HRM scores for plasmid controls and samples from adults with different stages of HIV disease.

<p>The box and whisker plots show the distribution of HRM scores for six regions in the HIV genome in control plasmids (n = 5; Subtype B) and in adults with acute (n = 20), recent (n = 102), and non-recent (n = 67) HIV infection (see text). For each column, the median (closed square), interquartile range (IQR, box), lower inner fence (first quartile [Q1] – [1.5 X IQR]) and upper inner fence (third quartile [Q3] + [1.5 X IQR], whiskers), outliers (greater than [Q3] + [1.5 x IQR], open circle) and extremes (greater than [Q3] + [3 x IQR], asterisk) are shown.</p

Comparison of HRM scores from adults with recent vs. non-recent HIV infection^*.

<p>*Abbreviations: HRM: high resolution melting. CI: confidence intervals. Recent samples were collected a median of 189 days after a negative HIV test (range: 14–540 days); non-recent samples were from individuals infected >2 years (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027211#s2" target="_blank">Methods</a>). Results from adults with acute HIV infection were not included in this analysis. Statistically significant values are shown in bold text.</p>a<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027211#pone-0027211-g001" target="_blank">Figure 1</a>.</p>b<p>Based on multivariate logistic regression adjusting for all regions simultaneously.</p

Regions of the HIV genome analyzed using the HRM diversity assay.

<p>The relevant regions of the HIV genome are shown (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027211#pone.0027211-Kuiken1" target="_blank">[39]</a>). Numbers at the ends of each genomic segment correspond to coordinates in HXB2 (Genbank accession number: K03455). The amplicons used for HRM diversity analysis (regions analyzed) are indicated by shaded boxes. (A) <i>Gag</i> and <i>pol</i> amplicons: The GAG1 amplicon includes a portion of the coding regions for <i>gag</i> p7 and <i>gag</i> p1. The GAG2 amplicon includes the coding regions for <i>gag</i> p1 and <i>gag</i> p6 and extends into the coding region for HIV protease (PR); this amplicon also corresponds to the transframe (TF) protein. The POL amplicon spans the junction between the coding regions of HIV protease and HIV reverse transcriptase (RT). (B) <i>Env</i> amplicons: The ENV1 amplicon includes the coding region for heptad repeat 1 (HR1) of gp41, as well as portions of the coding regions to either side of HR1. The ENV3 amplicon includes the coding region for heptad repeat 2 (HR2), as well as portions of the coding regions to either side of HR2. The ENV2 amplicon includes the coding region for immunodominant region (IDR) cluster I of gp41, as well as portions of the coding regions for HR1 and HR2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027211#pone.0027211-Dorn1" target="_blank">[33]</a>.</p

Use of the HRM diversity assay as part of a multi-assay algorithm for HIV incidence determination.

<p>Panel A shows one example of a multi-assay algorithm developed for HIV incidence determination. In this algorithm, samples from HIV-infected individuals are first tested using the BED-CEIA assay, using a high assay cutoff to indicate non-recent HIV infection (BED screen). Samples that are below the BED screen cutoff (BED recent samples) are then tested using a second serologic assay, such as one based on antibody avidity (avidity screen). Samples that are below the cutoff for the second serologic assay are considered to be “serologic recent” samples. Samples with low CD4 cell count test results are then excluded as non-recent (note that CD4 cell count test results are usually obtained for all HIV-infected individuals at the time of sample collection). Finally, samples that are not excluded based on CD4 cell count are tested using a viral load assay, and samples with low viral loads are excluded as non-recent. The remaining samples are characterized as recent for the purpose of estimating HIV incidence. Panel B shows an alternative multi-assay algorithm that incorporates the HRM diversity assay. In this algorithm, samples that are characterized as serologic recent based on two assays (BED screen and an avidity screen) are tested with a multi-region HRM diversity assay. Samples that have a high HRM score in at least one of the regions tested are excluded as non-recent. Samples that fail to amplify in all regions tested are also excluded as non-recent, based on the assumption that they have low viral loads; this could be confirmed with a viral load assay. Samples that have low HRM scores in all regions tested are characterized as recent for the purpose of estimating HIV incidence.</p

Comparison of HRM scores from recently infected individuals from the HIVNET 001 cohort and the EXPLORE cohort^*.

<p>*Abbreviations: HRM: high resolution melting. CI: confidence intervals.</p>a<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027211#pone-0027211-g001" target="_blank">Figure 1</a>.</p>b<p>HRM scores were obtained for samples collected from adults with recent HIV infection. Samples from the HIVNET 001 cohort were obtained from women and from men who likely acquired HIV through intravenous drug use. Samples from the EXPLORE cohort were obtained from men who likely acquired HIV infection through sexual exposure with other men. The median number of days between collection of the sample used for testing and the individual's last negative HIV tests was 165 days (range: 49–216) for the HIVNET 001 cohort and 189 days (range: 14–540) for the EXPLORE cohort. Median HRM scores and the first and third quartile HRM scores (in parentheses) are shown.</p>c<p>Wilcoxon Rank sum test.</p

Regions of the HIV genome analyzed using the high resolution melting (HRM) diversity assay.

a<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027211#pone-0027211-g001" target="_blank">Figure 1</a>.</p>b<p>Mixed nucleotides were present at some positions (W: A/T; K: G/T; R: A/G; Y: C/T; D: A/G/T; S: G/C).</p

Relationship between HRM scores for the ENV1, ENV2, and GAG2 regions.

<p>Scatter plots are shown for HRM scores for adults with acute, recent, and non-recent infection: (A) ENV1 vs. GAG2, (B) ENV3 vs. GAG2, and (C) ENV1 vs. ENV3.</p