First prospective comparison of genotypic versus phenotypic tropism assays in predicting virologic responses to maraviroc in a phase 3 study.

Maraviroc (MVC, a CCR5 antagonist) is only fully active against CCR5 tropic [R5] HIV-1, and tropism testing is required prior to initiating treatment. The MODERN study prospectively compared genotypic (GTT) and phenotypic (Trofile®) tropism testing with treatment-naive HIV-1-infected participants randomized 1:1 to either GTT or Trofile tropism assessments. Participants with R5 virus were randomized 1:1 to receive darunavir/ritonavir (DRV/r) with either MVC or tenofovir/emtricitabine. Screening samples were also retrospectively tested using the alternative assay. Positive predictive values (PPVs) for each assay were estimated using both the observed MVC+DRV/r response rate (HIV-1 RNA <50 copies/mL at Week 48) and model-based response estimates. The observed MVC+DRV/r response rate was 146/181 (80.7%) for GTT versus 160/215 (74.4%) for Trofile, with a stratification adjusted difference of 6.6% (95% CI, -1.5% to 14.7%) in favor of GTT. The model-based PPV estimates (±standard error) were 80.5% (±2.38) and 78.0% (±2.35) for GTT and Trofile, respectively (difference, 2.5%; 95% CI, -2.0% to 7.0%). Most participants had R5 results using both assays (285/396; 72%) and, of those, 79.3% (226/285) had HIV-1 RNA <50 copies/mL at Week 48. Both the genotypic and phenotypic tropism assays evaluated can effectively predict treatment response to MVC.

Clonal analysis of HIV-1 genotype and function associated with virologic failure in treatment-experienced persons receiving maraviroc: Results from the MOTIVATE phase 3 randomized, placebo-controlled trials.

Detailed clonal phenotypic/genotypic analyses explored viral-escape mechanisms during maraviroc-based therapy in highly treatment-experienced participants from the MOTIVATE trials. To allow real-time assessment of samples while maintaining a blind trial, the first 267 enrolled participants were selected for evaluation. At failure, plasma samples from 20/50 participants (16/20 maraviroc-treated) with CXCR4-using virus and all 38 (13 maraviroc-treated) with CCR5-tropic virus were evaluated. Of those maraviroc-treated participants with CXCR4-using virus at failure, genotypic and phenotypic clonal tropism determinations showed >90% correspondence in 14/16 at Day 1 and 14/16 at failure. Phylogenetic analysis of clonal sequences detected pre-treatment progenitor CXCR4-using virus, or on-treatment virus highly divergent from the Day 1 R5 virus, excluding possible co-receptor switch through maraviroc-mediated evolution. Re-analysis of pre-treatment samples using the enhanced-sensitivity Trofile® assay detected CXCR4-using virus pre-treatment in 16/20 participants failing with CXCR4-using virus. Post-maraviroc reversion of CXCR4-use to CCR5-tropic occurred in 7/8 participants with follow-up, suggesting selective maraviroc inhibition of CCR5-tropic variants in a mixed-tropic viral population, not emergence of de novo mutations in CCR5-tropic virus, as the main virologic escape mechanism. Maraviroc-resistant CCR5-tropic virus was observed in plasma from 5 treated participants with virus displaying reduced maximal percent inhibition (MPI) but no evidence of IC50 change. Env clones with reduced MPI showed 1-5 amino acid changes specific to each V3-loop region of env relative to Day 1. However, transferring on-treatment resistance-associated changes using site-directed mutagenesis did not always establish resistance in Day 1 virus, and key 'signature' mutation patterns associated with reduced susceptibility to maraviroc were not identified. Evolutionary divergence of the CXCR4-using viruses is confirmed, emphasizing natural selection not influenced directly by maraviroc; maraviroc simply unmasks pre-existing lineages by inhibiting the R5 virus. For R5-viral failure, resistance development through drug selection pressure was uncommon and manifested through reduced MPI and with virus strain-specific mutational patterns.

