Performance of in-house assay versus prototype kit.

<p>The normalized MFI values (n) and avidity index (a) for the in-house assay and kit were compared for drug naïve, known recent (infected <6 months) and long-term (infected >1 year) specimens. The boxes represent the 25^th to 75^th percentile of reactivity, while the middle lines represent the median values. Black dots indicate the 5^th to 95^th percentile of reactivity. </p

Inter-laboratory kit performance.

<p>The normalized MFI values (n) and avidity index (a) of known recent and long-term specimens, as measured by the prototype kits, were compared for each laboratory. </p

Changes towards population consensus.

<p>Top Row: The proportion of polymorphic sites where a mutant allele represents a change towards the (subtype-specific) population consensus. Bottom Row: The bias in changes towards population consensus. This is the ratio of the proportion of changes that are towards (subtype-specific) population consensus, compared to the expected proportion in the absence of selection, measured at polymorphic sites and with an assumed mutational transition to transversion ratio of two. A bias of 1 means the proportion of changes that are towards population consensus matches the expectation. The error bars give the 5 and 95 percentiles from 10,000 bootstraps of the individual data. Black, all changes; Blue, synonymous changes; Red, nonsynonymous changes.</p

Contribution to the mismatch in evolutionary rates if founder-like virus has a transmission advantage.

<p>Each point represents the mean contribution of the 34 individuals to the mismatch in phylogenetic rates if transmission occurs during the given time period, and where each viral sequence has a transmission fitness <i>w</i>_<i>d</i> = <i>e</i>^{−<i>α d</i>}. The contribution to the mismatch for each sampling time point for each individual was calculated as the ratio of the mean number of mutations from the founder population, <i>μ</i>, to the expected mean distance of transmitted virus from the founder population, <i>μT</i>, giving <i>m</i> = <i>μ</i>/<i>μT</i>. A mismatch of 1 therefore indicates the case where the within- and between-host rates of evolution are expected to be the same. We show results for a moderate (<i>α</i> = 1), large (<i>α</i> = 2), and very large (<i>α</i> = 3) transmission advantage. The error bars give the 5 and 95 percentiles from bootstrapping over the individuals 100,000 times. Black, all mutations; Blue, only synonymous mutations are considered when calculating the expected mismatch; Red, only nonsynonymous mutations are considered when calculating the expected mismatch (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007167#sec011" target="_blank">Methods</a>).</p

Diversity and divergence over time for 34 individuals for both p24 and gp41.

<p>Top Row: Mean pairwise diversity at first, second, and third codon positions over time for individuals (represented in yellow, pink, and light blue, respectively). The average change in mean pairwise diversity over time was inferred by linear regression. Bottom Row: Mean nonsynonymous (purple) and synonymous divergence (blue) over time for individuals.</p

Summary information for the 34 individuals and corresponding samples analysed in this study.

<p>Summary information for the 34 individuals and corresponding samples analysed in this study.</p

Evolutionary rates for p24 and gp41 at the within- and between-hosts scales measured in substitutions per site per year (subs/site/year).

<p>The within-host nonsynonymous (filled circles) and synonymous (open circles) substitution rates were estimated for p24 and gp41 for all 34 individuals, which are ordered from left to right (subtype A, pink; subtype C, green; subtype D, yellow). Blue and white columns correspond to the individual estimates. The estimates in the grey background indicate the between-host substitution rates for subtypes A, C, and D. The vertical lines represent the 95% credible intervals (solid lines, nonsynonymous; dashed lines, synonymous).</p

Number of samples classified as assay positive using the LAg-Avidity assay alone.

<p>Samples from the MACS, ALIVE, and HIVNET 001 cohorts (N = 1,780) were tested using the LAg-Avidity assay (LAg). Four assay cutoffs were evaluated: 0.5, 1.0, 1.5, and 3.0 optical density units (OD-n); samples were classified as assay positive if they were below the assay cutoff. The number and percentage of samples that were assay positive are presented separately for individuals with different durations of HIV infection (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082772#s2" target="_blank">Methods</a>). N indicates the number of samples in each group.</p

Performance characteristics of MAAs that include the LAg-Avidity assay and HIV viral load, with and without CD4 cell count.

<p>Samples from the MACS, ALIVE, and HIVNET 001 cohorts (N = 1,780) were tested using MAAs that included the LAg-Avidity assay and HIV viral load, with and without CD4 cell count. The cutoffs used for the LAg-Avidity assay (1.0 or 1.5 normalized optical density units [OD-n]) and the cutoff used for CD4 cell count (200 cells/mm³) are recommended by the assay manufacturer. The cutoff used for HIV viral load (VL, 1,000 copies/mL) was previously suggested for use with the Lag-Avidity assay along with self-report of antiretroviral treatment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082772#pone.0082772-Brookmeyer6" target="_blank">[35]</a>. Samples were classified as MAA positive if they met the criteria of each component assay. In the table, CD4 cell count testing is listed first in the MAA, since that testing must be performed at the time of sample collection. The number and percentage of samples that were MAA positive are presented separately for individuals with different durations of HIV infection (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082772#s2" target="_blank">Methods</a>). N indicates the number of samples in each group. The mean window period and shadow for each MAA are shown.</p

Proportion of samples classified as assay positive using the LAg-Avidity assay alone or with HIV viral load, as a function of the duration of HIV infection.

<p>Probability curves were generated by analyzing samples from three cohort studies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082772#s2" target="_blank">Methods</a>). (A) Probability curves generated using the LAg-Avidity assay with four different assay cutoffs (0.5, 1.0, 1.5, and 3.0 normalized optical density units [OD-n]). Samples were classified as assay positive if the LAg-Avidity assay result was below the assay cutoff. (B) Probability curves generated using the MAAs shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082772#pone-0082772-g002" target="_blank">Figure 2</a>. Samples were classified as MAA positive if results from each of the component assays met the requirements of the MAA.</p