Protease Resistance Mutations Research Articles

Exposition and sequencing to antiretrovirals in HIV-1 patients with triple-class antiretroviral failure, and harbouring protease resistance mutations

Address: 1University Hospital Germans Trias, Barcelona, Spain, 2Hospital Sant Pau, Barcelona, Spain, 3Hospital Carlos III, Madrid, Spain, 4University Hospital Germans Trias, Badalona, Spain, 5Hospital Xeral Cies, Vigo, Spain, 6Hospital Virgen del Rosell, Cartagena, Spain, 7Hospital Virgen de la Luz, Cuenca, Spain, 8Hospital Clinico Universitario San Cecilio, Granada, Spain and 9Hospital Juan Canalejo, A Coruna, Spain * Corresponding author

HIV‐1 replication capacity and genotype changes in patients undergoing treatment interruption or lamivudine monotherapy

The objective of this study was to investigate the mechanisms underlying the virological and immunological changes occurring in failing HIV-1 infected patients undergoing treatment interruption or lamivudine monotherapy (the E-184V Study). Associations were sought between the de-selection of individual reverse transcriptase and protease resistance mutations and replication capacity recovery, HIV-RNA changes, and immunological changes. The replication capacity recovery was defined as the ratio between the replication capacity at weeks 24 or 48, and that measured at baseline. The replication capacity recovery, which was evaluable in 21 patients at week 24 and in 18 at week 48, was significantly higher in the treatment interruption than in the lamivudine group at week 24 (P = 0.002). Forty-eight week replication capacity recovery was greater when the 184V (P = 0.023), the 41L (P = 0.02), or the 215Y mutation (P = 0.037) were deselected at week 12. A greater reduction in the CD4+/CD8+ ratio at week 48 (P = 0.038) was observed as the 184V mutation was deselected and the de-selection of the 184V mutation at week 12 was the only independent predictor of the change of the CD4+/CD8+ ratio at week 48 from baseline at multivariable analysis (F-value = 6.72, P = 0.021). In conclusion, among patients undergoing treatment interruption or lamivudine monotherapy, the recovery of HIV-1 replication capacity was associated with the de-selection of reverse transcriptase mutations. The de-selection of the 184V mutation predicts independently a reduction in the CD4+/CD8+ ratio.

Impact of baseline protease genotype and phenotype on the response to darunavir outside clinical trials

Impact of baseline protease genotype and phenotype on the response to darunavir outside clinical trials

Human Immunodeficiency Virus Type 1 Drug Resistance Mutations in Peripheral Blood Mononuclear Cell Proviral DNA among Antiretroviral Treatment-Naive and Treatment-Experienced Patients from Pune, India

The prevalence of HIV drug resistance (HIVDR) mutations in the HIV protease (PR) and reverse transcriptase (RT) genes was estimated from peripheral blood mononuclear cells (PBMCs) in a study population of 25 antiretroviral (ARV) therapy-naive and 50 ARV-experienced chronically infected patients from Pune city, Maharashtra State, western India. Of the 75 study HIV-1 sequences, 73 belonged to subtype C and 2 to subtype A1. On phylogenetic analysis, the study subtype C sequences sub clustered randomly with different Indian and non-Indian subtype C sequences, emphasizing the HIV-1 subtype C pol gene diversity. The heterosexual route was the most common route of transmission (74.67%). There were no observable HIVDR mutations in ARV-naive patients. The ARV-experienced patients had a history of exposure to nucleoside reverse transcriptase inhibitor and nonnucleoside reverse transcriptase inhibitor combinations. At least one HIVDR mutation in RT was observed in 29 (80.55%) of ARV-experienced patients with evidence of failing therapy. M184V was the most common observed HIVDR mutation. No PR major mutations were observed among ARV-experienced patients. A higher prevalence of proviral HIVDR mutations in PBMCs was associated with irregular adherence to therapy (p < 0.05) and HIV-1 RNA levels > 1000 copies/ml (p < 0.001).

