RESUMO
OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.
Assuntos
Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Inibidores de Integrase de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Estudos Transversais , Europa (Continente)/epidemiologia , Feminino , Variação Genética , Genótipo , Infecções por HIV/virologia , Integrase de HIV/genética , Inibidores de Integrase de HIV/farmacologia , HIV-1/genética , Humanos , Masculino , Vigilância da População , Fatores de Risco , Análise de Sequência de DNA , Carga ViralRESUMO
OBJECTIVES: The EuResist expert system is a novel data-driven online system for computing the probability of 8-week success for any given pair of HIV-1 genotype and combination antiretroviral therapy regimen plus optional patient information. The objective of this study was to compare the EuResist system vs. human experts (EVE) for the ability to predict response to treatment. METHODS: The EuResist system was compared with 10 HIV-1 drug resistance experts for the ability to predict 8-week response to 25 treatment cases derived from the EuResist database validation data set. All current and past patient data were made available to simulate clinical practice. The experts were asked to provide a qualitative and quantitative estimate of the probability of treatment success. RESULTS: There were 15 treatment successes and 10 treatment failures. In the classification task, the number of mislabelled cases was six for EuResist and 6-13 for the human experts [mean±standard deviation (SD) 9.1±1.9]. The accuracy of EuResist was higher than the average for the experts (0.76 vs. 0.64, respectively). The quantitative estimates computed by EuResist were significantly correlated (Pearson r=0.695, P<0.0001) with the mean quantitative estimates provided by the experts. However, the agreement among experts was only moderate (for the classification task, inter-rater κ=0.355; for the quantitative estimation, mean±SD coefficient of variation=55.9±22.4%). CONCLUSIONS: With this limited data set, the EuResist engine performed comparably to or better than human experts. The system warrants further investigation as a treatment-decision support tool in clinical practice.
Assuntos
Sistemas Inteligentes , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Bases de Dados Factuais , Feminino , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Humanos , Masculino , Probabilidade , Resultado do Tratamento , Carga ViralRESUMO
A better understanding of human immunodeficiency virus type 1 drug-resistance evolution under the selective pressure of combination treatment is important for the design of long-term effective treatment strategies. We applied Bayesian network learning to sequences from patients treated with the reverse transcriptase inhibitor combination of zidovudine (AZT) and lamivudine (3TC) to identify the role of many treatment-selected mutations in the development of resistance. Based on the Bayesian network structure, an in vivo fitness landscape was built, reflecting the necessary selective pressure under treatment, to evolve naive sequences to sequences obtained from patients treated with the combination. This landscape, combined with an evolutionary model, was used to predict resistance evolution in longitudinal sequence pairs. In our analysis, mutations 41L, 70R, 184V and 215F/Y were identified as major resistance mutations to the combination of AZT and 3TC, as they were associated directly with treatment experience. The network also suggested a possible role in resistance development for a number of novel mutations. Estimated fitness, using the landscape, correlated significantly with in vitro resistance phenotype in genotype-phenotype pairs (R(2)=0.70). Variation in predicted evolution under selective pressure correlated significantly with observed in vivo evolution during AZT plus 3CT treatment. In conclusion, we confirmed current knowledge on resistance development to the combination of AZT and 3CT, but additional novel mutations were identified. Moreover, a model to predict resistance evolution during AZT and 3CT treatment has been built and validated.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Lamivudina/uso terapêutico , Zidovudina/uso terapêutico , Fármacos Anti-HIV/farmacologia , Quimioterapia Combinada , Evolução Molecular , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Humanos , Lamivudina/farmacologia , Mutação de Sentido Incorreto , Mutação Puntual , RNA Viral/genética , Seleção Genética , Zidovudina/farmacologiaRESUMO
