Prevalence and impact of minority variant drug resistance mutations in primary HIV-1 infection.

To evaluate minority variant drug resistance mutations detected by the oligonucleotide ligation assay (OLA) but not consensus sequencing among subjects with primary HIV-1 infection.Observational, longitudinal cohort study. Consensus sequencing and OLA were performed on the first available specimens from 99 subjects enrolled after 1996. Survival analyses, adjusted for HIV-1 RNA levels at the start of antiretroviral (ARV) therapy, evaluated the time to virologic suppression (HIV-1 RNA<50 copies/mL) among subjects with minority variants conferring intermediate or high-level resistance.Consensus sequencing and OLA detected resistance mutations in 5% and 27% of subjects, respectively, in specimens obtained a median of 30 days after infection. Median time to virologic suppression was 110 (IQR 62-147) days for 63 treated subjects without detectable mutations, 84 (IQR 56-109) days for ten subjects with minority variant mutations treated with ≥3 active ARVs, and 104 (IQR 60-162) days for nine subjects with minority variant mutations treated with <3 active ARVs (p = .9). Compared to subjects without mutations, time to virologic suppression was similar for subjects with minority variant mutations treated with ≥3 active ARVs (aHR 1.2, 95% CI 0.6-2.4, p = .6) and subjects with minority variant mutations treated with <3 active ARVs (aHR 1.0, 95% CI 0.4-2.4, p = .9). Two subjects with drug resistance and two subjects without detectable resistance experienced virologic failure.Consensus sequencing significantly underestimated the prevalence of drug resistance mutations in ARV-naïve subjects with primary HIV-1 infection. Minority variants were not associated with impaired ARV response, possibly due to the small sample size. It is also possible that, with highly-potent ARVs, minority variant mutations may be relevant only at certain critical codons

Comparison of Oligonucleotide Ligation Assay and Consensus Sequencing for Detection of Drug-Resistant Mutants of Human Immunodeficiency Virus Type 1 in Peripheral Blood Mononuclear Cells and Plasma

Drug-resistant mutants of human immunodeficiency virus type 1 (HIV-1) recede below the limit of detection of most assays applied to plasma when selective pressure is altered due to changes in antiretroviral treatment (ART). Viral variants with different mutations are selected by the new ART when replication is not suppressed or wild-type variants with greater replication fitness outgrow mutants following the cessation of ART. Mutants selected by past ART appear to persist in reservoirs even when not detected in the plasma, and when conferring cross-resistance they can compromise the efficacy of novel ART. Oligonucleotide ligation assay (OLA) of virus in plasma and peripheral blood mononuclear cells (PBMC) was compared to consensus sequence dideoxynucleotide chain terminator sequencing for detection of 91 drug resistance mutations that had receded below the limit of detection by sequencing of plasma. OLA of plasma virus detected 27.5% (95% confidence interval [CI], 19 to 39%) of mutant genotypes; consensus sequencing of the PBMC amplicon from the same specimen detected 23.1% (95% CI, 14 to 34%); and OLA of PBMC detected 53.8% (95% CI, 44 to 64%). These data suggest that concentrations of drug-resistant mutants were greater in PBMC than in plasma after changes in ART and indicate that the OLA was more sensitive than consensus sequencing in detecting low levels of select drug-resistant mutants

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

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

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

Multiple Viral Genetic Analyses Detect Low-Level Human Immunodeficiency Virus Type 1 Replication during Effective Highly Active Antiretroviral Therapy

To evaluate human immunodeficiency virus type 1 (HIV-1) replication and selection of drug-resistant viruses during seemingly effective highly active antiretroviral therapy (HAART), multiple HIV-1 env and pol sequences were analyzed and viral DNA levels were quantified from nucleoside analog-experienced children prior to and during a median of 5.1 (range, 1.8 to 6.4) years of HAART. Viral replication was detected at different rates, with apparently increasing sensitivity: 1 of 10 by phylogenetic analysis; 2 of 10 by viral evolution with increasing genetic distances from the most recent common ancestor (MRCA) of infection; 3 of 10 by selection of drug-resistant mutants; and 6 of 10 by maintenance of genetic distances from the MRCA. When four- or five-drug antiretroviral regimens were given to these children, persistent plasma viral rebound did not occur despite the accumulation of highly drug-resistant genotypes. Among the four children without genetic evidence of viral replication, a statistically significant decrease in the genetic distance to the MRCA was detected in three, indicating the persistence of a greater number of early compared to recent viruses, and their HIV-1 DNA decreased by ≥0.9 log(10), resulting in lower absolute DNA levels (P = 0.007). This study demonstrates the variable rates of viral replication when HAART has suppressed plasma HIV-1 RNA for years to a median of <50 copies/ml and that combinations of four or five antiretroviral drugs suppress viral replication even after short-term virologic failure of three-drug HAART and despite ongoing accumulation of drug-resistant mutants. Furthermore, the decrease of cellular HIV-1 DNA to low absolute levels in those without genetic evidence of viral replication suggests that monitoring viral DNA during HAART may gauge low-level replication