Analysis of Clinical HIV-1 Strains with Resistance to Maraviroc Reveals Strain-Specific Resistance Mutations, Variable Degrees of Resistance, and Minimal Cross-Resistance to Other CCR5 Antagonists.

Maraviroc (MVC) is an allosteric inhibitor of human immunodeficiency virus type 1 (HIV-1) entry, and is the only CCR5 antagonist licensed for use as an anti-HIV-1 therapeutic. It acts by altering the conformation of the CCR5 extracellular loops, rendering CCR5 unrecognizable by the HIV-1 envelope (Env) glycoproteins. This study aimed to understand the mechanisms underlying the development of MVC resistance in HIV-1-infected patients. To do this, we obtained longitudinal plasma samples from eight subjects who experienced treatment failure with phenotypically verified, CCR5-tropic MVC resistance. We then cloned and characterized HIV-1 Envs (n = 77) from plasma of pretreatment (n = 36) and treatment failure (n = 41) samples. Our results showed variation in the magnitude of MVC resistance as measured by reductions in maximal percent inhibition of Env-pseudotyped viruses, which was more pronounced in 293-Affinofile cells compared to other cells with similar levels of CCR5 expression. Amino acid determinants of MVC resistance localized to the V3 Env region and were strain specific. We also observed minimal cross-resistance to other CCR5 antagonists by MVC-resistant strains. We conclude that 293-Affinofile cells are highly sensitive for detecting and measuring MVC resistance through a mechanism that is CCR5-dependent yet independent of CCR5 expression levels. The strain-specific nature of resistance mutations suggests that sequence-based diagnostics and prognostics will need to be more sophisticated than simple position scoring to be useful for managing resistance in subjects taking MVC. Finally, the minimal levels of cross-resistance suggests that recognition of the MVC-modified form of CCR5 does not necessarily lead to recognition of other antagonist-modified forms of CCR5.

Social support and support groups among people with HIV/AIDS in Ghana.

HIV/AIDS, a chronic burden in Ghana, poses social and health outcome concerns to those infected. Examining the Medical Outcome Study Social Support Survey (MOS-SSS) instrument among 300 Ghanaians from a cross-sectional design, Principal Component Analysis yielded four factors (positive interaction, trust building, information giving, and essential support), which accounted for 85.73% of the total variance in the MOS-SSS. A logistic regression analysis showed that essential support was the strongest predictor of the length of time an individual stayed in the support group, whereas positive interaction indicated negative association. The study's implications for policy, research, and practice were discussed.

Characterizing the Diverse Mutational Pathways Associated with R5-Tropic Maraviroc Resistance: HIV-1 That Uses the Drug-Bound CCR5 Coreceptor.

UNLABELLED: Entry inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to preexisting virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use a drug-bound CCR5 coreceptor. To understand this R5 resistance pathway, we analyzed >500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2, in which treatment-experienced patients received maraviroc plus optimized background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis showed that the C2-V3-C3 region tends to genotypically correspond to the recombinant's phenotype, indicating its primary importance in conferring resistance. Between patients, there was a notable lack of commonality in the specific sites conferring resistance, confirming the unusual nature of R5-tropic resistance. We used coevolutionary and positive-selection analyses to characterize the genotypic determinants of resistance and found that (i) there are complicated covariation networks, indicating frequent coevolutionary/compensatory changes in the context of protein structure; (ii) covarying sites under positive selection are enriched in resistant viruses; (iii) CD4 binding sites form part of a unique covariation network independent of the V3 loop; and (iv) the covariation network formed between the V3 loop and other regions of gp120 and gp41 intersects sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context dependent and thus inherently unpredictable. IMPORTANCE: The entry inhibitor drug maraviroc makes the cell coreceptor CCR5 unavailable for use by HIV-1 and is now used in combination antiretroviral therapy. Treatment failure with drug-resistant virus is particularly interesting because it tends to be rare, with lack of sensitivity usually associated with the presence of CXCR4-using virus (CXCR4 is the main alternative coreceptor HIV-1 uses, in addition to CD4). We analyzed envelope sequences from HIV-1, obtained from 20 patients who enrolled in maraviroc clinical trials and experienced treatment failure, without detection of CXCR4-using virus. Evolutionary analysis was employed to identify molecular changes that confer maraviroc resistance. We found that in these individuals, resistant viruses form a distinct population that evolved once and was successful as a result of drug pressure. Further evolutionary analysis placed the complex network of interdependent mutational changes into functional groups that help explain the impediments to the emergence of maraviroc-associated R5 drug resistance.