MOTIVATION: HIV-1 antiviral resistance is a major cause of antiviral treatment failure. The in vivo fitness landscape experienced by the virus in presence of treatment could in principle be used to determine both the susceptibility of the virus to the treatment and the genetic barrier to resistance. We propose a method to estimate this fitness landscape from cross-sectional clinical genetic sequence data of different subtypes, by reverse engineering the required selective pressure for HIV-1 sequences obtained from treatment naive patients, to evolve towards sequences obtained from treated patients. The method was evaluated for recovering 10 random fictive selective pressures in simulation experiments, and for modeling the selective pressure under treatment with the protease inhibitor nelfinavir. RESULTS: The estimated fitness function under nelfinavir treatment considered fitness contributions of 114 mutations at 48 sites. Estimated fitness correlated significantly with the in vitro resistance phenotype in 519 matched genotype-phenotype pairs (R(2) = 0.47 (0.41 - 0.54)) and variation in predicted evolution under nelfinavir selective pressure correlated significantly with observed in vivo evolution during nelfinavir treatment for 39 mutations (with FDR = 0.05). AVAILABILITY: The software is available on request from the authors, and data sets are available from http://jose.med.kuleuven.be/~kdforc0/nfv-fitness-data/.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Evolução Biológica , Farmacorresistência Viral/genética , Variação Genética/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Seleção Genética , Mapeamento Cromossômico/métodos , Simulação por Computador , Variação Genética/efeitos dos fármacos , Modelos Genéticos , Mutação/efeitos dos fármacos , Mutação/genéticaRESUMO
Human Immunodeficiency Virus-1 (HIV-1) antiviral resistance is a major cause of antiviral therapy failure and compromises future treatment options. As a consequence, resistance testing is the standard of care. Because of the high degree of HIV-1 natural variation and complex interactions, the role of resistance mutations is in many cases insufficiently understood. We applied a probabilistic model, Bayesian networks, to analyze direct influences between protein residues and exposure to treatment in clinical HIV-1 protease sequences from diverse subtypes. We can determine the specific role of many resistance mutations against the protease inhibitor nelfinavir, and determine relationships between resistance mutations and polymorphisms. We can show for example that in addition to the well-known major mutations 90M and 30N for nelfinavir resistance, 88S should not be treated as 88D but instead considered as a major mutation and explain the subtype-dependent prevalence of the 30N resistance pathway.
Assuntos
Teorema de Bayes , Farmacorresistência Viral/fisiologia , Produtos do Gene pol/química , Produtos do Gene pol/genética , HIV-1/genética , Modelos Estatísticos , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Substituição de Aminoácidos , Análise Mutacional de DNA , Produtos do Gene pol/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Reconhecimento Automatizado de Padrão/métodos , Alinhamento de Sequência/métodos , Relação Estrutura-AtividadeRESUMO
Interpretation of Human Immunodeficiency Virus 1 (HIV-1) genotypic drug resistance is still a major challenge in the follow-up of antiviral therapy in infected patients. Because of the high degree of HIV-1 natural variation, complex interactions and stochastic behaviour of evolution, the role of resistance mutations is in many cases not well understood. Using Bayesian network learning of HIV-1 sequence data from diverse subtypes (A, B, C, F and G), we could determine the specific role of many resistance mutations against the protease inhibitors (PIs) nelfinavir (NFV), indinavir (IDV), and saquinavir (SQV). Such networks visualize relationships between treatment, selection of resistance mutations and presence of polymorphisms in a graphical way. The analysis identified 30N, 88S, and 90M for nelfinavir, 90M for saquinavir, and 82A/T and 46I/L for indinavir as most probable major resistance mutations. Moreover we found striking similarities for the role of many mutations against all of these drugs. For example, for all three inhibitors, we found that the novel mutation 89I was minor and associated with mutations at positions 90 and 71. Bayesian network learning provides an autonomous method to gain insight in the role of resistance mutations and the influence of HIV-1 natural variation. We successfully applied the method to three protease inhibitors. The analysis shows differences with current knowledge especially concerning resistance development in several non-B subtypes.