Viral fitness: relation to drug resistance mutations and mechanisms involved: nucleoside reverse transcriptase inhibitor mutations

Nucleoside and nucleotide reverse transcriptase inhibitors constitute the backbone of highly active antiretroviral therapy in the treatment of HIV-1 infection. One of the major obstacles in achieving the long-term efficacy of anti-HIV-1 therapy is the development of resistance. The advent of resistance mutations is usually accompanied by a change in viral replicative fitness. This review focuses on the most common nucleoside reverse transcriptase inhibitor-associated mutations and their effects on HIV-1 replicative fitness. Recent studies have explained the two main mechanisms of resistance to nucleoside reverse transcriptase inhibitors and their role in HIV-1 replicative fitness. The first involves mutations directly interfering with binding or incorporation and seems to impact replicative fitness more adversely than the second mechanism, which involves enhanced excision of the newly incorporated analogue. Further studies have helped explain the antagonistic effects between amino acid substitutions, K65R, L74V, M184V, and thymidine analogue mutations, showing how viral replicative fitness influences the evolution of thymidine analogue resistance pathways. Nucleoside reverse transcriptase inhibitor resistance mutations impact HIV-1 replicative fitness to a lesser extent than protease resistance mutations. The monitoring of viral replicative fitness may help in the management of HIV-1 infection in highly antiretroviral-experienced individuals.

Dual Selection Pressure by Drugs and HLA Class I-Restricted Immune Responses on Human Immunodeficiency Virus Type 1 Protease

To determine the influence of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells on the development of drug resistance mutations in the HIV-1 protease, we analyzed protease sequences from viruses from a human leukocyte antigen class I (HLA class I)-typed cohort of 94 HIV-1-positive individuals. In univariate statistical analyses (Fisher's exact test), minor and major drug resistance mutations as well as drug-associated polymorphisms showed associations with HLA class I alleles. All correlations with P values of 0.05 or less were considered to be relevant without corrections for multiple tests. A subset of these observed correlations was experimentally validated by enzyme-linked immunospot assays, allowing the definition of 10 new epitopes recognized by CD8+ T cells from patients with the appropriate HLA class I type. Several drug resistance-associated mutations in the protease acted as escape mutations; however, cells from many patients were still able to generate CD8+ T cells targeting the escape mutants. This result presumably indicates the usage of different T-cell receptors by CD8+ T cells targeting these epitopes in these patients. Our results support a fundamental role for HLA class I-restricted immune responses in shaping the sequence of the HIV-1 protease in vivo. This role may have important clinical implications both for the understanding of drug resistance pathways and for the design of therapeutic vaccines targeting drug-resistant HIV-1.

Protease inhibitor associated mutations compromise the efficacy of therapy in human immunodeficiency virus – 1 (HIV-1) infected pediatric patients: a cross-sectional study

BackgroundAlthough the introduction of combined therapy with reverse transcriptase and protease inhibitors has resulted in considerable decrease in HIV related mortality; it has also induced the development of multiple drug-resistant HIV-1 variants.The few studies on HIV-1 mutagenesis in HIV infected children have not evaluated the impact of HIV-1 mutations on the clinical, virological and immunological presentation of HIV disease that is fundamental to optimizing the treatment regimens for these patients.ResultsA cross sectional study was conducted to evaluate the impact of treatment regimens and resistance mutation patterns on the clinical, virological, and immunological presentation of HIV disease in 41 children (25 male and 16 female) at the Robert Wood Johnson Pediatric AIDS Program in New Brunswick, New Jersey. The study participants were symptomatic and had preceding treatment history with combined ARV regimens including protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Fifteen (36.6%) children were treated with NRTI+NNRTI+ PI, 6 (14.6%) with NRTI+NNRTIs, 13 (31.7%) with NRTI+PIs, and the remaining 7 (17.1%) received NRTIs only.Combined ARV regimens did not significantly influence the incidence of NRTI and NNRTI associated mutations. The duration of ARV therapy and the child's age had no significant impact on the ARV related mutations. The clinico-immunological presentation of the HIV disease was not associated with ARV treatment regimens or number of resistance mutations. However, primary mutations in the protease (PR) gene increased the likelihood of plasma viral load (PVL) ≥ 10,000 copies/mL irrespective of the child's age, duration of ARV therapy, presence of NRTI and NNRTI mutation. Viremia ≥ 10,000 copies/mL was recorded in almost all the children with primary mutations in the PR region (n = 12/13, 92.3%) as compared with only 50.0% (n = 14/28) of HIV infected children without (PR-), P < 0.008. However, CD-4 T cells were not affected by the mutations in the PR gene of the HIV-1 isolates.ConclusionPrimary PR resistance mutations significantly increase the likelihood for high viral replication in pediatric patients with moderate/severe HIV-1 infection, which may affect the long-term clinical prognosis of the HIV infected children.