Genotypic analysis of the V3 region of HIV from virologic nonresponders to maraviroc-containing regimens reveals distinct patterns of failure.

Changes in HIV tropism from R5 to non-R5 or development of drug resistance is often associated with virologic failure in patients treated with maraviroc, a CCR5 antagonist. We sought to examine changes in HIV envelope sequences and inferred tropism in patients who did not respond to maraviroc-based regimens. We selected 181 patients who experienced early virologic failure on maraviroc-containing therapy in the MOTIVATE trials. All patients had R5 HIV by the original Trofile assay before entry. We used population-based sequencing methods and the geno2pheno algorithm to examine changes in tropism and V3 sequences at the time of failure. Using deep sequencing, we assessed whether V3 sequences observed at failure emerged from preexisting subpopulations. From population genotyping data at failure, 90 patients had R5 results, and 91 had non-R5 results. Of the latter group, the geno2pheno false-positive rate (FPR) value fell from a median of 20 at screening to 1.1 at failure. By deep sequencing, the median percentage of non-R5 variants in these patients rose from 1.4% to 99.5% after a median of 4 weeks on maraviroc. In 70% of cases, deep sequencing could detect a pretreatment CXCR4-using subpopulation, which emerged at failure. Overall, there were two distinct patterns of failure of maraviroc. Patients failing with R5 generally had few V3 substitutions and low non-R5 prevalence by deep sequencing. Patients with non-R5 HIV who were failing developed very-high-prevalence non-R5 HIV (median, 99%) and had very low geno2pheno values.

Correlation between genotypic (V3 population sequencing) and phenotypic (Trofile ES) methods of characterizing co-receptor usage of HIV-1 from 200 treatment-naïve HIV patients screened for Study A4001078.

Assessment of HIV-1 co-receptor usage is essential to identify patients who are likely to respond to maraviroc (MVC)-containing regimens. Co-receptor usage of plasma virus from all treatment-naïve patients screened for a MVC clinical trial was assessed using phenotypic and genotypic methodologies to evaluate concordance between testing methods and to assess the quantity of CXCR4-using (non-R5) virus in samples giving discordant results. Co-receptor usage was prospectively measured using the enhanced sensitivity Trofile assay (Trofile ES) to screen patients for enrollment in Study A4001078. Population and deep sequencing methodologies were utilized retrospectively to analyze all screening samples, with co-receptor usage determined using the geno2pheno algorithm. Concordance between methods was explored using descriptive statistics. The quantity of non-R5 virus in all samples was measured using deep sequencing. Trofile ES and matched genotype results were obtained for 199screening samples. Concordance of Trofile ES with population genotyping (5.75% false-positive rate [FPR]) and deep sequencing (3.5% FPR; 2% non-R5 threshold) was 91.7% and 89.6%, respectively. Population genotype data were available for all samples with non-reportable Trofile ES results; the distribution of co-receptor usage in this set was consistent with that in the overall population: 75% (12/16) R5 and 25% (4/16) non-R5. The majority of samples contained non-R5 plasma HIV-1 RNA estimated at either <1 log(10) (62.0%) or ⩾4 log(10) (30.5%) copies/mL; the absolute amount of detectable non-R5 virus remained stable between screening and baseline visits. Samples originally classified as non-R5 by Trofile ES but R5 by population sequencing had a relatively low absolute amount of non-R5 virus (mean 2.1%, median 0.1%). The determination of co-receptor usage using either Trofile ES or genotyping methodologies showed similar frequencies of R5 and non-R5 virus in this treatment-naïve study population. For both concordant and discordant samples, population sequencing appropriately identified R5 samples with low levels of non-R5-using virus.