Assuntos
Teorema de Bayes , Farmacorresistência Viral/genética , Infecções por HIV/virologia , Inibidores da Protease de HIV/farmacologia , HIV-1/genética , Mutação , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , Humanos , Indinavir/farmacologia , Indinavir/uso terapêutico , Dados de Sequência Molecular , Nelfinavir/farmacologia , Nelfinavir/uso terapêutico , Saquinavir/farmacologia , Saquinavir/uso terapêuticoRESUMO
Genotypic assays are used often to guide clinicians in decisions concerning the treatment of patients. An optimized sequence-based genotypic assay was used to determine the whole protease and reverse transcriptase (RT) gene, including the gag cleavage site region and RNase H region. Since non-B subtypes are increasing in countries where subtype B was the most prevalent subtype, and treatment becomes more available in developing countries where the epidemic is characterized by a high prevalence of non-B subtypes, it was important that the genotypic test was evaluated using a panel of different subtypes. Amplification was successful for different subtypes: A, B, C, D, F, G, H, J, CRF01_AE, CRF02_AG, CRF11_cpx, CRF13_cpx and an uncharacterized recombinant sample. The detection limit of the PCR was 1000 copies/ml, except for 1 subtype C sample (PL3) and 1 CRF02_AG sample (PL8). The detection limit for these samples was 5000 copies/ml. A sequence could be obtained in both directions for most of the samples.
Assuntos
Infecções por HIV/virologia , Protease de HIV/classificação , Transcriptase Reversa do HIV/classificação , HIV-1/classificação , HIV-1/genética , Reação em Cadeia da Polimerase/métodos , Primers do DNA , DNA Complementar/metabolismo , Farmacorresistência Viral/genética , Produtos do Gene gag/química , Produtos do Gene gag/metabolismo , Genótipo , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , RNA Viral/isolamento & purificação , Ribonuclease H/genéticaRESUMO
BACKGROUND: After the initial discovery of 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) and tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and thione (TIBO) derivatives, several other non-nucleoside reverse transcriptase (RT) inhibitors (NNRTI), including nevirapine (BI-RG-587), pyridinone derivatives (L-696,229 and L-697,661), delavirdine (U-90152), alpha-anilinophenylacetamides (alpha-APA) and various other classes of NNRTI have been described. The hallmark of NNRTI has been based on their ability to interact with a specific site ('pocket') of HIV-1 RT. OBJECTIVE: To investigate whether, in addition to HIV-1, different strains of HIV-2 (ROD and EHO) and SIV (mac251, agm3 and mndGB1) are sensitive to a selection of NNRTI i.e. delavirdine, the HEPT derivative I-EBU (MKC-442), 8-chloro-TIBO (tivirapine), alpha-APA (loviride), nevirapine and the pyridinone derivative L-697,661. METHODS AND RESULTS: The NNRTI tested inhibited the replication of the different strains of HIV-2 and SIV at micromolar concentrations. The inhibitory effects of the NNRTI on HIV-2-induced cytopathicity correlated well with their inhibitory effects on HIV-2 RT activity. Drug-resistant HIV-2 (EHO) variants containing the Ser102Leu and/or Glu219Asp mutations in their RT were selected after passaging the virus in MT-4 cells in the presence of increasing concentrations of delavirdine. The EHO virus mutants were at least 20-fold less susceptible to the antiviral effects of delavirdine. Some cross-resistance, depending on the mutant strain, was observed with the other NNRTI tested (i.e. MKC-442, tivirapine, loviride and pyridinone L-697,661). CONCLUSIONS: Our data demonstrate that NNRTI are not exclusively specific for HIV-1 but are also inhibitory to different HIV-2 and SIV strains. These observations will have important implications for the development of new NNRTI with higher activity against both HIV-1 and HIV-2. Furthermore, in view of their anti-SIV activity, NNRTI could be evaluated further for their in vivo anti-retrovirus efficacy in non-human primate models.