Successful Treatment with Atazanavir and Lopinavir/Ritonavir Combination Therapy in Protease Inhibitor-Susceptible and Protease Inhibitor-Resistant HIV-Infected Patients

The combination of atazanavir (ATV) plus lopinavir/ritonavir (LPV/r) has been used in practice. However, clinical data supporting its use are limited. The objective of this study was to evaluate the efficacy and tolerability of regimens with ATV + LPV/r in protease inhibitor (PI)-susceptible and PI-resistant patients. A retrospective review of 2703 charts was performed to identify all patients who received ATV + LPV/r. From June 2003 to January 2005, 33 patients received ATV + LPV/r with nucleoside reverse transcriptase inhibitors (NRTIs) for 3 months or more. Virologic success (HIV-RNA < 400 copies per milliliter) was achieved in 30 patients (91%) in a median of 10 weeks (range, 2-68). Nineteen of the 23 patients (83%) who had ultrasensitive viral load (VL) assays were nondetectable. Among patients with 6 or more protease resistance (PR) mutations (PI-resistant), 11 of 14 (79%) achieved virologic success. Eleven of those received phenotypic testing (10 Virtual Phenotype, VircoLab, Baltimore, MD). Despite predicted phenotypic resistance to ATV (6 patients) and LPV/r (7 patients), virologic success was achieved in 4 of 6 (67%) and 4 of 7 (57%), respectively. The 3 PI-resistant patients who were virologic failures had extensive prior LPV/r use, 8-11 PR mutations, and predicted phenotypic resistance to LPV/r, but 2 of 3 had CD4 increases with ATV + LPV/r. Overall, 28 patients (85%) continue to tolerate ATV + LPV/r for a median of 32 weeks follow-up (range, 12-76). Combination ATV + LPV/r with NRTIs appears safe, tolerable, and efficacious in PI-resistant patients (>/=6 PR mutations) and predicted phenotypic resistance to ATV and LPV/r. Further studies of ATV + LPV/r in HIV-treatment are warranted.

Frequency of Protease and Reverse Transcriptase Drug Resistance Mutations in Naïve HIV-Infected Patients

Infections with drug-resistant HIV viruses in naïve subjects may cause antiretroviral (ARV) treatment failure. The prevalence of ARV resistance mutations in HIV-1 transcripts of infected naïve patients from northeast Mexico was determined in this study. RNA was extracted from plasma samples of 42 naïve individuals who were diagnosed between February 2001 and September 2003 as HIV-1 infected. Both protease (Pr) and reverse transcriptase (RT) were sequenced in 30 patients. In six samples only the RT segment was sequenced and in three samples only the protease segment was analyzed. One of 36 isolates (2.8%) had the M184V resistance mutation to nucleoside retrotranscriptase inhibitors. In the Pr segment, only minor mutations were detected in 27/33 isolates (81.8%). In this first study, prevalence of major mutations associated with ARV resistance in naïve patients in northeast Mexico is low compared to other countries, perhaps due to a low level of exposure of this population to ARV drugs.

