Grazia Punzi

Co-receptor switch during HAART is independent of virological success.

The influence of antiretroviral therapy on co‐receptor tropism remains controversial. To verify if co‐receptor tropism shift was affected by HAART, the evolution of proviral DNA V3 genotype after 12 months of a new antiretroviral regimen was compared between responder and non‐responder patients. Baseline blood samples were collected from 36 patients infected with HIV‐1 subtype‐B (18 naïve and 18 experienced) for virus isolation and env V3 genotyping from plasma HIV‐1 RNA and PBMC DNA. DNA V3 genotyping was repeated after 12 months from initiating HAART. WebPSSM was used for categorizing V3 sequences into X4 or R5; for analysis purposes, dual/mixed viruses were considered as X4. From the 10 (28%) patients changing their proviral DNA V3 genotype during therapy, six shifted from R5‐to‐X4 and four from X4‐to‐R5. The lack of reaching virological suppression was not associated with an X4‐to‐R5 (P = 0.25) or R5‐to‐X4 (P = 0.14) shift; time‐to‐viral suppression and CD4 increase were similar in both groups. No association was found between tropism shift and patient baseline characteristics including age, sex, CDC stage, CD4 count, viral load, exposure and length of previous HAART, enfuvirtide use in the new regimen, number of reverse transcriptase and protease resistance‐associated mutations. Conversely, CD4 nadir was correlated to emergence of X4 virus in proviral DNA (mean 27.2 ± 30.6 in R5‐to‐X4 shifting patients vs. 161.6 ± 150.6 in non‐shifting patients, P = 0.02). The occurrence of a tropism shift in both directions was independent of HAART use, irrespective of its efficacy. The CD4 count nadir was the only baseline characteristic able to predict an R5‐to‐X4 viral shift. J. Med. Virol. 81:2036–2044, 2009.

Impact of Mutations Outside the V3 Region on Coreceptor Tropism Phenotypically Assessed in Patients Infected with HIV-1 Subtype B

ABSTRACT HIV coreceptor tropism (CTR) testing is a prerequisite for prescribing a coreceptor antagonist. CTR is increasingly deduced by analyzing the V3 loop sequence of gp120. We investigated the impact of mutations outside V3 on CTR as determined by the enhanced-sensitivity Trofile assay (ESTA). Paired ESTA and gp120 sequencing (population sequencing; from codon 32 of the conserved C1 to the variable V5 domains) were obtained from 60 antiretroviral treatment (ART)-naïve patients (15 with AIDS) infected with subtype B HIV-1. For gp120 sequence analysis, nucleotide mixtures were considered when the second highest electropherogram peak was >25%; sequences were translated into all possible permutations and classified as X4, dual/mixed (DM), and R5 based on coincident ESTA results. ESTA identified R5 and DM viruses in 72 and 28% of patients, respectively; no pure X4 was labeled. Forty percent of AIDS patients had R5 strains. Thirty-two positions, mostly outside V3, were significantly (P < 0.05) different between R5 and DM sequences. According to multivariate analysis, amino acid changes at 9 and 7 positions within the C1 to C4 and V1 to V5 regions, respectively, maintained a statistical significance, as did the net charge of V3 and C4. When analyzing only R5 sequences, 6 positions in the variable regions were found which, along with the V4 net charge, were significantly different for sequences from early- and end-stage disease patients. This study identifies specific amino acid changes outside V3 which contribute to CTR. Extending the analysis to include pure X4 and increasing the sample size would be desirable to define gp120 variables/changes which should be included in predictive algorithms.

Evolution of transmitted HIV-1 drug resistance in HIV-1-infected patients in Italy from 2000 to 2010

Prevalence and predictors of transmitted drug resistance (TDR), defined as the presence of at least one WHO surveillance drug resistance mutation (SDRM), were investigated in antiretroviral-naïve HIV-1-infected patients, with a genotypic resistance test (GRT) performed ≤6 months before starting cART between 2000 and 2010. 3163 HIV-1 sequences were selected (69% subtype B). Overall, the prevalence of TDR was 12% (13.2% subtype B, 9% non-B). TDR significantly declined overall and for the single drug classes. Older age independently predicted increased odds of TDR, whereas a more recent GRT, a higher HIV-RNA and C vs. B subtype predicted lower odds of TDR.

Prevalence of HIV-1 integrase mutations related to resistance to dolutegravir in raltegravir naïve and pretreated patients.

