Representative results of an experiment measuring genetic drift in HIV populations.

<p>A. The frequency of Vpr-FS in replicate cultures. Each point represents one of 12 independent cultures done for each dilution. B. Variance in frequency of Vpr-FS-StuI was calculated from data shown in panel A. Each point corresponds to average variance in frequency of Vpr-FS-StuI from initial 50%, adjusted for contribution from assay variability (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000431#s4" target="_blank">Materials and Methods</a>). The expected variance for an ideal population is shown as straight dashed line.</p

Figure 1

<p>A. Difference in length of the insertion in <i>vpr</i> gene of Vpr-FS and Vpr-FS-StuI clones allows measurements of their relative abundance in mixtures. Fragments of genomes containing insertions are amplified in RT-PCR reactions using fluorescently labeled primers. Relative abundance of products of different length can be quantitated from fluorescence intensity of the corresponding bands in GeneScan assay. B. Scheme of the experimental approach used to correlate the number of infected cells to the amount of genetic drift. Viral variants Vpr-FS and Vpr-FS-StuI are mixed in 1∶1 ratio. The mixture is serially diluted and used to infect multiple replicates of cell cultures (shown 6 replicates for each dilution). Cultures are scored as positive (black circles) or negative (white circles) for HIV infection. Cell-free virus is collected from virus-positive wells and the frequency of virions of each variant is measured by the GeneScan assay. The data is used to calculate the amount of genetic drift for each dilution. Dilutions containing positive and negative wells are used to calculate TCID50, which provides the measure of viral population size at the start of the experiment.</p

Genetic drift under a variety of culture conditions.

1<p>Fold increase was averaged for all population sizes in all experiments. SE is standard error.</p>2<p>Variance is significantly greater than the variance of the standard C8166 condition.</p>3<p>Variance is significantly less than the variance of the standard C8166 condition.</p>4<p>Adjusted for differences in population sizes. CI is 95% confidence intervals.</p

HIV-1 drug resistance detected by consensus sequencing and OLA in ARV-naïve subjects and virologic response to ARV therapy.

<p>In this table, the subset of subjects who received antiretroviral (ARV) therapy are grouped based on whether they had drug resistance detected by consensus sequencing (Group I), drug resistance detected by OLA but who received at least three active ARV agents (Group II), or drug resistance detected by OLA who received fewer than three active agents (Group III). ARVs are highlighted in grey if subjects had mutations conferring at least intermediate level resistance to that ARV. K70R, L74V, T215F, and V82S/T were not detected in any treated subjects.</p><p>CS: consensus sequencing; OLA: oligonucleotide ligation assay; VL: viral load (HIV-1 RNA level); ARV: antiretroviral; VF: virologic failure; IDV: indinavir, HU: hydroxyurea, ABC: abacavir, EFV: efavirenz, NVP: nevirapine, r-: ritonavir-boosted, LPV: lopinavir, ATZ: atazanavir; DNS: did not suppress.</p>1<p>Log₁₀ copies/mL.</p>2<p>Antiretroviral medications were switched on day 5 due to side effects.</p>3<p>Subject #69234 subsequently discontinued medications two months later due to adherence difficulties.</p>4<p>OLA probes did not test for M41L and T215D.</p>5<p>OLA on PBMC for subject 56710 yielded indeterminate results for T215Y.</p>6<p>DNS: did not suppress prior to discontinuing ARVs or study censorship. Subjects #26973, 44378, and 78882 were followed for 104, 44, and 63 days, respectively, while receiving ARVs.</p

HIV-1 drug resistance in ARV-naïve subjects with primary HIV-1 infection.

<p>ARV: antiretroviral; OLA: oligonucleotide ligation assay; PBMC: peripheral blood mononuclear cells.</p><p>+ = subjects with ≥1 mutation or mixture.</p><p>− = subjects without mutations or with indeterminate results.</p><p>Numbers represent subjects in whom HIV-1 drug resistance was/was not detected in plasma and PBMC specimens that had been obtained a median of 29 (IQR 19–66) and 31 (IQR 19–66) days after HIV-1 infection, respectively; all specimens were collected within six months of infection. McNemar's exact tests compare only subjects with discordant results (indicated in bold).</p

Characteristics of study subjects with primary HIV-1 infection evaluated for drug resistance mutations.

<p>OLA: oligonucleotide ligation assay; CS: consensus sequencing; IQR: interquartile range; NS: not significant at p = .05; ARV: antiretroviral; PI: protease inhibitor; NRTI: nucleoside reverse transcriptase inhibitor; NNRTI: non-nucleoside reverse transcriptase inhibitor therapy.</p><p>*differences between groups were not significant in analyses that were both unadjusted and adjusted for time from infection to the date of sampling.</p

Time to suppression of plasma HIV-1 RNA levels among previously ARV-naïve subjects with and without minority variant drug resistance mutations.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028952#pone-0028952-g001" target="_blank">Figure 1:</a> The median time to virologic suppression (HIV-1 RNA<50 copies/mL) was 110 (IQR 62–147) days for 63 treated subjects without detectable mutations (solid line), 84 (IQR 56–109) days for 10 subjects with minority variant mutations treated with ≥3 active ARVs (dashed line), and 104 (60–162) days for nine subjects with minority variant mutations treated with <3 active ARVs (dotted line) (p = .9).</p

Predictors of concurrent HIV-1 cell-free RNA detection in breast milk during PMTCT HAART.

<p>Notes. N, number of subjects in analysis. The association between breast milk HIV-1 RNA detection and each covariate is examined separately in an unadjusted model, and adjusted for days on HAART.</p><p>*Analysis for breast milk HIV-1 DNA was restricted to the first month postpartum because the majority of HIV-1 DNA measurements (154/163) were made during this time interval.</p><p>**In all models adjusted for time, days on HAART remained insignificant with an OR of approximately 1.0 (not shown).</p

Comparison of women by detection of breast milk HIV-1 RNA.

<p>Notes. Median (IQR) or frequency (n/N) are shown for each correlate. VL, viral load; p-d, person-days 95%CI, 95% confidence interval;</p><p>*At any time (includes antenatal and on HAART).</p><p>**Includes clinical mastitis, cracked nipples, localized swelling, and breast abscess.</p><p>***Measurements at delivery or first postpartum visit.</p

Detection of breast milk HIV-1 RNA in women receiving HAART for PMTCT.

<p>The detection of HIV-1 RNA is shown for 25 women on HAART postpartum. Visits were ranked by date, and are shown only for visits while HAART was being administered. *The first postpartum HIV-1 plasma RNA measurement is provided for each woman. M-586 and -538 did not have a plasma viral load measured within a week of delivery; plasma viral loads shown for these two women coincide with visit 1.</p