Molecular Dynamic and Free Energy Studies of Primary Resistance Mutations in HIV-1 Protease−Ritonavir Complexes

To understand the basis of drug resistance of the HIV-1 protease, molecular dynamic (MD) and free energy calculations of the wild-type and three primary resistance mutants, V82F, I84V, and V82F/I84V, of HIV-1 protease complexed with ritonavir were carried out. Analysis of the MD trajectories revealed overall structures of the protein and the hydrogen bonding of the catalytic residues to ritonavir were similar in all four complexes. Substantial differences were also found near the catalytic binding domain, of which the double mutant complex has the greatest impact on conformational changes of the protein and the inhibitor. The tip of the HIV-1 protease flap of the double mutant has the greater degree of opening with respect to that of the others. Additionally, the phenyl ring of Phe82 moves away from the binding pocket S1', and the conformational change of ritonavir subsite P1' consequently affects the cavity size of the protein and the conformational energy of the inhibitor. Calculations of binding free energy using the solvent continuum model were able to reproduce the same trend of the experimental inhibition constant. The results show that the resistance mutants require hydrophobic residues to maintain the interactions in the binding pocket. Changes of the cavity volume correlate well with free energy penalties due to the mutation and are responsible for the loss of drug susceptibility.

Performance of Six Different Ritonavir-Boosted Protease Inhibitor–Based Regimens in Heavily Antiretroviral-Experienced HIV-Infected Patients

Background: Regimens based on ritonavir-boosted protease inhibitors (PI/r) are often used as rescue interventions. It is unclear whether significant differences exist between distinct PI/r. Method: All HIV+ patients who had experienced PI failure at two HIV clinics and were rescued with a regimen based on saquinavir (SQV)/r 1000/100 mg bid, indinavir (IDV)/r 800/100 mg bid, lopinavir (LPV)/r 400/100 mg bid, amprenavir (APV)/r 600/100 mg bid, atazanavir (ATV)/r 300/100 mg qd, or tipranavir (TPV)/r 500/200 mg bid were retrospectively examined. A significant virological response (VR) was defined as >1 log reduction in plasma HIV-RNA or to <50 copies/mL at week 24. Results: A total of 389 patients were included in the analysis: 139 on SQV/r, 35 on IDV/r, 129 on LPV/r, 35 on APV/r, 29 on ATV/r, and 22 on TPV/r. No significant differences in HIV-RNA and CD4 counts at baseline were recognized between groups. In a multivariate analysis, only the total number of protease resistance mutations was associated with a lower VR (odds ratio [OR] = 0.77, 95% CI 0.68–0.87, p .001). The presence of <5 or ⩾5 protease resistance mutations at baseline was the best threshold to discriminate the achievement of VR in any treatment group. In an intent-to-treat analysis, for individuals with ⩾5 protease resistance mutations, the rates of VR were 64% with TPV/r, 47% with LPV/r, 46% with SQV/r, 33% with ATV/r, 25% with IDV/r, and 16% with APV/r. Adverse events leading to treatment withdrawal occurred more frequently using IDV/r (22.8%) than others (p = .03). Conclusion: The rate of VR in salvage therapy using PI/r-based regimens is relatively high in PI-experienced patients. The efficacy is greatly influenced by the number of baseline protease resistance mutations; 5 mutations is the best threshold to predict the chances of VR to any PI/r-based regimen.

A sensitive genotyping assay for detection of drug resistance mutations in reverse transcriptase of HIV-1 subtypes B and C in samples stored as dried blood spots or frozen RNA extracts

Exposure to antiretroviral drugs in resource-constrained settings is likely to result in the emergence of drug-resistant HIV-1 variants, limiting treatment options. Genotypic drug resistance testing assists clinical management and outcomes assessment, but a sensitive and reproducible genotypic assay feasible for resource-constrained settings is needed. A sensitive, reproducible genotyping assay to detect HIV-1 drug resistance mutations in reverse transcriptase and protease was developed and validated using blood stored as dried blood spots or as frozen RNA extracts from Zambian children infected with subtype C. HIV-1 genotypes derived from samples stored as dried blood spots were compared to those derived from paired liquid plasma samples in American young adults infected with HIV-1 subtype B. The method reproducibly amplified patient-specific sequences and detected drug resistance mutations from all of the dried blood spots or excess frozen RNA extracts with detectable viremia over a broad range of viral loads (193–3 million HIV-1 RNA copies/mL) in both HIV-1 subtypes B and C infection. This method captured the genetic variation typical of HIV-1 infection, including mutations at usual sites of drug resistance, polymorphisms, and mixtures. This sensitive and reproducible genotypic assay is feasible for detection of antiretroviral resistance in resource-constrained settings.