Population-based sequencing of the V3-loop can predict the virological response to maraviroc in treatment-naive patients of the MERIT trial.

BACKGROUND: MERIT was a randomized trial comparing maraviroc (MVC) + Combivir versus efavirenz (EFV) + Combivir in drug-naive patients screened as having R5 HIV-1 by the original Trofile assay (OTA). We retrospectively evaluated treatment response after rescreening for viral tropism using population-based V3-loop sequencing. METHODS: HIV env V3-loop was amplified in triplicate using reverse transcriptase-polymerase chain reaction from stored screening plasma and sequenced. Automated base calling was performed using custom software (RECall) and tropism inferred by geno2pheno (5.75% false-positive rate). Tropism results by genotype were compared with those of OTA and Enhanced Sensitivity Trofile assay (ESTA), where all results were available (n = 876). RESULTS: Approximately 8% of patients screened as having R5 virus by OTA were classified as having non-R5 virus by V3-loop genotyping. These patients were less likely to have early or sustained week-48 treatment response to MVC, but not EFV. When restricted to patients with R5 virus by genotype, reanalysis of the primary study endpoint (plasma viral load <50 copies/mL at week 48) showed noninferiority of MVC twice daily to EFV (67% vs. 68%). Rescreening by genotype and ESTA had 84% concordance; patients receiving MVC twice daily rescreened as having R5 virus had greater than 1 log10 copies per milliliter decrease in viral load over those rescreened as having non-R5 virus. Where genotype and ESTA screening results were discordant outcomes were similar. CONCLUSIONS: The exclusion of ∼8% of patients with CXCR4-using virus by population-based sequencing would likely have resulted in noninferior responses in the MVC twice-daily and EFV arms. Rescreening by ESTA and population-based sequencing predicted similar virological response.

Characterization of resistance to the nonnucleoside NS5B inhibitor filibuvir in hepatitis C virus-infected patients.

Filibuvir (PF-00868554) is an investigational nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural 5B (NS5B) RNA-dependent RNA polymerase currently in development for treating chronic HCV infection. The aim of this study was to characterize the selection of filibuvir-resistant variants in HCV-infected individuals receiving filibuvir as short (3- to 10-day) monotherapy. We identified amino acid M423 as the primary site of mutation arising upon filibuvir dosing. Through bulk cloning of clinical NS5B sequences into a transient-replicon system, and supported by site-directed mutagenesis of the Con1 replicon, we confirmed that mutations M423I/T/V mediate phenotypic resistance. Selection in patients of an NS5B mutation at M423 was associated with a reduced replicative capacity in vitro relative to the pretherapy sequence; consistent with this, reversion to wild-type M423 was observed in the majority of patients following therapy cessation. Mutations at NS5B residues R422 and M426 were detected in a small number of patients at baseline or the end of therapy and also mediate reductions in filibuvir susceptibility, suggesting these are rare but clinically relevant alternative resistance pathways. Amino acid variants at position M423 in HCV NS5B polymerase are the preferred pathway for selection of viral resistance to filibuvir in vivo.

An integrated transcriptomic and meta-analysis of hepatoma cells reveals factors that influence susceptibility to HCV infection.