The prevalence of HIV-1 integrase mutations related to resistance to the next-generation integrase inhibitor (INI), dolutegravir (DTG), was assessed in 440 INI-naïve subjects and in 120 patients failing a raltegravir (RTG)-containing regimen. Of the mutations selected by DTG in vitro, S153FY was not detected in any isolate while L101I and T124A were highly prevalent in both groups and significantly associated with non-B subtype. RTG-selected double and triple mutants, mostly the G140S/Q148H variant, were detected in only 32 (26.7%) RTG-treated patients. As L101I and T124A do not appear to exert any major effect in vivo and double and triple mutants resistant to DTG are infrequently selected by RTG, DTG can be effectively used in INI-naïve patients and may retain activity in many patients failing RTG.

Prevalence of mutations and determinants of genotypic resistance to etravirine (TMC125) in a large Italian resistance database (ARCA).

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)].

Antiretroviral Genotypic Resistance in Plasma RNA and Whole Blood DNA in HIV-1 Infected Patients Failing HAART

The extent to which HIV‐1 proviral DNA mutations cause clinically relevant antiretroviral resistance is still controversial. Paired plasma HIV‐1 RNA and whole blood DNA were compared in patients failing HAART to investigate if the additional knowledge of archived mutations could improve the selection of potentially active drugs. Seventy‐three HIV‐1‐infected patients with first/second HAART failure were studied before starting a new regimen based on RNA genotyping. Follow‐up data after a 12‐week therapy were available. DNA genotyping was retrospectively performed on stored whole blood samples and mutational profiles were compared to those from RNA. The mean number of IAS pol mutations was significantly higher in RNA (4.45 ± 2.76) than in DNA (2.88 ± 2.47) (P < 0.001). DNA genotyping provided a 6% increase in detection of resistance‐associated mutations. Among 64/73 patients showing discordant DNA/RNA profiles, 54 (84%) also differed for predicted active drugs. 16/73 (22%) patients had ≥1 mutation revealed by DNA genotyping alone, probably affecting therapy success in 2/16. However, neither RNA/DNA discordance nor detection of isolated DNA mutations were statistically associated with outcome. In conclusion, plasma RNA remains the elective choice for HIV genotyping in patients with therapy failure, even if the detection of proviral resistance‐associated mutations, not simultaneously found in RNA, is a frequent event. Therefore, in some cases DNA plus RNA genotyping might assist in choosing more accurately subsequent antiretroviral regimens. J. Med. Virol. 80:1695–1706, 2008.

The role of baseline HIV-1 RNA, drug resistance, and regimen type as determinants of response to first-line antiretroviral therapy

The factors influencing virological response to first‐line combined antiretroviral therapy (cART) in an Italian cohort of HIV‐1‐infected patients were examined. Eligible patients were those enrolled in a national prospective observational cohort (Antiretroviral Resistance Cohort Analysis), starting first‐line cART between 2001 and 2011 and who had at least one follow‐up of HIV‐1 RNA. The primary endpoint was virological success, defined as the first viral load <50 copies/ml. Time to events were analyzed by Kaplan–Meier analysis and Cox proportional hazard model. One thousand three hundred five patients met the study inclusion criteria. In a multivariable model adjusting for transmission mode, presence of transmitted drug resistance, baseline CD4+ cell count, viral subtype, and type of NRTI backbone employed, independent predictors of virological success were higher baseline viral load (≥500,000 vs. <100,000 HR 0.52; P < 0.001), a weighted genotypic susceptibility score (wGSS) <3 (HR 0.58; P = 0.003), male sex (HR 0.76 P = 0.001), and type of initial third drug employed (integrase inhibitor vs. boosted protease inhibitors HR 3.23; P < 0.001). In the subset with HIV‐1 RNA >100,000 copies/ml, virologic success was only associated with the use of integrase inhibitors in the first cART regimen. Independent predictors of immunological success were baseline CD4+ cell count and wGSS <3. High baseline HIV‐1 RNA, predicted activity of the first‐line regimen based on genotypic resistance testing, gender, and use of new agents were found to predict time to achieve virological success. The type of initial nucleoside analog backbone was not found to predict virological response. J. Med. Virol. 86: 1648–1655, 2014.

