Analysis of HIV Using a High Resolution Melting (HRM) Diversity Assay: Automation of HRM Data Analysis Enhances the Utility of the Assay for Analysis of HIV Incidence

<div><h3>Background</h3><p>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.</p> <h3>Methods</h3><p>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.</p> <h3>Results</h3><p>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.</p> <h3>Conclusion</h3><p>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.</p> </div

Correlation between HRM scores calculated with the optimal DivMelt analysis protocols and HRM scores calculated using the manual method ^a.

a<p>Pearson’s correlation coefficient with manual HRM score. The region used to optimize each protocol is shown in the column on the left; the regions analyzed are shown in the headers for the six other columns.</p>b<p>Detailed region-specific analysis protocol descriptions are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051359#pone-0051359-t001" target="_blank">Table 1</a>.</p>c<p>HIV genome regions are described in the footnote of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051359#pone-0051359-t001" target="_blank">Table 1</a>.</p

Percentage of samples excluded by the internal quality control for each of the optimal DivMelt analysis protocols^a.

a<p>All values are reported as percentages. The region used to optimize each protocol is shown in the column on the left; the regions analyzed are shown in the headers for the six other columns.</p>b<p>Detailed region-specific analysis protocol descriptions are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051359#pone-0051359-t001" target="_blank">Table 1</a>.</p>c<p>HIV genome regions are described in the footnote of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051359#pone-0051359-t001" target="_blank">Table 1</a>.</p

Fluorescence vs. Temperature and -dFluorescence/Temperature vs. Temperature plots.

<p>DivMelt generates PDF plots to allow visualization of the melting data. The plate file name is given at the top of each plot. Below the file name is the sample location information. This information includes the sample well number (X1; ranging 1–96), the row letter (X2; ranging A–H), and the column designation (X3; ranging 1–12). The sample name is presented in bold above the Fluorescence vs. Temperature plot (top panel). The Fluorescence vs. Temperature plot tracks the decline in fluorescence as the DNA melts in response to the temperature increase. To the right of this panel, the settings used in the analysis are noted. The lower panel consists of the melting peak or –dFluorescence/dT vs. Temperature plot. To the right of the lower panel are the analysis results (marked on the melting peak in equivalent colors). These include the T1 and T2 values (in blue) and the temperature that corresponds to the peak (in purple). Up to three alternative T1 and T2 values may be given if they are present in the analysis (in green). These alternatives are additional values that met the theta value criteria for T1 and T2 identification but were not selected by DivMelt. The HRM score (Score), the diversity output of the assay, is noted between the top and bottom panels of the figure. This value is calculated by subtracting T1 from T2 and corresponds to the peak width.</p

Description of the shoulder height threshold tool.

<p>The shoulder height threshold tool allows selective inclusion or exclusion of small peaks that are connected to the principal melting peak (shoulders). The image indicates the variation in T1 selection based on the specifications of the shoulder height threshold tool and the height of the shoulder.</p

Univariate linear associations of albumin and other selected participant characteristics with body mass, fat mass and fat free mass index.

<p>CD4 counts are cells/µl; Asterisks represent p-values for association within the column HIV/CD4 group (*<0.05, **<0.01, ***<0.001);</p>1<p>Categorized as none, 1–3, 3–5, >5, treated as continuous variable;</p>2<p>Aspartate amino transferase <i>or</i> alanine amino transferase >35 mg/dl.</p

Univariate variables and Change in CD4 count at 6, 12, and 24 months from pre-ART visit.

<p>Abbreviations: ART = antiretroviral therapy, BMI = body mass index, FFMI = fat free mass index, FMI = fat mass index, RWF =  Rwandan Francs, WHO = World Health Organization.</p

Baseline characteristics of study participants (n = 895).

a<p>P-value is for comparing all four HIV/CD4 groups.</p>b<p>n(%) for categorical variables or mean±standard-deviation for continuous variables.</p>c<p>Alanine aminotransferase or aspartate aminotransferase>35 mg/dL.</p

Description of ART usage among 371 women who initiated treatment.

<p>Proportions of HIV+ ART initiators: 26.7% used AZT, 62.5% used D4T, 89.1% used NRTI total and 1.3% used NRTI other than AZT or D4T.</p><p>Description of ART usage among 371 women who initiated treatment.</p