Long-Term (120-Week) antiviral efficacy and tolerability of fosamprenavir/ritonavir once daily in therapy-naive patients with HIV-1 infection: An uncontrolled, open-label, single-arm follow-on study

Background: In the SOLO study (APV30002), once-daily antiretroviral treatment with the protease inhibitor fosamprenavir (FPV) 1400 mg boosted by ritonavir (r) 200 mg plus abacavir/lamivudine (ABC/3TC) was found to be noninferior to nelfinavir plus ABC/3TC over 48 weeks in treatment-naive patients with HIV -1 infection. Objective: This interim report presents antiviral efficacy and tolerability data from 211 patients who received FPV/r QD for at least 48 weeks in SOLO and continued this treatment in the follow-on study (APV30005) for up to 120 weeks. Methods: APV30005 is an international, multicenter, uncontrolled, open-label, follow-on study conducted to provide continued access to FPV in patients with HIV-1 infection who had participated in previous FPV studies, including SOLO, and to obtain longer-term data on the antiviral response and tolerability of an FPV-containing regimen. Patients who had completed at least 48 weeks of FPV/r therapy in the SOLO study were eligible to enter the follow-on study and continue receiving FPV/r 1400/200 QD, with study visits every 12 weeks. Their background regimens were chosen at the investigators' discretion and could be changed at any time. Antiviral response end points included plasma HIV-1 RNA levels <400 and <50 copies/mL, median plasma HIV-1 RNA levels, median and absolute changes from baseline in the CD4 cell count, and the frequency of HIV disease progression. Genotype and phenotype analyses were performed for patients meeting the criterion for virologic failure (defined as plasma HIV -1 RNA >1000 copies/mL on 2 consecutive occasions on or after week 12). Tolerability was assessed in terms of adverse-event reports evaluated by the primary investigator and changes in laboratory values. Assessments were conducted at 12-week intervals during the follow-on study. Data from the baseline visit (day 1 of SOLO) were compared with data from the follow-on study through March 31, 2004, when all patients had completed at least 120 weeks of therapy with FPV/r QD. Because this was a rollover study, no significance testing was performed and all reported results are descriptive. Results: The demographic and baseline characteristics of the patients who received FPV/r QD in this follow on study (N = 211) were similar to those of the 322 patients randomized to receive FPV/r QD in the SOLO study. Their median age was 36 years, 72% were male, 49% were white, and 39% were black. The median baseline plasma HIV 1 RNA level was 4.82 log 10 copies/ mL, and the median baseline CD4+ cell count was 168 cells/mm 3. The median duration of exposure to FPV/r QD from SOLO baseline through the cutoff date was 996 days (142 weeks), ranging from 372 to 1226 days (53–175 weeks). At week 120, plasma HIV-1 RNA levels <400 and <50 copies/mL were achieved and maintained in 75% (159) and 66% (139) of patients, respectively, when missing data and discontinuations were counted as failures. The median CD4+ cell count at week 120 was 451 cells/mm 3, a median change from baseline of 292 cells/mm3. In 14 patients with no baseline resistance who met the criterion for virologic failure, no viral protease resistance mutations were detected. Extended treatment was generally well tolerated. The most frequently reported drug-related grade 2-4 adverse events were diarrhea (22 [10%]), nausea (17 [8%]), drug hypersensitivity (14 [7%], all cases attributed to ABC, which was a study drug in SOLO), and increased triglycerides (14 [7%]). The nature of adverse events reported after 48 weeks of therapy was comparable to that reported before week 48. Adverse events occurred at a similar or lower frequency between weeks 48 and 120 compared with before week 48. Similarly, laboratory abnormalities seen by week 120 were comparable to those seen by week 48, although they were less frequent. Conclusions: Extended treatment (120 weeks) with FPV/r QD in these antiretroviral therapy-naive, HIV-1-infected patients was associated with sustained antiviral response and immunologic improvement. Adverse events had generally developed by 48 weeks of therapy and did not occur at a higher frequency through 120 weeks of treatment.

