Claudia Balotta

Prevalence of Drug-Resistant HIV-1 Variants in Untreated Individuals in Europe: Implications for Clinical Management

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.

Transmission of Drug-Resistant HIV-1 Is Stabilizing in Europe

The SPREAD Programme investigated prospectively the time trend from September 2002 through December 2005 of transmitted drug resistance (TDR) among 2793 patients in 20 European countries and in Israel with newly diagnosed human immunodeficiency virus type 1 (HIV-1) infection. The overall prevalence of TDR was 8.4% (225 of 2687 patients; 95% confidence interval [CI], 7.4%-9.5%), the prevalence of nucleoside reverse-transcriptase inhibitor (NRTI) resistance was 4.7% (125 of 2687 patients; 95% CI, 3.9%-5.5%), the prevalence of nonucleoside reverse-transcriptase inhibitor (NNRTI) resistance was 2.3% (62 of 2687 patients; 95% CI, 1.8%-2.9%), and the prevalence of protease inhibitor (PI) resistance was 2.9% (79 of 2687 patients; 95% CI, 2.4%-3.6%). There was no time trend in the overall TDR or in NRTI resistance, but there was a statistically significant decrease in PI resistance (P = .04) and in NNRTI resistance after an initial increase (P = .02). We found that TDR appears to be stabilizing in Europe, consistent with recent reports of decreasing drug resistance and improved viral suppression in patients treated for HIV-1 infection.

Risk of failure in patients with 215 HIV-1 revertants starting their first thymidine analog-containing highly active antiretroviral therapy.

Objective: To investigate the impact of 215 HIV-1 revertants on the risk of virological failure of the first thymidine analog-containing highly active antiretroviral therapy (HAART). Design: The study included 491 HIV-1 subjects of the Italian Cohort Naive for Antiretrovirals, 405 of whom received a genotypic assay before therapy and had a virological follow-up. Methods: Pre-treatment genotypic resistance was assessed by sequencing of the whole protease (PR) and reverse transcriptase (RT) region. Results: Three (3.2%) and 13 (3.3%) individuals with recent (n = 95) and chronic (n = 396) HIV-1 infection carried an HIV-1 strain with 215 revertants (215D/C/E/A/V), respectively. In contrast, nucleoside associated mutations were higher in the former (15.8%) compared with the latter group (6.8%) (P = 0.005). A multivariable regression model, considering pre-HAART viral load levels, use of saquinavir-hard gel as the only PI, use of zidovudine, number of other RT and PR mutations, indicated that patients carrying 215 revertants had an increased risk of virological failure compared with those not carrying such mutants (adjusted relative hazard = 2.97 95% confidence interval, 1.11–7.94, P = 0.03). Among patients with 215 revertants, who experienced virological failure, four out of seven showed the emergence of the 215Y resistant mutation. The probability of 215Y occurrence was different between patients carrying 215 revertants compared with those who did not carried these mutants (P = 0.006). Conclusions: HIV-1 215 revertants with an increased ability for selecting 215Y mutation are associated with a higher risk of virological failure and may lead to the appearance of virus carrying 215Y/F mutation in vivo. These findings suggest that 215 revertant viruses may compromise the efficacy of the first thymidine analog-containing regimen.

Tracing the HIV-1 subtype B mobility in Europe: a phylogeographic approach

BackgroundThe prevalence and the origin of HIV-1 subtype B, the most prevalent circulating clade among the long-term residents in Europe, have been studied extensively. However the spatial diffusion of the epidemic from the perspective of the virus has not previously been traced.ResultsIn the current study we inferred the migration history of HIV-1 subtype B by way of a phylogeography of viral sequences sampled from 16 European countries and Israel. Migration events were inferred from viral phylogenies by character reconstruction using parsimony. With regard to the spatial dispersal of the HIV subtype B sequences across viral phylogenies, in most of the countries in Europe the epidemic was introduced by multiple sources and subsequently spread within local networks. Poland provides an exception where most of the infections were the result of a single point introduction. According to the significant migratory pathways, we show that there are considerable differences across Europe. Specifically, Greece, Portugal, Serbia and Spain, provide sources shedding HIV-1; Austria, Belgium and Luxembourg, on the other hand, are migratory targets, while for Denmark, Germany, Italy, Israel, Norway, the Netherlands, Sweden, Switzerland and the UK we inferred significant bidirectional migration. For Poland no significant migratory pathways were inferred.ConclusionSubtype B phylogeographies provide a new insight about the geographical distribution of viral lineages, as well as the significant pathways of virus dispersal across Europe, suggesting that intervention strategies should also address tourists, travellers and migrants.

