HIV-1 subtype C phylogenetic relationship and diversity of HIV non-structural genes is consistent with the multiplicity of HIV-1 infection determined by analysis of the <i>env</i>/<i>gag</i> genes.

<p>A maximum likelihood phylogenetic tree was reconstructed using Fastree2 (Price <i>et al</i>., 2010) using the GTR+G model for nucleotide substitution and visualized in Figtree v.1.1.3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035491#pone.0035491-Rambaut1" target="_blank">[54]</a>. Alternative likelihood ratio tests <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035491#pone.0035491-Anisimova1" target="_blank">[55]</a> >0.95 are shown by an asterisk. Subjects infected with multiple viral variants are colored red. Patient B and D subtrees (individual trees on grey background) show branching topology of earliest sampling (0–90 days p/s) and represent examples of single (subject B) and multiple (subject D) HIV-1 transmission.</p

HIV-1 subtyping by analysis of phylogenetic relationships of HIV-1 non-structural genes.

<p>The analyzed region of HIV-1 genome corresponded to nucleotide positions 5,041 to 6,310 in HXB2. Three sequences were randomly selected for each study subject (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035491#s2" target="_blank">Methods</a>). A phylogenetic tree was inferred by Mr. Bayes using GTR model. The convergence was reached after 10 M MCMC run. The consensus tree was visualized in Figtree v.1.3.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035491#pone.0035491-Rambaut1" target="_blank">[54]</a>. Clade credibility values of >0.95 shown by asterisk, Subtype D cluster showed the support of 0.93 indicated by + symbol. HIV-1 subtype C reference sequences are shown as blue circles. All non-subtype C group M reference sequences are shown at the bottom of the phylogenetic tree. SIV sequence (CPZ.CM98.CAM3.AF115393) was used as an outgroup.</p

Individual distribution of pairwise distances for each of the non-stuctural genes,<i>vif</i> (HXB2 start 5041 to 5619), <i>vpr</i> (HXB2 start 5559 to 5850), <i>vpu</i> (HXB2 start 6062 to 6310), <i>tat</i> exon 1 (HXB2 start 5831 to 6045), and <i>rev</i> exon 1(HXB2 start 5970 to 6045).

<p>Individual distribution of pairwise distances for each of the non-stuctural genes,<i>vif</i> (HXB2 start 5041 to 5619), <i>vpr</i> (HXB2 start 5559 to 5850), <i>vpu</i> (HXB2 start 6062 to 6310), <i>tat</i> exon 1 (HXB2 start 5831 to 6045), and <i>rev</i> exon 1(HXB2 start 5970 to 6045).</p

HIV-1C diversity, mean and 95% confidence intervals for non-structural genes <i>vif</i>, <i>vpr</i>, <i>vpu</i>, <i>tat</i> exon 1 and <i>rev</i> exon 1 over the first 500 days p/s.

<p>Viral diversity for each subject was calculated using maximum composite likelihood model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035491#pone.0035491-Tamura2" target="_blank">[56]</a>.</p

Shows a reduction in the relative false-recency rate (rFRR) when viral load information is added to the combination BED plus PwD screening procedure.

<p>The figure shows how additional biomarker information can be used to improve the combination screening procedure for the 180-day cut-off. We hypothesize that treatment naïve participants with viral loads ≤1000 copies/mL are more likely to be recently infected with HIV. Results show an rFRR estimate of 31.6% (95% CI: 11–63.1) at a 90% sensitivity level. Since we are interested in the reduction of the rFRR by the PwD assay, we subtract this estimate from 100%. Thus, the PwD assay reduces the rFRR by 68.4% (or by at least 36.9% given the upper bound of the 95% CI) while maintaining a BED sensitivity of 90% for the subsample of VL >1000 copies/mL specimens. The figure displays both ROC curves for the viral load covariate and the corresponding rFRR estimates (displayed by the dotted vertical lines).</p

Shows a reduction in the relative false-recency rate when viral load information is added to the combination LAg plus PwD screening.

<p>The figure shows how additional biomarker information can be used to improve the combination screening procedure for the 130-day cut-off. We hypothesize that treatment naïve participants with viral loads ≤1000 copies/mL are more likely to be recently infected with HIV. Results show an rFRR estimate of 38.1% (95% CI: 15.8–88.6) at a 90% sensitivity level. Since we are interested in the reduction of the rFRR by the PwD assay, we subtract this estimate from 100%. Thus, the PwD assay reduces the rFRR by 61.9% (or by at least 11.4% given an upper bound of the 95% CI) while maintaining a LAg sensitivity of 90% for the subsample of VL <1000 copies/mL specimens. The figure displays both ROC curves for the viral load covariate and the corresponding rFRR estimates (displayed by the dotted vertical lines).</p

Combination assay screening to identify HIV infection recency for the 130 and 180-day cut-offs periods.

<p>Combination assay screening to identify HIV infection recency for the 130 and 180-day cut-offs periods.</p

Participant and covariate characteristics.

<p>Participant and covariate characteristics.</p

Area under the curve (AUC) of a receiver-operator characteristics (ROC) graph comparing the accuracy of the PwD, BED, and LAg assays in identifying HIV infection recency.

<p>Area under the curve (AUC) of a receiver-operator characteristics (ROC) graph comparing the accuracy of the PwD, BED, and LAg assays in identifying HIV infection recency.</p