Sensitivity and Specificity of the ViroSeq Human Immunodeficiency Virus Type 1 (HIV-1) Genotyping System for Detection of HIV-1 Drug Resistance Mutations by Use of an ABI PRISM 3100 Genetic Analyzer

The ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system is an integrated system for identification of drug resistance mutations in HIV-1 protease and reverse transcriptase (RT). Reagents are included for sample preparation, reverse transcription, PCR amplification, and sequencing. Software is provided to assemble and edit sequence data and to generate a drug resistance report. We determined the sensitivity and specificity of the ViroSeq system for mutation detection using an ABI PRISM 3100 genetic analyzer with a set of clinical samples and recombinant viruses. Twenty clinical plasma samples (viral loads, 1,800 to 10,500 copies/ml) were characterized by cloning and sequencing individual viral variants. Twelve recombinant-virus samples (viral loads, approximately 2,000 to 5,000 copies/ml) were also prepared. Eleven recombinant-virus samples contained drug resistance mutations as 40% mixtures. One recombinant-virus sample contained an insertion at codon 69 in RT (100% mutant). Plasma and recombinant-virus samples were analyzed using the ViroSeq system. Each sample was analyzed on three consecutive days at each of three testing laboratories. The sensitivity of mutation detection was 99.65% for the clinical plasma samples and 99.7% for the recombinant-virus preparations. The specificity of mutation detection was 99.95% for the clinical samples and 100% for the recombinant-virus mixtures. The base calling accuracy of the 3100 instrument was 99.91%. Mutations in clinical plasma samples and recombinant-virus samples were detected with high sensitivity and specificity, including mutations present as mixtures. This report supports the use of the ViroSeq system for identification of drug resistance mutations in HIV-1 protease and RT genes.

Human immunodeficiency virus type 1 (HIV-1) genotyping in Rio de Janeiro, Brazil: assessing subtype and drug-resistance associated mutations in HIV-1 infected individuals failing highly active antiretroviral therapy

In order to assess the human immunodeficiency virus type 1 (HIV-1) drug resistance mutation profiles and evaluate the distribution of the genetic subtypes in the state of Rio de Janeiro, Brazil, blood samples from 547 HIV-1 infected patients failing antiretroviral (ARV) therapy, were collected during the years 2002 and 2003 to perform the viral resistance genotyping at the Renageno Laboratory from Rio de Janeiro (Oswaldo Cruz Foundation). Viral resistance genotyping was performed using ViroSeq Genotyping System (Celera Diagnostic-Abbott, US). The HIV-1 subtyping based on polymerase (pol) gene sequences (protease and reverse transcriptase-RT regions) was as follows: subtype B (91.2%), subtype F (4.9%), and B/F viral recombinant forms (3.3%). The subtype C was identified in two patients (0.4%) and the recombinant CRF_02/AG virus was found infecting one patient (0.2%). The HIV-1 genotyping profile associated to the reverse transcriptase inhibitors has shown a high frequency of the M184V mutation followed by the timidine-associated mutations. The K103N mutation was the most prevalent to the non-nucleoside RT inhibitor and the resistance associated to protease inhibitor showed the minor mutations L63P, L10F/R, and A71V as the more prevalent. A large proportion of subtype B was observed in HIV-1 treated patients from Rio de Janeiro. In addition, we have identified the circulation of drug-resistant HIV-1 subtype C and are presenting the first report of the occurrence of an African recombinant CRF_02/AG virus in Rio de Janeiro, Brazil. A clear association between HIV-1 subtypes and protease resistance mutations was observed in this study. The maintenance of resistance genotyping programs for HIV-1 failing patients is important to the management of ARV therapies and to attempt and monitor the HIV-1 subtype prevalence in Brazil.