Homozygous Δ32 deletion of the CCR-5 chemokine receptor gene in an HIV-1-infected patient

Background:Recent research has found that entry of non-syncytium-inducing (NSI), monocyte-macrophage-tropic HIV-1 isolates requires binding to both CD4 and CCR5 receptors, and that Δ32/Δ32 homozygous individuals are protected against infection. Objective:To analyse the polymorphism of CCR-5 gene in HIV-1-infected and uninfected subjects. Design and methods:CCR-5 sequences were amplified by polymerase chain reaction (PCR) from DNA of peripheral blood mononuclear cells. Samples from 152 HIV-1-infected subjects and 122 uninfected controls were tested for the detection of the 32 base-pair deletion. HIV-1 phenotype was determined by viral isolation and MT-2 evaluation. Results:The wild-type/Δ32 heterozygous and Δ32/Δ32 homozygous conditions were represented in 10.7 and 0.8% of healthy controls and in 9.8 and 0.7% of HIV-1-infected subjects, respectively. Of note, the Δ32/Δ32 deletion of the CCR-5 gene was detected by PCR and sequencing confirmed in a patient with progressive infection harbouring a clade B virus with SI phenotype. Conclusions:Δ32/Δ32 homozygosity for the CCR-5 gene does not confer absolute protection against HIV-1 infection, suggesting that either macrophage-tropic viral strains could use coreceptors other than CCR-5 or infect independently of the presence of a functional CCR-5 coreceptor. Alternatively, primary infection sustained by T-cell-tropic isolates, although exceptional, may occur.

Secondary Mutations in the Protease Region of Human Immunodeficiency Virus and Virologic Failure in Drug-Naive Patients Treated with Protease Inhibitor-Based Therapy

The role of mutations in protease (PR) and reverse-transcriptase (RT) of human immunodeficiency virus (HIV) in predicting virologic failure was assessed in 248 antiretroviral-naive HIV-positive patients who began a PR inhibitor-containing antiretroviral regimen. Genotypic testing was performed on plasma samples stored before the start of therapy. Twenty-seven patients (10.9%) had mutations in the RT, 5 (2%) carried primary mutations in the PR, and 131 (52.8%) showed only secondary PR mutations. Virologic failure at week 24 occurred in 62 (25.0%) of 248 patients. There was a statistically significant correlation between virologic failure and the number of PR mutations (P= .04, chi(2) test). Mutations at codons 10 and 36 of PR (present in 39.3% and 40.0% of patients in whom treatment failed, respectively) were identified by stepwise logistic regression as the strongest predictors of virologic failure (odds ratio, 2.20; 95% confidence interval, 1.30-3.75; P= .004). If confirmed in independent studies, this result may justify the increased use of HIV genotyping in drug-naive patients requiring antiretroviral therapy.

Susceptibility to PNU-140690 (tipranavir) of human immunodeficiency virus type 1 isolates derived from patients with multidrug resistance to other protease inhibitors.

ABSTRACT In our study we examined the anti-human immunodeficiency virus type 1 (anti-HIV-1) activity of a novel HIV-1 protease inhibitor, PNU-140690 (tipranavir), against patient-derived isolates resistant to multiple other protease inhibitors (PIs). The aim of our experiments was to investigate the genotypes and the in vitro phenotypes of drug resistance of PNU-140690. We carried out drug susceptibility tests with peripheral blood mononuclear cells and a fixed amount of infectious virus (1,000 50% tissue culture infective doses) to determine the 50% inhibitory concentration (IC50) and IC90, PCR assays for the detection of drug resistance mutations in RNA in plasma, and direct sequencing of PCR products. Phenotypic resistance to PIs was invariably related to genotypic mutations. The substitutions among the amino acid residues of the protease included L10I, K20R, L24I, M36I, N37D, G48V, I54V, L63P, I64V, A71V, V77I, V82A, I84V, and L90M. Isolates from all of the patients had developed a maximal degree of resistance to indinavir, ritonavir, and nelfinavir (IC50s, >0.1 μM). We also compared these mutations with the amino acid changes previously described in association with in vivo tipranavir administration. The mutations included the following: I15V, E35D, N37D, R41K, D60E, and A71T. Infections with IIIB, 14aPre, and N70 were inhibited by an average drug IC90 of 0.18 ± 0.02 μM in multiple experiments. The average mean ± standard error of mean IC90 for the entire group of multidrug-resistant isolates derived from the mean values for two culture wells with p24 antigen supernatant appeared to be 0.619 ± 0.055 μM (range, 0.31 to 0.86 μM). Tipranavir retained a sustained antiviral activity against PI-MDR clinical isolates and might be useful in combination regimens with other antiretroviral agents for patients who have already failed other PI-containing therapies.

