV-uninfected individuals, factors associated with higher adjusted odds of recent infection were living in Nairobi (adjusted odds ratio [AOR] 11.37; confidence interval [CI] 2.64–48.87) and Nyanza (AOR 4.55; CI 1.39–14.89) provinces compared to Western province; being widowed (AOR 8.04; CI 1.42–45.50) or currently married (AOR 6.42; CI 1.55–26.58) compared to being never married; having had ≥ 2 sexual partners in the last year (AOR 2.86; CI 1.51–5.41); not using a condom at last sex in the past year (AOR 1.61; CI 1.34–1.93); reporting a sexually transmitted infection (STI) diagnosis or symptoms of STI in the past year (AOR 1.97; CI 1.05–8.37); and being aged <30 years with: 1) HSV-2 infection (AOR 8.84; CI 2.62–29.85), 2) male genital ulcer disease (AOR 8.70; CI 2.36–32.08), or 3) lack of male circumcision (AOR 17.83; CI 2.19–144.90). Compared to HIV-uninfected persons, factors associated with higher adjusted odds of long-standing infection included living in Coast (AOR 1.55; CI 1.04–2.32) and Nyanza (AOR 2.33; CI 1.67–3.25) provinces compared to Western province; being separated/divorced (AOR 1.87; CI 1.16–3.01) or widowed (AOR 2.83; CI 1.78–4.45) compared to being never married; having ever used a condom (AOR 1.61; CI 1.34–1.93); and having a STI diagnosis or symptoms of STI in the past year (AOR 1.89; CI 1.20–2.97). Factors associated with lower adjusted odds of long-standing infection included using a condom at last sex in the past year (AOR 0.47; CI 0.36–0.61), having no HSV2-infection at aged <30 years (AOR 0.38; CI 0.20–0.75) or being an uncircumcised male aged <30 years (AOR 0.30; CI 0.15–0.61).</p><p>Conclusion</p><p>We identified factors associated with increased risk of recent and longstanding HIV infection using a RITA applied to blood specimens collected in a nationally representative survey. Though some false-recent cases may have been present in our sample, the correlates of recent infection identified were epidemiologically and biologically plausible. These methods can be used as a model for other countries with similar epidemics to inform targeted combination prevention strategies aimed to drastically decrease new infections in the population.</p></div

Toward elimination of mother-to-child transmission of HIV in Malawi: Findings from the Malawi Population-based HIV Impact Assessment (2015-2016)

Malawi encabezó el desarrollo y la implementación de la Opción B+ para la prevención de la transmisión maternoinfantil del VIH (PTMI), brindando TAR de por vida a todas las mujeres embarazadas y lactantes seropositivas. Utilizamos datos de la Evaluación del impacto del VIH basada en la población de Malawi (MPHIA, por sus siglas en inglés) de 2015-2016 para estimar el progreso hacia los objetivos 90-90-90 (el 90 % de las personas con VIH saben que son seropositivos; de estas, el 90 % está recibiendo TAR). ; y de estos, el 90% tiene supresión de la carga viral [VLS]) para mujeres VIH positivas que reportaron un nacimiento vivo en los 3 años anteriores. Métodos MPHIA fue una encuesta de hogares representativa a nivel nacional; Se entrevistó a mujeres elegibles de 15 a 64 años de edad que dieron su consentimiento sobre embarazos y resultados, incluido el estado del VIH durante su embarazo más reciente, aceptación de la PTMI y pruebas de diagnóstico infantil temprano (EID). Los análisis descriptivos se ponderaron para tener en cuenta el complejo diseño de la encuesta. Los resultados de la carga viral (VL) se clasificaron por VLS (<1000 copias/mL) y VL indetectable (objetivo no detectado/por debajo del límite de detección). Resultados De las 3.153 mujeres incluidas en nuestro análisis, 371 (10,1 %, intervalo de confianza [IC] del 95 %: 8,8 %-11,3 %) dieron positivo en la prueba del VIH en la encuesta. La mayoría de las mujeres seropositivas (84,2 %, IC del 95 %: 79,9 %-88,6 %) informaron conocer su estado serológico; de estos, el 94,9 % (IC 95 %: 91,7 %-98,2 %) estaban recibiendo TAR; y de estos, el 91,2% (IC 95%: 87,4%-95,0%) tenían VLS. Entre las 371 mujeres con VIH, el 76,0 % (IC 95 %: 70,4 %-81,7 %) tenía VLS y el 66,5 % (IC 95 %: 59,8 %-73,2 %) tenía LV indetectable. Entre 262 niños expuestos al VIH, el 50,8 % (IC 95 %: 42,8 %-58,8 %) se sometió a la prueba EID dentro de los 2 meses posteriores al nacimiento, mientras que el 17,9 % (IC 95 %: 11,9 %-23,8 %) no recibió la prueba EID. De 190 niños expuestos al VIH con un resultado de la prueba del VIH notificado, el 2,1 % (IC del 95 %: 0,0 %-4,6 %) tuvo resultados positivos. Conclusiones Los datos de MPHIA demuestran una alta captación de PTMI a nivel de población. Sin embargo, nuestros resultados identifican algunas brechas en VLS en mujeres posparto y pruebas de EID.Q1Malawi spearheaded the development and implementation of Option B+ for prevention of mother-to-child transmission of HIV (PMTCT), providing life-long ART for all HIV-positive pregnant and breastfeeding women. We used data from the 2015-2016 Malawi Population-based HIV Impact Assessment (MPHIA) to estimate progress toward 90-90-90 targets (90% of those with HIV know their HIV-positive status; of these, 90% are receiving ART; and of these, 90% have viral load suppression [VLS]) for HIV-positive women reporting a live birth in the previous 3 years. Methods MPHIA was a nationally representative household survey; consenting eligible women aged 15-64 years were interviewed on pregnancies and outcomes, including HIV status during their most recent pregnancy, PMTCT uptake, and early infant diagnosis (EID) testing. Descriptive analyses were weighted to account for the complex survey design. Viral load (VL) results were categorized by VLS (<1,000 copies/mL) and undetectable VL (target not detected/below the limit of detection). Results Of the 3,153 women included in our analysis, 371 (10.1%, 95% confidence interval [CI]: 8.8%-11.3%) tested HIV positive in the survey. Most HIV-positive women (84.2%, 95% CI: 79.9%-88.6%) reported knowing their HIV-positive status; of these, 94.9% (95% CI: 91.7%-98.2%) were receiving ART; and of these, 91.2% (95% CI: 87.4%-95.0%) had VLS. Among the 371 HIV-positive women, 76.0% (95% CI: 70.4%-81.7%) had VLS and 66.5% (95% CI: 59.8%-73.2%) had undetectable VL. Among 262 HIV-exposed children, 50.8% (95% CI: 42.8%-58.8%) received EID testing within 2 months of birth, whereas 17.9% (95% CI: 11.9%-23.8%) did not receive EID testing. Of 190 HIV-exposed children with a reported HIV test result, 2.1% (95% CI: 0.0%-4.6%) had positive results. Conclusions MPHIA data demonstrate high PMTCT uptake at a population level. However, our results identify some gaps in VLS in postpartum women and EID testing.N/AS

Estimated days of mean duration of recent infection (MDRI) and standard error, in parentheses, for BED and BRAI incidence bioassays.

<p>A total of 858 measurements from 209 subjects of 4 cohorts and 749 measurements from 162 subjects of 3 cohorts were used to estimate BED and BRAI MDRI, respectively. Results for five estimation procedures are given.</p