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Objective: The aim of the present study was to evaluate the virological response to a new antiretroviral treatment (ART2) after failure of a nonnucleoside reverse transcriptase inhibitor (NNRTI) plus two nucleoside reverse transcriptase inhibitors (NRTIs)-containing regimen., Design: Retrospective observational study based on the Italian ARCA cohort database. Adult patients were included if they had a virological failure (defined as plasma viral load above 500 copies/ml in two subsequent visits) while on a treatment with one NNRTI plus 2 NRTIs, had an available HIV genotype., Results: Patients on ART2 were followed up for 791 person/year and median follow up was 10.8 months(IQR 5.2-26). Variables associated with reduced risk of ART2 virological failure at univariable analysis had started the treatment in recent years (HR 0.90; 95% CI 0.86-0.94, p < 0.0001) and duration of previous NNRTI treatment (HR 0.995; 95%CI 0.990-0.990, p=0.045). Variables associated with increased risk of virological failure of ART2 were a higher plasma viral load (pVL) at baseline(HR 1.2; 95% CI 1.07-1.34, p=0.002) and the type of treatment, in particular an unboosted PI-containing regimen vs. a boosted PI-containing regimen(HR 1.6; 95%CI 1.25-2.04 p < 0.0001) and a non-PI-containing vs. a boosted PI-containing regimen (HR 1.56; 95% CI 1.25-1.96, p < 0.0001). At multivariable analysis, year of ART2 start, pVL at NNRTI failure as well as using a boosted PI remained statistically significant predictors., Conclusion: This study highlights the role of drugs with high genetic barrier, such as boosted PI as a cornerstone to build a new antiretroviral treatment in patients failing a NNRTI based regimen.

Novel integrase inhibitors are in advanced clinical development, and cross-resistance data are needed to consider the possibility to plan a sequential usage within this class of antiretroviral drugs. Ex vivo phenotypic assays were conducted on 11 wild-type and 27 fully replicating recombinant viruses obtained from 11 patients failing previous raltegravir-containing regimens. Dolutegravir maintained its activity in vitro on viruses with mutations in position 143 and 155. However, viruses with mutation Q148R associated with secondary mutations and the combination Q148H+G140S were instead associated with a reduced level of susceptibility to dolutegravir in vitro.

The objective of this study was to address the evolution of human immunodeficiency virus type 1 (HIV-1) mutations resistant to the integrase inhibitor raltegravir after drug interruption. Thirteen HIV-1 infected patients undergoing virological failure due to the selection of raltegravir-resistant variants, who had interrupted raltegravir treatment, were enrolled. For all patients, the virological failure was associated with the selection of variants, with mutations conferring resistance to all of the drugs present in their regimens. Patients were prospectively monitored at baseline (raltegravir interruption) and every 4-24 weeks for clinical, virological and immunological parameters, including HIV-1 viraemia, CD4(+) T-cell counts, and sequence analysis of the HIV-1 integrase sequence. Reversion to the wild-type HIV-1 integrase sequence genotype was observed between 4 and 36 weeks after raltegravir withdrawal in eight out of the 13 patients. Reversion was not observed in three patients. In two patients, reversion was partial at week 24 from raltegravir interruption. These results highlight that in eight out of 13 patients under treatment with raltegravir and experiencing a virological failure, HIV-1 variants harbouring mutations associated with raltegravir resistance become undetectable after drug interruption within a few weeks (in some cases, very rapidly). This occurs under different therapy regimens and in patients receiving 3TC mono-therapy. In the other patients, complete reversion of the integrase sequence is not observed, and either primary or secondary resistance mutations are fixed in the replication competent viral population in vivo also for long time, suggesting that other factors may influence this dynamic process., (2010 The Authors. Clinical Microbiology and Infection; 2010 European Society of Clinical Microbiology and Infectious Diseases.)

Objectives: To evaluate whether etravirine (TMC125) might be effective in patients failing therapy with current nonnucleoside reverse transcriptase inhibitors (NNRTIs), we analysed the prevalence of TMC125 mutations and the possible determinants of genotypic resistance to this drug among sequences reported to a large database in Italy [Antiretroviral Resistance Cohort Analysis (ARCA)]., Methods: We analysed the prevalence of TMC125 resistance-associated mutations (RAMs) and the TMC125 weighted genotypic score (WGS) together with the determinants of genotypic resistance. A total of 5011 sequences from 2955 patients failing NNRTI therapy were evaluated., Results: Among the sequences in ARCA, 68% had at least one and 9.8% at least three TMC125 RAMs, whereas 31% had a WGS>2. Frequent RAMs were Y181C, G190A, K101E and A98G, whereas V179F, Y181V and G190S appeared in <5% of sequences. Multivariate analysis revealed a higher risk of developing at least three TMC125 RAMs associated with both nevirapine and efavirenz exposure, whereas CD4 counts > or = 200 cells/microL retained their protective effect. An increased risk of WGS>2 was linked to higher HIV RNA values (maximum risk at >5 log(10) copies/mL) and nevirapine exposure; CD4 counts > or = 200 cells/microL were protective., Conclusions: The prevalence of TMC125 resistance mutations in the ARCA cohort was 68%. The DUET studies showed that at least three TMC125-associated mutations were required to impair the efficacy of the drug and Y181C/V, V179F and G190S had the greatest effect on response. The prevalence of these mutations among the patients examined in our study was low. However, WGS>2 was found for one-third of our sequences. Previous nevirapine exposure was associated with an increased risk of having WGS>2 (adjusted odds ratio 1.76).