An outbreak of HIV-1 BC recombinants in Southern Italy

BACKGROUND In Western Europe, a previously subtype B HIV-1 restricted area, BC recombinants have been rarely reported. OBJECTIVE To describe an outbreak of HIV-1 BC recombinants in southern Italy. STUDY DESIGN We analyzed pol (protease/reverse transcriptase) sequences from 135 newly diagnosed HIV-1-infected patients during the years 2009-2011. For phylogenetic relationships, sequences were aligned to the most recent reference data set from the Los Alamos database using BioEdit (version 7.1.3). The resulting alignment was analyzed with the Phylip package (version 3.67) building a neighbor-joining tree based on the Kimura two-parameter substitution model. The reliability of the tree topology was assessed through bootstrapping using 1000 replicates. The recombination pattern was characterized using SimPlot 3.5.1 and SplitsTree 4. RESULTS At phylogenetic analysis, 22 (16.2%) isolates whose sequences were not unequivocally assigned to a pure subtype or known CRF, formed a distinct monophyletic clade (100% of bootstrap value). For these isolates, the recombination analysis identified a BC mosaic pattern with two breakpoints at positions 2778±5 and 3162±8 (HXB2 numbering) which differed from those of known BC CRFs. All patients from whom these sequences were derived were highly educated youth Italians, 91% males and 82% MSM. Sequences of pol integrase, gp120 and gp41 from these same patients were classified as C subtype. CONCLUSIONS This outbreak which further reflects the increasing heterogeneity of HIV epidemic in our country is the first report of an Italian outbreak of a BC recombinant, possibly a novel candidate CRF.

Factors associated with virological success with raltegravir-containing regimens and prevalence of raltegravir-resistance-associated mutations at failure in the ARCA database

Raltegravir (RAL) is the only licensed human immunodeficiency virus (HIV) integrase inhibitor. The factors associated with the virological response to RAL-containing regimens and the prevalence of integrase mutations associated with RAL failure deserve further investigation. From the Antiretroviral Resistance Cohort Analysis database, we selected triple-class-experienced subjects failing their current treatment with complete treatment history available. Selection criteria included HIV-RNA, CD4 count and HIV genotype within 3 months of RAL initiation. Factors associated with 24-week response were analysed; genotypic sensitivity scores (GSS) and weighted-GSS were evaluated. Virological response was achieved in 74.3% of 105 subjects. Mutations associated with RAL failure were detected in 12/24 subjects with an integrase genotype, with the prevalence of Q148H + G140S. Each extra unit of GSS (p 0.05, OR 2.62; 95% CI 1.00-6.87). was found to be a associated with response. Weighted-GSS had borderline statistical significance (p 0.063, OR 2.04; 95% CI 0.96-4.33) When stratifying for different cut-offs (<1 as reference, 1-1.49, ≥1.5), a borderline significant increase in the probability of response appeared for GSS ≥1.5 (p 0.053, OR 4.00; 95% CI 0.98-16.25). GSS ≥1 showed the highest sensitivity, 82.6%. Receiver operating characteristic curves depicted the widest area under the curve (0.663, p 0.054) of GSS ≥1. Unresponsiveness to RAL-containing regimens among triple-class-experienced subjects was low. The activity of the background regimen was strongly associated with response. Although few integrase genotypes were available at failure, half of these were without integrase resistance mutations. The substantial rate of RAL failure in the absence of known RAL-resistance mutations may be associated with adherence issues and this issue warrants further analysis in longer observations.

Low rate of virological failure and maintenance of susceptibility to HIV-1 protease inhibitors with first-line lopinavir/ritonavir-based antiretroviral treatment in clinical practice.

Protease inhibitor (PI)‐resistant HIV‐1 has hardly ever been detected at failed boosted PI‐based first‐line antiretroviral regimens in clinical trials. However, this phenomenon has not been investigated in clinical practice. To address this gap, data from patients starting a first‐line lopinavir/ritonavir (LPV/rtv)‐based therapy with available baseline HIV‐1 RNA load, a viral genotype and follow‐up viral load after 3 and 6 months of treatment were extracted from the Italian Antiretroviral Resistance Cohort Analysis (ARCA) observational database. Based on survival analysis, 39 (7.1%) and 43 (7.8%) of the 548 examined patient cases had an HIV‐1 RNA >500 and >50 copies/ml, respectively, after 6 months of treatment. Cox proportional hazard models detected baseline HIV‐1 RNA (RH 1.79, 95%CI 1.10–2.92 per 1 − log10 increase, P = 0.02) and resistance to the nucleoside backbone (RH 1.04, 95%CI 1.02–1.06 per 10‐point increase using the Stanford HIVdb algorithm, P < 0.001) as independent predictors of HIV‐1 RNA at >500 copies/ml, but not at the >50 copies/ml cutoff criteria. Higher baseline viral load, older patient age, heterosexual route of infection and use of tenofovir/emtricitabine were predictors of failure at month 3 using the 50‐copy and/or 500‐copy threshold. Resistance to LPV/rtv did not occur or increase in any of the available 36 follow‐up HIV‐1 genotypes. Resistance to the nucleoside backbone (M184V) developed in four cases. Despite the likely differences in patient population and adherence, both the low rate of virological failure and the lack of development of LPV/rtv resistance documented in clinical trials are thus confirmed in clinical practice. J. Med. Virol. 82:1996–2003, 2010.