The Calculated Genetic Barrier for Antiretroviral Drug Resistance Substitutions Is Largely Similar for Different HIV-1 Subtypes

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_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.

HIV-1 subtype distribution and its demographic determinants in newly diagnosed patients in Europe suggest highly compartmentalized epidemics

BackgroundUnderstanding HIV-1 subtype distribution and epidemiology can assist preventive measures and clinical decisions. Sequence variation may affect antiviral drug resistance development, disease progression, evolutionary rates and transmission routes.ResultsWe investigated the subtype distribution of HIV-1 in Europe and Israel in a representative sample of patients diagnosed between 2002 and 2005 and related it to the demographic data available. 2793 PRO-RT sequences were subtyped either with the REGA Subtyping tool or by a manual procedure that included phylogenetic tree and recombination analysis. The most prevalent subtypes/CRFs in our dataset were subtype B (66.1%), followed by sub-subtype A1 (6.9%), subtype C (6.8%) and CRF02_AG (4.7%). Substantial differences in the proportion of new diagnoses with distinct subtypes were found between European countries: the lowest proportion of subtype B was found in Israel (27.9%) and Portugal (39.2%), while the highest was observed in Poland (96.2%) and Slovenia (93.6%). Other subtypes were significantly more diagnosed in immigrant populations. Subtype B was significantly more diagnosed in men than in women and in MSM > IDUs > heterosexuals. Furthermore, the subtype distribution according to continent of origin of the patients suggests they acquired their infection there or in Europe from compatriots.ConclusionsThe association of subtype with demographic parameters suggests highly compartmentalized epidemics, determined by social and behavioural characteristics of the patients.

Prevalence of transmitted HIV-1 drug resistance and the role of resistance algorithms - Data from seroconverters in the CASCADE collaboration from 1987 to 2003

Objectives:To examine factors influencing the rate of transmitted drug resistance (TDR) among seroconverters, with particular emphasis on 3 widely used genotypic drug resistance algorithms. Methods:The study used data from CASCADE (Concerted Action on Seroconversion to AIDS and Death in Europe), a collaboration of seroconverter cohorts in Europe and Canada. Genotypic resistance data were derived within 18 months of the last seronegative test or date of laboratory evidence of acute infection and before the initiation of antiretroviral therapy. The Stanford algorithm was used to analyze each individuals nucleotide sequence. A multivariate logistic model was used to assess independent relationships between the presence of TDR and exposure category, sex, age at seroconversion, and year of seroconversion. The paper also describes 3 alternative definitions of resistance: the Stanford algorithm, the key resistance mutations defined by the International AIDS Society, and the Agence Nationale de Recherches sur le Sida (ANRS) algorithm. Results:Forty-five of 438 patients (10.3%) seroconverting between 1987 and 2003 were infected with a drug-resistant HIV-1 variant. Forty patients (9.1%) showed resistance mutations to only 1 class of antiretroviral drugs, 2 (0.5%) to 2 classes, and 3 (0.7%) to 3 classes of antiretroviral therapy. It was suggested that individuals seroconverting later in calendar time were more likely to have TDR (relative risk 3.89 and 95% CI: 0.84 to 18.02, and relative risk 4.69 and 95% CI: 1.03 to 21.31, for 1996-1999 and 2000-2003, respectively, compared with pre-1996; P trend = 0.08). This trend was apparent regardless of the definition of TDR used. The total estimated proportion of individuals with TDR varied between 10.3% and 15.5% according to which definition was used. Conclusions:Evidence was found for the rise of TDR over time. A specific definition of what constitutes TDR rather than a simple list of mutations is needed.