Objectives: To understand the dynamic viral evolution observed during failure on raltegravir-containing regimens, we studied the genotypic and phenotypic patterns of resistance to raltegravir and the residual replication capacity (rRC) of HIV-1 variants selected in vivo., Methods: Clonal genotypic analyses were performed on sequential HIV-1 integrase sequences amplified from 11 failing patients and sampled every 4-24 weeks for up to 64 weeks. Fully replicating recombinant viruses were generated using modified vectors in which selected viral integrase genes amplified from patients' plasma were cloned. rRC was measured by a novel multiple cycle competition assay. Resistance to raltegravir and the rRC of resistant HIV-1 variants selected in vivo were evaluated in purified CD4+ T cells., Results: In all of the patients but one, failure was associated with selection of mutations in positions 143, 148 or 155. Unlike mutations at position 143 (Y143S/K/R), identified alone or in combination with others, mutations at position 148 and 155 were always found in combination. A wide range of resistance levels to raltegravir [from 10- to 770-fold change in 50% inhibitory concentration (IC(50)) compared with baseline] was observed using recombinant viral clones. Finally, rRC was not significantly altered in highly resistant variants., Discussion: Two patterns of viral evolution were observed in the resistant viral populations, driving the variants towards a fast (most of them with G140S + Q148H mutations) or progressive increase in resistance to raltegravir. These results may have implications either for the evaluation of genotypic results, or for the correct clinical use of the compound.

Objective: : Evaluate HIV-1 subtype B integrase gene evolution in patients failing raltegravir (RAL)-based savage regimens by clonal analysis of the replicating viral quasispecies., Design: : Seven triple class failure HIV-1 (subtype B)-infected patients, followed at San Raffaele Hospital and enrolled in the RAL Expanded Access Program (MK0518-023), were evaluated. Patients were followed up for 24-48 weeks and due to the absence of other active drugs, RAL was maintained in their regimens even if resistance mutations were detected., Methods: : Immunologic and virologic parameters were recorded every 4 weeks, and amplification and clonal analysis of viral populations were performed at baseline and every 4-12 weeks in all patients., Results: : Resistance to RAL appeared initially associated with selection of single variants (Y143R, Q148R N155H) in the majority of patients; however, in three patients, complex patterns of viral mutations were observed. The clonal analysis of viral quasispecies allowed to describe the evolution of each viral population and the progressive accumulation of RAL resistance-associated mutations and polymorphisms associated with therapy failure., Conclusion: : The complex patterns of resistance mutations observed, including novel variants evolved under continuous RAL pressure, suggesting that they are the result of the equilibrium between drug resistance and enzyme function. Despite the efficacy of this compound, our data discourage its use in a functional monotherapy and maintaining RAL even in presence of RAL resistance-associated mutations may lead to the progressive formation of viral reservoirs with multiple integrase inhibitor-resistant variants that may limit the future efficacy of other integrase inhibitors due to cross-resistance.

The genotypic inhibitory quotient (GIQ) is the ratio between drug concentration and the number of resistance mutations. However, as different resistance scores can be calculated for the same protease inhibitor, the ability of a GIQ to predict the virological outcome may vary depending on the resistance score used as the denominator. Forty-four highly treatment-experienced HIV-infected patients failing on their current regimen were treated with a lopinavir/ritonavir-containing combination for at least 48 weeks. Three GIQs were calculated for each patient: the denominator of the first (GIQ-A) was the lopinavir/ritonavir score calculated using the mutations listed by IAS-USA as being related to lopinavir/ritonavir resistance; the denominator of the second (GIQ-B) was the total number of mutations related to resistance to any protease inhibitor as reported by IAS-USA; and the denominator of the third (GIQ-C) was the lopinavir/ritonavir score proposed by Parkin et al. The median (IQR) of the GIQ-A, B, and C was 4.69 (3.83-9.76), 0.97 (0.74-1.62), and 1.02 (0.68-2.52), respectively. At week 48, the median decrease in HIV-RNA was 1.09 (0.32-2.34) log(10) copies/ml (P < 0.0001), with 13 subjects (29.5%) attaining undetectable levels. All of the GIQs independently predicted the change in viral load from baseline and undetectable HIV-RNA at week 48. The partial R(2) of GIQ-C was greater than that of GIQ-B, which was greater than that of GIQ-A. All of the GIQs were independent predictors of the 48-week virological response. The predictive value of the GIQ for lopinavir/ritonavir may vary depending on the algorithm used to score drug resistance., ((c) 2006 Wiley-Liss, Inc.)