Predictive value of drug levels, HIV genotyping, and the genotypic inhibitory quotient (GIQ) on response to saquinavir/ritonavir in antiretroviral-experienced HIV-infected patients

Plasma levels of HIV protease inhibitors (PI) are often close to IC50 values of wild-type viruses when administered without ritonavir boosting. The impact of drug levels, resistance mutations, and the genotypic inhibitory quotient (GIQ) were examined on the response to saquinavir/ritonavir (SQV/r)-based salvage therapy. Patients with prior exposure to PI other than SQV and currently failing virologically were recruited prospectively in a multicenter trial. All patients began SQV/r 1000/100 mg bid plus another two antiretrovirals. A total of 139 patients was enrolled. At month 12, virological response (VR), defined as plasma HIV-RNA decline >1 log, was recorded in 68.2% of patients on treatment (41.7% in the intent-to-treat analysis). The median baseline number of protease resistance mutations was three. The VR was significantly lower in patients with >5 protease resistance mutations and in those with plasma SQV Cmin<0.1 microg/ml. At week 48, the VR was seen in 77.1% of patients with a GIQ>0.04 compared to 18.2% of those with a lower GIQ (P=0.001). In the multivariate analysis, <or=5 protease resistance mutations and SQV Cmin>0.1 microg/ml were independently associated with VR. Interestingly, drug levels had their highest predictive value of the VR at week 12, since low SQV plasma levels often permitted ruling out poorly adherent patients. In contrast, the number of protease resistance mutations had the highest impact on the VR at week 24, suggesting that for those taking the drugs, the VR is highly dependent of the presence of resistance mutations. At any time, nevertheless, the GIQ provided the most accurate prediction of the VR.

Natural resistance-associated mutations to Enfuvirtide (T20) and polymorphisms in the gp41 region of different HIV-1 genetic forms from T20 naive patients

The natural occurrence of primary resistance mutations in reverse transcriptase (RT) and protease (PR) genes of HIV-1 isolates from untreated patients has been reported and it may have important implications for the response to drug treatment. It is predictable that the same occurs in the HR1 region of gp41 sequence from patients who have never received T20 therapy, and in this regard it would be important to know not only the mutation frequencies at HR1 region but also the natural polymorphisms at resistance-associated positions present in the absence of this drug. The objectives of this study are to investigate the existence of natural resistance-associated mutations to T20 in HR1 gp41 region corresponding to different HIV-1 genetic forms from T20 naive patients and to determine their prevalence. Two hundred HIV-1 gp41 sequences were included: subtype B: 164 (81.3%); subtype A: 15 (8.2%); subtype G: 10 (4.6%); subtype F: 6 (3.5%); subtype C: 3 (1.8%); subtype K: 1 (0.6%); and subtype D: 1 (0.6%). We analyzed the resistance-associated mutations previously described: Q32H/R, G36D/S, I37V, V38A/M, Q39R/H, Q40H, N42T/D/Q/H, N43D/S/K/Q, L44M, L45M, R46M and V69I. Natural resistance mutations to T20 were found at a high frequency: 10.5%, corresponding to 9.1% in subtype B and 16.7% in non-B subtype samples. Polymorphisms were more frequent in non-B and recombinant forms than in subtype B (p<0.001). Different substitutions were related to subtypes: N42S in subtypes A, B, G and C, but not in F, Q56R in subtype A from CRF02_AG, and L54M in subtype B from CRF14_BG. To our knowledge this is the first study describing natural-resistance to T20 among different HIV-1 subtypes, warranting a study of the biological significance of this mutations and their clinical relevance. The detection of differences between subtypes may have an influence on the rate and patterns of resistance in patients undergoing T20 treatment.

Resistance to HIV protease inhibitors: mechanisms and clinical consequences.