Hepatitis C virus (HCV) is a global problem. To better understand HCV infection researchers employ in vitro HCV cell-culture (HCVcc) systems that use Huh-7 derived hepatoma cells that are particularly permissive to HCV infection. A variety of hyper-permissive cells have been subcloned for this purpose. In addition, subclones of Huh-7 which have evolved resistance to HCV are available. However, the mechanisms of susceptibility or resistance to infection among these cells have not been fully determined. In order to elucidate mechanisms by which hepatoma cells are susceptible or resistant to HCV infection we performed genome-wide expression analyses of six Huh-7 derived cell cultures that have different levels of permissiveness to infection. A great number of genes, representing a wide spectrum of functions are differentially expressed between cells. To focus our investigation, we identify host proteins from HCV replicase complexes, perform gene expression analysis of three HCV infected cells and conduct a detailed analysis of differentially expressed host factors by integrating a variety of data sources. Our results demonstrate that changes relating to susceptibility to HCV infection in hepatoma cells are linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication. This work identifies both known and novel host factors that may influence HCV infection. Our findings build upon current knowledge of the complex interplay between HCV and the host cell, which could aid development of new antiviral strategies.

Deep V3 sequencing for HIV type 1 tropism in treatment-naive patients: a reanalysis of the MERIT trial of maraviroc.

BACKGROUND: Deep sequencing is a highly sensitive technique that can detect and quantify the proportion of non-R5 human immunodeficiency virus (HIV) variants, including small minorities, that may emerge and cause virologic failure in patients who receive maraviroc-containing regimens. We retrospectively tested the ability of deep sequencing to predict response to a maraviroc-containing regimen in the Maraviroc versus Efavirenz in Treatment-Naive Patients (MERIT) trial. Results were compared with those obtained using the Enhanced Sensitivity Trofile Assay (ESTA), which is widely used in clinical practice. METHODS: Screening plasma samples from treatment-naive patients who received maraviroc and efavirenz in the MERIT trial were assessed. Samples were extracted, and the V3 region of HIV type 1 glycoprotein 120 was amplified in triplicate and combined in equal quantities before sequencing on a Roche/454 Genome Sequencer-FLX (n = 859). Tropism was inferred from third variable (V3) sequences, with samples classified as non-R5 if ≥2% of the viral population scored ≤3.5 using geno2pheno. RESULTS: Deep sequencing distinguished between responders and nonresponders to maraviroc. Among patients identified as having R5-HIV by deep sequencing, 67% of maraviroc recipients and 69% of efavirenz recipients had a plasma viral load <50 copies/mL at week 48, similar to the ESTA results: 68% and 68%, respectively. CONCLUSIONS: Reanalysis of the MERIT trial using deep V3 loop sequencing indicates that, had patients originally been screened using this method, the maraviroc arm would have likely been found to be noninferior to the efavirenz arm.

Mutations in gp41 are correlated with coreceptor tropism but do not improve prediction methods substantially.

BACKGROUND: The main determinants of HIV-1 coreceptor usage are located in the V3-loop of gp120, although mutations in V2 and gp41 are also known. Incorporation of V2 is known to improve prediction algorithms; however, this has not been confirmed for gp41 mutations. METHODS: Samples with V3 and gp41 genotypes and Trofile assay (Monogram Biosciences, South San Francisco, CA, USA) results were taken from the HOMER cohort (n=444) and from patients screened for the MOTIVATE studies (n=1,916; 859 with maraviroc outcome data). Correlations of mutations with tropism were assessed using Fisher's exact test and prediction models trained using support vector machines. Models were validated by cross-validation, by testing models from one dataset on the other, and by analysing virological outcome. RESULTS: Several mutations within gp41 were highly significant for CXCR4 usage; most strikingly an insertion occurring in 7.7% of HOMER-R5 and 46.3% of HOMER-X4 samples (MOTIVATE 5.7% and 25.2%, respectively). Models trained on gp41 sequence alone achieved relatively high areas under the receiver-operating characteristic curve (AUCs; HOMER 0.713 and MOTIVATE 0.736) that were almost as good as V3 models (0.773 and 0.884, respectively). However, combining the two regions improved predictions only marginally (0.813 and 0.902, respectively). Similar results were found when models were trained on HOMER and validated on MOTIVATE or vice versa. The difference in median log viral load decrease at week 24 between patients with R5 and X4 virus was 1.65 (HOMER 2.45 and MOTIVATE 0.79) for V3 models, 1.59 for gp41-models (2.42 and 0.83, respectively) and 1.58 for the combined predictor (2.44 and 0.86, respectively). CONCLUSIONS: Several mutations within gp41 showed strong correlation with tropism in two independent datasets. However, incorporating gp41 mutations into prediction models is not mandatory because they do not improve substantially on models trained on V3 sequences alone.

