Summary statistics of sequencing experiments, read mapping, and error rates.

<p>For all four experiments, the total number of reads obtained and those overlapping amino acids 10 to 93 of the protease are reported. All 454/Roche reads mapping to this region were used in the haplotype reconstruction. For the Illumina Genome Analyzer, only those mapping to the region of highest entropy were considered. The last column reports mean and standard deviation of the sequencing error rate (1 – θ, where the parameter θ is estimated during haplotype reconstruction).</p

Performance of local haplotype reconstruction.

<p>For all four experiments, we report the total number of predicted haplotypes (column Reconstructed), the number of correct haplotypes (true positives, TP), the number of reconstructed haplotypes that do not match any of the original clones (false positives, FP), and the number of missed haplotypes (false negatives, FN). This number is equal to 10 – TP, because ten is the total number of haplotypes present in the sample. Sensitivity is defined as TP/(TP+FN) and specificity as TP/(TP+FP). Local haplotype reconstruction was performed on the 252 bp region of the HIV <i>pol</i> gene coding for protease amino acids 10 to 93 for the 454/Roche data, and on the 35 bp subregion of highest entropy for the Illumina reads.</p

Frequencies of all perfectly reconstructed haplotypes.

<p>Reported are, for all four experiments, the relative frequencies in percent of the reconstructed haplotypes matching exactly one of the original clones (named 07-56681, …, 08-04512) as estimated by direct mapping and by ShoRAH. Undetected haplotypes are indicated by a dash (‘—’).</p

Diversity of the protease region measured on the multiple sequence alignments.

<p>The plot shows the Shannon entropy of each column of the multiple sequence alignment of all mapped reads (orange bars) and its moving average in a window of 35 bp (blue lines). Numbering of bases follows the nucleotide position on the protease, i.e., position 1 corresponds to position 2253 on HXB2. As a reference, the top subfigure shows the diversity of the mixture of the original ten clones assuming equal frequencies. The remaining subfigures refer to the four sequencing experiments using either 454/Roche or Illumina GA and PCR amplification or not.</p

Global haplotype reconstruction at low diversity.

<p>Same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047046#pone-0047046-g002" target="_blank">Figure 2</a>, but the mean distance between clones is 1.9%.</p

HIV-1 Tropism Testing in Subjects Achieving Undetectable HIV-1 RNA: Diagnostic Accuracy, Viral Evolution and Compartmentalization

<div><p>Background</p><p>Technically, HIV-1 tropism can be evaluated in plasma or peripheral blood mononuclear cells (PBMCs). However, only tropism testing of plasma HIV-1 has been validated as a tool to predict virological response to CCR5 antagonists in clinical trials. The preferable tropism testing strategy in subjects with undetectable HIV-1 viremia, in whom plasma tropism testing is not feasible, remains uncertain.</p><p>Methods & Results</p><p>We designed a proof-of-concept study including 30 chronically HIV-1-infected individuals who achieved HIV-1 RNA <50 copies/mL during at least 2 years after first-line ART initiation. First, we determined the diagnostic accuracy of 454 and population sequencing of gp120 V3-loops in plasma and PBMCs, as well as of MT-2 assays before ART initiation. The Enhanced Sensitivity Trofile Assay (ESTA) was used as the technical reference standard. 454 sequencing of plasma viruses provided the highest agreement with ESTA. The accuracy of 454 sequencing decreased in PBMCs due to reduced specificity. Population sequencing in plasma and PBMCs was slightly less accurate than plasma 454 sequencing, being less sensitive but more specific. MT-2 assays had low sensitivity but 100% specificity. Then, we used optimized 454 sequence data to investigate viral evolution in PBMCs during viremia suppression and only found evolution of R5 viruses in one subject. No <i>de novo</i> CXCR4-using HIV-1 production was observed over time. Finally, Slatkin-Maddison tests suggested that plasma and cell-associated V3 forms were sometimes compartmentalized.</p><p>Conclusions</p><p>The absence of tropism shifts during viremia suppression suggests that, when available, testing of stored plasma samples is generally safe and informative, provided that HIV-1 suppression is maintained. Tropism testing in PBMCs may not necessarily produce equivalent biological results to plasma, because the structure of viral populations and the diagnostic performance of tropism assays may sometimes vary between compartments. Thereby, proviral DNA tropism testing should be specifically validated in clinical trials before it can be applied to routine clinical decision-making.</p></div

Selection of a CXCR4-using variant above the 454 sequencing error threshold during persistent viremia suppression in Subject 26.

