Use of a High Resolution Melting Assay to Analyze HIV Diversity in HIV-infected Ugandan Children
and ††Department of Paediatrics and Child Health, School of Medicine, Makerere University, Kampala, Uganda. The Pediatric Infectious Disease Journal
(Impact Factor: 2.72).
07/2012; 31(11):e222-e228. DOI: 10.1097/INF.0b013e3182678c3f
We used a novel high resolution melting (HRM) diversity assay to analyze HIV diversity in Ugandan children (age 0.6-12.4 years) who were enrolled in an observational study of antiretroviral treatment (ART). Children were maintained on ART if they were clinically and immunologically stable.
HIV diversity was measured before ART (baseline) in 76 children and after 48 or 96 weeks of ART in 14 children who were not virally suppressed. HIV diversity (expressed as HRM scores) was measured in 6 regions of the HIV genome (2 in gag, 1 in pol, 3 in env).
Higher baseline HRM scores were significantly associated with older age (≥2 years, P ≤ 0.001 for all 6 regions). HRM scores from different regions were weakly correlated. Higher baseline HRM scores in 3 regions (1 in gag, 2 in env) were associated with ART failure. HIV diversity was lower in 4 regions (2 in gag, 1 in pol, 1 in env) after 48-96 weeks of nonsuppressive ART compared with baseline.
Higher levels of HIV diversity were observed in older children before ART, and higher levels of diversity in some regions of the HIV genome were associated with ART failure. Prolonged exposure to nonsuppressive ART was associated with a significant decrease in viral diversity in selected regions of the HIV genome.
Available from: Deborah J Donnell
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ABSTRACT: We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology.
Available from: Matthew M. Cousins
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ABSTRACT: HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data.
DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2-T1 = HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves.
HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method.
DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies.
Available from: Lynne M Mofenson
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ABSTRACT: The Post-exposure Prophylaxis in Infants (PEPI)-Malawi trial evaluated infant antiretroviral regimens for prevention of post-natal HIV transmission. A multi-assay algorithm (MAA) that includes the BED capture immunoassay, an avidity assay, CD4 cell count, and viral load was used to identify women who were vs. were not recently infected at the time of enrollment (MAA recent, N = 73; MAA non-recent, N = 2,488); a subset of the women in the MAA non-recent group known to have been HIV infected for at least 2 years before enrollment (known non-recent, N = 54). Antibody maturation and viral diversification were examined in these women.
Samples collected at enrollment (N = 2,561) and 12-24 months later (N = 1,306) were available for serologic analysis using the BED and avidity assays. A subset of those samples was used for analysis of viral diversity, which was performed using a high resolution melting (HRM) diversity assay. Viral diversity analysis was performed using all available samples from women in the MAA recent group (61 enrollment samples, 38 follow-up samples) and the known non-recent group (43 enrollment samples, 22 follow-up samples). Diversity data from PEPI-Malawi were also compared to similar data from 169 adults in the United States (US) with known recent infection (N = 102) and known non-recent infection (N = 67).
In PEPI-Malawi, results from the BED and avidity assays increased over time in the MAA recent group, but did not change significantly in the MAA non-recent group. At enrollment, HIV diversity was lower in the MAA recent group than in the known non-recent group. HRM diversity assay results from women in PEPI-Malawi were similar to those from adults in the US with known duration of HIV infection.
Antibody maturation and HIV diversification patterns in African women provide additional support for use of the MAA to identify populations with recent HIV infection.
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