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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. [ABSTRACT FROM AUTHOR]

Background: In HIV-infected children, viral diversity tends to increase with age in the absence of antiretroviral treatment (ART). We measured HIV diversity in African children (ages 6–36 months) enrolled in a randomized clinical trial comparing two ART regimens (Cohort I of the P1060 trial). Children in this cohort were exposed to single dose nevirapine (sdNVP) at birth. Methods: HIV diversity was measured retrospectively using a high resolution melting (HRM) diversity assay. Samples were obtained from 139 children at the enrollment visit prior to ART initiation. Six regions of the HIV genome were analyzed: two in gag, one in pol, and three in env. A single numeric HRM score that reflects HIV diversity was generated for each region; composite HRM scores were also calculated (mean and median for all six regions). Results: In multivariable median regression models using backwards selection that started with demographic and clinical variables, older age was associated with higher HRM scores (higher HIV diversity) in pol (P = 0.005) and with higher mean (P = 0.014) and median (P<0.001) HRM scores. In multivariable models adjusted for age, pre-treatment HIV viral load, pre-treatment CD4%, and randomized treatment regimen, higher HRM scores in pol were associated with shorter time to virologic suppression (P = 0.016) and longer time to study endpoints (virologic failure [VF], VF/death, and VF/off study treatment; P<0.001 for all measures). Conclusions: In this cohort of sdNVP-exposed, ART-naïve African children, higher levels of HIV diversity in the HIV pol region prior to ART initiation were associated with better treatment outcomes. [ABSTRACT FROM AUTHOR]

Background: Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual. Methods: HIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14-540 days), and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env. Results: Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection. Conclusions: The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined. [ABSTRACT FROM AUTHOR]

Background: The level of viral diversity in an HIV-infected individual can change during the course of HIV infection, reflecting mutagenesis during viral replication and selection of viral variants by immune and other selective pressures. Differences in the level of viral diversity in HIV-infected infants may reflect differences in viral dynamics, immune responses, or other factors that may also influence HIV disease progression. We used a novel high resolution melting (HRM) assay to measure HIV diversity in Ugandan infants and examined the relationship between diversity and survival through 5 years of age. Methods: Plasma samples were obtained from 31 HIV-infected infants (HIVNET 012 trial). The HRM assay was used to measure diversity in two regions in the gag gene (Gag1 and Gag2) and one region in the pol gene (Pol). Results: HRM scores in all three regions increased with age from 6-8 weeks to 12-18 months (for Gag1: P = 0.005; for Gag2: P = 0.006; for Pol: P = 0.016). Higher HRM scores at 6-8 weeks of age (scores above the 75th percentile) were associated with an increased risk of death by 5 years of age (for Pol: P = 0.005; for Gag1/Gag2 (mean of two scores): P = 0.003; for Gag1/ Gag2/Pol (mean of three scores): P = 0.002). We did not find an association between HRM scores and other clinical and laboratory variables. Conclusions: Genetic diversity in HIV gag and pol measured using the HRM assay was typically low near birth and increased over time. Higher HIV diversity in these regions at 6-8 weeks of age was associated with a significantly increased risk of death by 5 years of age. [ABSTRACT FROM AUTHOR]

Background: In HIV-infected children, viral diversity tends to increase with age in the absence of antiretroviral treatment (ART). We measured HIV diversity in African children (ages 6–36 months) enrolled in a randomized clinical trial comparing two ART regimens (Cohort I of the P1060 trial). Children in this cohort were exposed to single dose nevirapine (sdNVP) at birth. Methods: HIV diversity was measured retrospectively using a high resolution melting (HRM) diversity assay. Samples were obtained from 139 children at the enrollment visit prior to ART initiation. Six regions of the HIV genome were analyzed: two in gag, one in pol, and three in env. A single numeric HRM score that reflects HIV diversity was generated for each region; composite HRM scores were also calculated (mean and median for all six regions). Results: In multivariable median regression models using backwards selection that started with demographic and clinical variables, older age was associated with higher HRM scores (higher HIV diversity) in pol (P = 0.005) and with higher mean (P = 0.014) and median (P<0.001) HRM scores. In multivariable models adjusted for age, pre-treatment HIV viral load, pre-treatment CD4%, and randomized treatment regimen, higher HRM scores in pol were associated with shorter time to virologic suppression (P = 0.016) and longer time to study endpoints (virologic failure [VF], VF/death, and VF/off study treatment; P<0.001 for all measures). Conclusions: In this cohort of sdNVP-exposed, ART-naïve African children, higher levels of HIV diversity in the HIV pol region prior to ART initiation were associated with better treatment outcomes.