Patients infected with HIV-1 with more than 1000 HIV-1 RNA copies/mL, who were genotyped within 3 months before starting stavudine and treated for at least 3 months with a stable stavudine-containing highly active antiretroviral therapy, were selected from our database to identify the determinants of response to stavudine. Nonresponse was defined as a failure to achieve HIV-1 RNA level of less than 400 copies/mL or a reduction of more than 2 log10 by week 12. Univariate logistic analysis was used to elicit the failure-associated reverse transcriptase mutations (scored 1 to develop a genotype score). Eighty-one patients were eligible for the analysis, including 75 (93%) who previously received zidovudine. Thirty-five (43%) were nonresponders. Univariate logistic analysis revealed the following failure-associated mutations: 41L (P = 0.0001), 44D (P = 0.02), 118I (P = 0.0006), 184V (P = 0.04), 210W (P = 0.0004), and 215Y (P = 0.002) for a median stavudine score of 2. Failure was observed in 7 (18.9%) of 37 patients with a score less than 2, compared with 28 (63.6%) of 44 patients with a score of 2 or greater (P < 0.0001). The multivariable analysis showed that the 118I mutation (P = 0.04) was the only independent genotypic predictor of failing on a stavudine- containing highly active antiretroviral therapy.

Background: The genetic barrier, defined as the number of mutations required to overcome drug-selective pressure, is an important factor for the development of HIV drug resistance. Because of high variability between subtypes, particular HIV-1 subtypes could have different genetic barriers for drug resistance substitutions. This study compared the genetic barrier between subtypes using some 2000 HIV-1 sequences (>600 of non-B subtype) isolated from anti-retroviral-naive patients in Europe., Methods: The genetic barrier was calculated as the sum of transitions (scored as 1) and/or transversions (2.5) required for evolution to any major drug resistance substitution. In addition, the number of minor protease substitutions was determined for every subtype., Results: Few dissimilarities were found. An increased genetic barrier was calculated for I82A (subtypes C and G), V108I (subtype G), V118I (subtype G), Q151M (subtypes D and F), L210W (subtypes C, F, G, and CRF02&#95;AG), and P225H (subtype A) (P < 0.001 compared with subtype B). A decreased genetic barrier was found for I82T (subtypes C and G) and V106M (subtype C) (P < 0.001 vs subtype B). Conversely, minor protease substitutions differed extensively between subtypes., Conclusions: Based on the calculated genetic barrier, the rate of drug resistance development may be similar for different HIV-1 subtypes. Because of differences in minor protease substitutions, protease inhibitor resistance could be enhanced in particular subtypes once the relevant major substitutions are selected.

To investigate the dynamics of the K103N mutation following the withdrawal of non-nucleoside reverse transcriptase inhibitors (NNRTIs), we selected the Human Immunodeficiency Virus (HIV)-infected patients with the mutation at the time or after the failure of an NNRTI-containing regimen from an observational database. Of 62 patients fulfilling the inclusion criteria, 39 continued antiretroviral treatment without NNRTIs (group A), whereas 23 discontinued all antiretrovirals after NNRTI failure (group B). A total of 149 tests were analysed, with a median (IQR) of two (2-3) tests/patient. The overgrowth of wild-type virus at position 103 was observed in 14 subjects in group A (36%) and nine in group B (39%). No significant trend was found in relation to the disappearance of K103N variants in either group, but patients tested while receiving antiretrovirals had a significantly higher probability of retaining the K103N mutation over 24 months than those tested during treatment interruption (P = 0.007). In conclusion, following NNRTI discontinuation, HIV variants carrying the K103N mutation are not overgrown for long by wild-type quasispecies at this position in the majority of patients, although treatment interruption favours their disappearance. This suggests that the K103N mutation per se has little impact on viral fitness in vivo.