Baseline CD4(+) T-cell counts and weighted background susceptibility scores strongly predict response to maraviroc regimens in treatment-experienced patients.

BACKGROUND: Maraviroc-containing regimens are known to achieve virological suppression in many treatment-experienced patients. This study aimed to evaluate a more rigorous methodological approach to resistance-response analysis in large clinical studies and to better establish which subpopulations of patients were most likely to benefit from maraviroc by refining and extending previous subgroup analyses from the MOTIVATE studies. METHODS: Individual weighted optimized background therapy (OBT) susceptibility scores were calculated by combining genotypic or phenotypic resistance testing with prior drug use information. Virological response (HIV-1 RNA<50 copies/ml at week 48) using each of these methods was compared with a commonly used method of counting active drugs. Baseline predictors of virological response, including weighted or unweighted scoring, maraviroc use, baseline CD4(+) T-cell count, HIV-1 plasma viral load and tropism, were assessed by logistic regression modelling. RESULTS: Genotypic or phenotypic weighted methods were similarly predictive of virological response and better than counting active drugs. Weighted scoring and baseline CD4(+) T-cell count were the strongest predictors of virological response (P<0.0001): ≈70% of maraviroc patients with a weighted score ≥2 had a virological response, rising to ≈80% when the baseline CD4(+) T-cell count was ≥50 cells/mm(3). CONCLUSIONS: Approximately 80% of patients with a CD4(+) T-cell count ≥50 cells/mm(3) receiving maraviroc with the equivalent of at least two fully active agents achieved HIV-1 RNA<50 copies/ml at week 48 in the MOTIVATE studies. Genotypic and phenotypic weighted scores were similarly predictive of virological response.

Deep sequencing to infer HIV-1 co-receptor usage: application to three clinical trials of maraviroc in treatment-experienced patients.

BACKGROUND: The Maraviroc versus Optimized Therapy in Viremic Antiretroviral Treatment-Experienced Patients (MOTIVATE) studies compared maraviroc versus placebo in treatment-experienced patients with CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1), screened using the original Trofile assay. A subset with non-R5 HIV infection entered the A4001029 trial. We retrospectively examined the performance of a genotypic tropism assay based on deep sequencing of the HIV env V3 loop in predicting virologic response to maraviroc in these trials. METHODS: V3 amplicons were prepared from 1827 screening plasma samples and sequenced on a Roche/454 GS-FLX to a depth of >3000 sequences/sample. Samples were considered non-R5 if ≥2% of their viral population scored greater than or equal to -4.75 or ≤3.5 using the PSSM(x4/R5) or geno2pheno algorithms, respectively. RESULTS: Deep sequencing identified more than twice as many maraviroc recipients as having non-R5 HIV, compared with the original Trofile. With use of genotyping, we determined that 49% of maraviroc recipients with R5 HIV at screening had a week 48 viral load <50 copies/mL versus 26% of recipients with non-R5. Corresponding percentages were 46% and 23% with screening by Trofile. In cases in which screening assays differed, median week 8 log10 copies/mL viral load decrease favored 454. Other parameters predicted by genotyping included likelihood of changing to non-R5 tropism. CONCLUSIONS: This large study establishes deep V3 sequencing as a promising tool for identifying treatment-experienced individuals who could benefit from CCR5-antagonist-containing regimens.