<p><b><i>Panel A, antiretroviral treatment history, virological and immunological evolution</i></b><b>.</b> Continuous line, HIV-1 RNA levels; dashed line, CD4+ counts; horizontal bars, time period during which a given antiretroviral drug was prescribed. Vertical lines indicate the timepoints when 454 sequencing was performed. LPVr, lopinavir/ritonavir; AZT, zidovudine; ddI, didanosine; RAL, raltegravir. <b><i>Panel B, maximum likelihood nucleotide-based phylogenetic tree</i></b> including V3-loop haplotypes present at a frequency ≥0.6% in the virus population in plasma (triangles), PBMCs before therapy initiation (circles) and PBMCs after persistent viremia suppression (squares). The tree is rooted at the most frequent plasma sequence before antiretroviral treatment initiation. Filled symbols show predicted CXCR4-using viruses; open symbols show predicted CCR5-using viruses. Symbol size increases proportionally to the V3-loop haplotype frequency in the virus population in 10% intervals. Node reliability was tested using 1000 bootstraps; bootstrap values ≥50% are shown. The V3-loop aminoacid sequence translation is shown next to each taxon. Aminoacid changes relative to the predominant sequence in plasma are highlighted in bold and underlined. Gaps correspond to aminoacid indeterminations. A Geno2Pheno _[coreceptor] false positive rate (FPR) equal or lower than 10% was used to define CXCR4 use. The actual false positive rate of each sequence is shown. *Sequence #2 was identical to one detected in 0.04% of PBMC-associated viruses, below the error threshold, before treatment initiation.</p

Prevalence of CXCR4-using viruses using different tropism assays and settings.

<p>Bar plot showing the mean and 95% confidence intervals of the prevalence of subjects with CXCR4-using viruses using different tropism assays and settings. The Geno2Pheno_[coreceptor] clinical model was only used in pre-treatment bulk sequences derived from plasma RNA; otherwise, the clonal model was used. ESTA, Enhanced-Sensitivity Trofile™ Assay; FPR, Geno2Pheno_[coreceptor] false positive rate used to assign tropism; MT-2, Direct cocultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells. * p-value<0.05, two-sided exact binomial tst.</p

Accuracy of Tropism Assays Relative to the Enhanced-Sensitivity Trofile™ Assay.^{^a}

a<p>Values are mean percentages (95% confidence interval of the mean), calculated assuming a binomial distribution of the data.</p>b<p><i>G2P FPR</i>, Geno2Pheno_[coreceptor] false positive rate used for population and 454 sequencing to assign CXCR4 use . The Geno2Pheno_[coreceptor] clonal model was always used.</p>c<p><i>MT-2</i>, Direct cocultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells.</p>d<p><i>PPV</i>, Positive Predictive Value.</p>e<p><i>NPV</i>, Negative Predictive Value.</p>f<p>“Accuracy” is defined as: (True positives + True negatives)/Total.</p

Longitudinal tropism testing results per subject.^{^a^,^b^,^c}

a<p><i>ESTA</i>, <i>Enhanced-Sensitivity Trofile™ Assay</i>; <i>Pop Seq</i>, population sequencing of the V3-loop; <i>454</i>, 454 sequencing of the V3-loop; <i>MT2</i>, direct co-cultivation of patient-derived peripheral blood mononuclear cells with MT-2 cells. HIV-1 RNA levels are in copies/mL; CD4+ cell counts are in cells/mm³.</p>b<p>Population and 454 sequencing data shown here used the Geno2Pheno_[coreceptor] false positive rate cut-off providing highest accuracy when assigning HIV-1 tropism, i.e.: 20% and 10%, respectively. Based on internal error controls, only V3 forms present in ≥0.6% of viruses were considered for tropism prediction with 454 sequencing.</p>c<p>Tests detecting CXCR4-using HIV are reported as “<i>dual-mixed, DM</i>” for ESTA, <i>“X4”</i> for population sequencing, “<i>percent of X4 viruses”</i> for 454, and “<i>syncytium-inducing, SI</i>” for MT-2 assays; for clarity, viruses only using CCR5 are shown as dashes; NA, tropism test result not available due to lack of amplification.</p