Background: Infection with drug-resistant human immunodeficiency virus type 1 (HIV-1) can impair the response to combination therapy. Widespread transmission of drug-resistant variants has the disturbing potential of limiting future therapy options and affecting the efficacy of postexposure prophylaxis., Methods: We determined the baseline rate of drug resistance in 2208 therapy-naive patients recently and chronically infected with HIV-1 from 19 European countries during 1996-2002., Results: In Europe, 1 of 10 antiretroviral-naive patients carried viruses with > or = 1 drug-resistance mutation. Recently infected patients harbored resistant variants more often than did chronically infected patients (13.5% vs. 8.7%; P=.006). Non-B viruses (30%) less frequently carried resistance mutations than did subtype B viruses (4.8% vs. 12.9%; P<.01). Baseline resistance increased over time in newly diagnosed cases of non-B infection: from 2.0% (1/49) in 1996-1998 to 8.2% (16/194) in 2000-2001., Conclusions: Drug-resistant variants are frequently present in both recently and chronically infected therapy-naive patients. Drug-resistant variants are most commonly seen in patients infected with subtype B virus, probably because of longer exposure of these viruses to drugs. However, an increase in baseline resistance in non-B viruses is observed. These data argue for testing all drug-naive patients and are of relevance when guidelines for management of postexposure prophylaxis and first-line therapy are updated.

The redistribution of mutations related to protease inhibitor (PI) resistance after a PI-sparing regimen in human immunodeficiency virus (HIV)-infected, highly PI-experienced patients was prospectively assessed. Twenty-five patients failing a PI-including regimen were given PI-sparing antiretroviral therapy, and then followed for 24 weeks after PI resumption. Genotyping was performed by direct sequencing before and during the PI-sparing regimen. The median (interquartile range, IQR) baseline CD4+ T-lymphocyte count was 198 (120-255) cells/microl, and the median HIV-RNA level was 82,000 (41,000-300,000) copies/ml. Patients had experienced a median of 4.5 (4-5.25) PIs. The median number of PI mutations was eight (6-9). The PI-sparing regimen consisted of a median of three (3-4) drugs and lasted for a median of 53 (24-67) weeks. At the end of the study, the median number of PI mutations was 6.5 (6-9). The median change in the number of PI mutations was -1 (IQR from -1 to 0). A reduction from baseline was observed in 13 cases (52%); nine (36%) showed no change and three (12%) showed an increased number of PI substitutions. In highly PI-experienced patients, a PI-sparing regimen may lead to a reduction, no change, or increase in the number of PI mutations. The reduction is negligible in most cases.

The objective of this study was to evaluate virological and pharmacological determinants of a 24-week virological response to unboosted atazanavir (ATV) in highly drug-experienced HIV-infected patients. Among patients enrolled in the ATV Expanded Access Program, those with HIV-RNA >1000 copies/mL, a genotype performed within three months from the baseline (BL), and who completed 24 weeks of treatment, were included. They received at least three antiretrovirals, including ATV 400 mg once daily without boosting. ATV plasma levels were evaluated after four weeks of treatment by high performance liquid chromatography (HPLC). ATV genotypic inhibitory quotient (GIQ) was calculated as the ratio between ATV Ctrough and the number of the BL ATV-related protease resistance mutations (among the following: 10I/V/F, 20R/M/I, 24I, 331/F/V, 36I/L/V, 46I/L, 48V, 54V/L, 63P, 71V/T/I, 73C/S/T/A, 82A/F/S/T, 84V, and 90M). Thirty-five subjects were included. At baseline, median (interquartile range) CD4+ T-lymphocytes, HIV-RNA, and ATV resistance mutations were 232.5 (106-303)/microL, 4.7 (4.2-5.1) log10 copies/mL, 2 (1-6), respectively. Thirteen (37.1%) subjects were off-therapy and 11 (31.4%) showed no PI mutation at baseline. Median steady-state ATV Ctrough was 230 ng/mL (87-520), for an ATV GIQ of 86.5 (25.5-165.5). Median HIV-RNA changes from baseline at weeks 4, 12 and 24 were -1.76 (from -0.44 to -2.12), -1.41 (from -0.41 to -2.81) and -1.44 (from -0.42 to -2.71) log10, respectively. The HIV-RNA changes were correlated to the number of ATV resistance mutations at each time point (P < 0.05), whereas no correlation was found between ATV Ctrough or ATV GIQ and HIV-RNA changes. In conclusion, the number of ATV resistance mutations is the only correlate to virological response through 24 weeks of treatment with unboosted atazanavir 400 mg once daily.

To understand the evolutionary characteristics of HIV-1 variants resistant to protease inhibitors (PI), the replicating plasma virus was analyzed in three patients shifted to PI-sparing regimen after virological failure. The dynamic features of carryover mutations associated with PI resistance in the absence of selective pressure on the protease gene indicate that viral variants resistant to reverse transcriptase inhibitors and bearing mutations of the protease sequence can maintain efficient replication capacity in vivo.