The evolutionary analysis of emerging low frequency HIV-1 CXCR4 using variants through time--an ultra-deep approach.

Large-scale parallel pyrosequencing produces unprecedented quantities of sequence data. However, when generated from viral populations current mapping software is inadequate for dealing with the high levels of variation present, resulting in the potential for biased data loss. In order to apply the 454 Life Sciences' pyrosequencing system to the study of viral populations, we have developed software for the processing of highly variable sequence data. Here we demonstrate our software by analyzing two temporally sampled HIV-1 intra-patient datasets from a clinical study of maraviroc. This drug binds the CCR5 coreceptor, thus preventing HIV-1 infection of the cell. The objective is to determine viral tropism (CCR5 versus CXCR4 usage) and track the evolution of minority CXCR4-using variants that may limit the response to a maraviroc-containing treatment regimen. Five time points (two prior to treatment) were available from each patient. We first quantify the effects of divergence on initial read k-mer mapping and demonstrate the importance of utilizing population-specific template sequences in relation to the analysis of next-generation sequence data. Then, in conjunction with coreceptor prediction algorithms that infer HIV tropism, our software was used to quantify the viral population structure pre- and post-treatment. In both cases, low frequency CXCR4-using variants (2.5-15%) were detected prior to treatment. Following phylogenetic inference, these variants were observed to exist as distinct lineages that were maintained through time. Our analysis, thus confirms the role of pre-existing CXCR4-using virus in the emergence of maraviroc-insensitive HIV. The software will have utility for the study of intra-host viral diversity and evolution of other fast evolving viruses, and is available from http://www.bioinf.manchester.ac.uk/segminator/.

Population-based V3 genotypic tropism assay: a retrospective analysis using screening samples from the A4001029 and MOTIVATE studies.

BACKGROUND: The MOTIVATE-1 and 2 studies compared maraviroc (MVC) along with optimized background therapy (OBT) vs. placebo along with OBT in treatment-experienced patients screened as having R5-HIV (original Monogram Trofile). A subset screened with non-R5 HIV were treated with MVC or placebo along with OBT in a sister safety trial, A4001029. This analysis retrospectively examined the performance of population-based sequence analysis of HIV-1 env V3-loop to predict coreceptor tropism. METHODS: Triplicate V3-loop sequences were generated using stored screening plasma samples and data was processed using custom software ('ReCall'), blinded to clinical response. Tropism was inferred using geno2pheno ('g2p'; 5% false positive rate). Primary outcomes were viral load changes after starting maraviroc; and concordance with prior screening Trofile results. RESULTS: Genotype and Trofile results were available for 1164 individuals with virological outcome data (N = 169 non-R5 by Trofile). Compared with Trofile, V3 genotyping had a specificity of 92.6% and a sensitivity of 67.4% for detecting non-R5 virus. However, when compared with clinical outcome, virological responses were consistently similar between Trofile and V3 genotype at weeks 8 and 24 following the initiation of therapy for patients categorized as R5. CONCLUSION: Despite differences in sensitivity for predicting non-R5 HIV, week 8 and 24 week virological responses were similar in this treatment-experienced population. These findings suggest the potential utility of V3 genotyping as an accessible assay to select patients who may benefit from maraviroc treatment. Optimization of the predictive tropism algorithm may lead to further improvement in the clinical utility of HIV genotypic tropism assays.

Group-based randomized trial of contingencies for health and abstinence in HIV patients.