HIV-1 protease is an aspartic protease composed by two identical monomers, 99 amino acids in length. Drug resistance is mainly mediated by structural changes in the substrate cleft that result in a reduction in drug binding affinity. Sequence analysis of drug resistance clones has shown mutations not only within the protease but also at several of the protease cleavage sites. Changes at more than 20 positions within the HIV-1 genome have been associated with PI resistance. The spectrum of mutations selected during therapy with indinavir, nelfinavir, saquinavir, ritonavir, amprenavir and atazanavir has been well characterized. Specific changes are characteristically linked to resistance to each of these compounds (i.e., D30N for nelfinavir, I50L for atazanavir or I50V for amprenavir). In contrast, for drugs such as lopinavir and tipranavir, which always are used boosted with low-dose ritonavir, combinations of multiple protease mutations rather than few specific changes seem to be necessary for causing significant drug resistance. Something similar happens when other PIs are equally boosted with ritonavir. Overall, when more than 5 protease resistance mutations are present, the response to any boosted-PI should be expected to be compromised.

Predictors of Virological Response to Atazanavir in Protease Inhibitor-Experienced Patients

Background: Atazanavir (ATV) is the latest approved HIV protease inhibitor (PI). Even though it is very convenient (only two capsules once a day), concerns have risen about its potency. Method: The clinical performance of ATV 400 mg once a day was examined in all PI-experienced patients who were included in the ATV expanded access program conducted in a single institution. The predictive value of baseline drug resistance HIV genotypes, ATV plasma trough levels, and the genotypic inhibitory quotient (GIQ) on the virological response at week 24 was assessed. Results: Data from 92 patients were analyzed. ATV was prescribed as part of a rescue intervention (45%), a simplification strategy (11%), or an attempt to ameliorate hyperlipidemias (23%) or other toxicities (16%). Tenofovir (TDF) was concomitantly used with ATV in 78% of patients. None received ritonavir boosting. In patients with detectable viremia at baseline (65%), the median HIV RNA drop was 0.7 logs. The median ATV Cmin was 0.12 μg/mL (IQR, 0.05-0.22 μg/mL), which is clearly above the IC90 (90% inhibitory concentration) for ATV in wild-type viruses. The virological response did not correlate significantly with ATV Cmin. The median number of protease resistance mutations was lower in patients showing virological response than in nonresponders (1 vs. 5; p = .07). A higher HIV RNA drop was associated with a higher GIQ (p = .02; β = –5.4; 95% CI, –10 to –1). Only 4 patients (4%) discontinued treatment due to ATV-related toxicities (hyperbilirubinemia in 1). Bilirubin levels were associated with ATV plasma concentrations (p = .05; β = 3.2; 95% CI, –0.1 to 6.5). The rate of hypertriglyceridemia and hypercholesterolemia declined significantly with respect to baseline. Conclusion: ATV is relatively safe and provides significant virological response in PI-experienced patients, mainly among those with a low number of protease resistance mutations. The GIQ predicts accurately the virological response in patients receiving ATV. Hyperbilirubinemia is associated with higher ATV plasma levels.

Detection of human immunodeficiency virus type 1 antiretroviral resistance mutations by high-density DNA probe arrays.

Genotypic resistance testing has become an important tool in the clinical management of patients infected with human immunodeficiency virus type 1 (HIV-1). Standard sequencing methodology and hybridization-based technology are the two principal methods used for HIV-1 genotyping. This report describes an evaluation of a new hybridization-based HIV-1 genotypic test of 99 clinical samples from patients infected mostly with HIV-1 subtype B and receiving treatment. This test combines RNA extraction with magnetic silica particles, amplification by nested reverse transcriptase PCR, and detection with high-density probe arrays designed to detect 204 antiretroviral resistance mutations simultaneously in Gag cleavage sites, protease, reverse transcriptase, integrase, and gp41. The nested reverse transcriptase PCR success rates at viral loads exceeding 1,000 copies/ml were 98% for the 2.1-kb amplicon that covers the Gag cleavage sites and the protease and reverse transcriptase genes, 92% for the gp41 amplicon, and 100% for the integrase amplicon. We analyzed 4,465 relevant codons with the HIV-1 DNA chip genotyping assay and the classic sequence-based method. Key resistance mutations in protease and reverse transcriptase were identified correctly 95 and 92% of the time, respectively. This test should be a valuable alternative to the standard sequence-based system for HIV-1 drug resistance monitoring and a useful diagnostic tool for simultaneous multiple genetic analyses.