OBJECTIVE: Contingency management (CM) treatments are usually applied individually for drug abstinence, but CM can also be targeted toward health behaviors and implemented in groups. This study evaluated effects of a group-based CM intervention that focused on reinforcing health behaviors. METHOD: HIV-positive patients with cocaine or opioid use disorders (n = 170) were randomized to weekly CM or 12-step (TS) groups for 24 weeks (mean attendance was 10.8 +/- 8.1 sessions for CM participants and 9.0 +/- 6.9 session for TS participants). During the treatment period, both groups received compensation for attendance ($10 per session) and submission of urine samples (about $2 per sample). In addition, participants received $25 for submitting samples and completing evaluations at Months 1, 3, 6, 9, and 12; 65-75 of the 81 participants assigned to TS and 71-80 of the 89 participants assigned to CM completed these evaluations. During the treatment period, patients in the CM group received chances to win prizes contingent upon completing health activities and submitting substance-free specimens (M = $260, SD = $267). RESULTS: Mean attendance was 10.8 +/- 8.1 sessions for CM participants and 9.0 +/- 6.9 sessions for TS participants. CM participants submitted a significantly greater number of consecutive drug-free specimens than did TS participants (5.2 +/- 6.0 vs. 3.7 +/- 5.6), but proportions of negative samples did not differ between groups during treatment or at follow-up evaluations. From pre- to posttreatment, CM participants showed greater reductions in viral loads and HIV-risk behaviors than did TS participants, but these effects were not maintained throughout the follow-up period. CONCLUSIONS: These data suggest the efficacy of group-based CM for HIV-positive substance abusers, but more research is needed to extend the long-term benefits.

Detection of low-frequency pretherapy chemokine (CXC motif) receptor 4 (CXCR4)-using HIV-1 with ultra-deep pyrosequencing.

OBJECTIVE: Identification of low-frequency variants is of clinical importance in the identification of preexisting drug resistance. Using 'ultra-deep' sequencing, we address the detection of potential resistance to the chemokine (C-C motif) receptor 5 (CCR5) antagonist, maraviroc, due to the pretreatment presence of low levels of chemokine (CXC motif) receptor 4 (CXCR4)-using virus. METHODS: We present a novel protocol for the phenotyping of HIV based on '454' pyrosequence data and apply this to two large data sets comprised of 104 628 (before treatment, day 1) and 191 637 (after treatment, day 11) reads from the envelope region. We study resistance in the context of the evolutionary history of the intrapatient viral population. Variation was also investigated both within and outside the V3 region, the region associated with the receptor switch. RESULTS: CXCR4-using virus can be detected at low frequency prior to maraviroc treatment ( approximately 0.5%) and at high frequency after failure of monotherapy ( approximately 81%). Inferring an evolutionary tree from the 1674 unique reads that span the V3 region confirms that the CXCR4-using population emerged from low-frequency CXCR4-using variants present before treatment. Changes in the frequency of amino acid residues used at individual sites were found in regions outside the V3 region, indicative of other potential sites associated with receptor usage. CONCLUSION: We have provided a high-resolution snapshot of intrapatient viral variation, prior and after treatment with maraviroc, and detected preexisting CXCR4-using variants present at an extremely low frequency. The evolutionary analysis demonstrates the extent of diversity present at a single time point within an infected individual and the rapid effect of drug pressure on the structure of a viral population.

Cocaine-exposed infant behavior during Still-Face: risk factor analyses.

Prenatal cocaine exposure and the role of gender were evaluated using risk factor analyses to determine whether 6-month-old cocaine-exposed male infants demonstrated greater disruptions in infant-caregiver socioemotional interactions during a Still-Face test. Overall, non-cocaine-exposed infants spent more time looking at toys, compared with cocaine-exposed infants; nonexposed female infants spent more time scanning the environment, compared with nonexposed male infants. When caregiver behavior during the Still-Face was evaluated, differences emerged in amount of time the caregiver spent vocalizing to the infant. She vocalized more to a cocaine-exposed infant compared with a nonexposed one; she reduced vocalizing more during the test if the cocaine-exposed infant was female. An exposure by gender interaction emerged in the amount of change in caregiver vocalizations; however, the overarching hypothesis that male cocaine-exposed infants are at higher risk than nonexposed male, nonexposed female, and cocaine-exposed female infants was not supported. Because this interaction was evident in this cohort at 24 months, future research is needed to determine at what age an interaction begins to emerge in this cohort.