Assuntos
Fármacos Anti-HIV/farmacologia , Delavirdina/farmacologia , HIV-2/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Sequência de Aminoácidos , Células Cultivadas/virologia , Efeito Citopatogênico Viral , Resistência Microbiana a Medicamentos , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , HIV-2/fisiologia , Dados de Sequência Molecular , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Vírus da Imunodeficiência Símia/fisiologiaRESUMO
OBJECTIVES: To investigate the genotypic and phenotypic effects of in vitro resistance selection with lamivudine and/or the second generation non-nucleoside reverse transcriptase inhibitor (NNRTI) quinoxaline HBY097 using HIV-1 isolates carrying the multi-nucleoside resistance pattern linked to the Q151M mutation. METHODS: Virus strains were selected in C8166 cells in the presence of increasing concentrations of lamivudine or HBY097. In parallel control experiments, the virus was cultured in C8166 cells in the absence of drugs. The entire reverse transcriptase encoding region was amplified using polymerase chain reaction and was subsequently sequenced. Antiviral activities of drugs were evaluated in C8166 cells. RESULTS: High-level resistant viruses were selected rapidly in the presence of lamivudine and quinoxaline (less than 10 passages). The multi-nucleoside resistance mutations were stable during in vitro resistance selection. Lamivudine elicited the acquisition of the M184I mutation. Phenotypic resistance to all nucleoside-analog reverse transcriptase inhibitors (NRTIs) was increased when M184I was added to the multi-nucleoside resistance background in the absence of NNRTI-resistance mutations. In most cases of HBY097 resistance selection, at least two mutations associated with NNRTI resistance resulted in high-level NNRTI resistance. The NNRTI resistance-related mutations partially reversed the phenotypic resistance to most NRTIs, except to abacavir. The addition of the M184I mutation to the NNRTI-multi-nucleoside resistance set abolished this antagonizing effect for didanosine, zalcitabine and lamivudine, but further potentiated the phenotypic reversal for zidovudine and stavudine. CONCLUSION: Changes in the non-nucleoside binding pocket must affect the conformation of residues at the dNTP binding site, and can result in a partial phenotypic reversal of the multi-nucleoside resistance phenotype.
Assuntos
Fármacos Anti-HIV/farmacologia , Antivirais/farmacologia , Resistência a Múltiplos Medicamentos/genética , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Lamivudina/farmacologia , Mutação , Quinoxalinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/metabolismo , Antivirais/metabolismo , Sítios de Ligação , Resistência Microbiana a Medicamentos , Genótipo , Infecções por HIV/tratamento farmacológico , HIV-1/classificação , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Lamivudina/metabolismo , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , Fenótipo , Quinoxalinas/metabolismo , Inibidores da Transcriptase Reversa/metabolismoRESUMO
OBJECTIVE: To study the prevalence of multiple dideoxynucleoside (ddN)-resistant (MddNR) HIV-1 in European patients under treatment with multiple ddN analogues, and to characterize MddNR strains genotypically and phenotypically. DESIGN AND METHODS: Blood samples from patients after > or = 6 months of treatment with multiple ddN were screened for the MddNR mutation Q151M. After confirmation of MddNR in 15 patients from five European countries, genotypic resistance was evaluated by DNA sequencing of the reverse transcriptase (RT) gene. Phenotypic resistance was measured by the recombinant virus assay. Results were compared with the clinical evolution of the patients. RESULTS: The prevalence of MddNR strains in European patients treated with multiple ddN analogues was 3.5%. Viruses typically contained amino acid substitutions V75F, F77L, F116Y and Q151M in the RT gene. A new mutation, S68G, was frequently associated with MddNR. Phenotypically, viruses displayed high-level resistance to zidovudine (ZDV), didanosine (ddl), zalcitabine (ddC), stavudine (d4T) and partial resistance to lamivudine (3TC) once multiple mutations were present. Under in-vivo treatment pressure, some MddNR strains additionally developed resistance to protease inhibitors or non-nucleoside RT inhibitors (NNRTI). Clinically, most patients had advanced HIV disease with low CD4 cell counts, high viral loads and a rapid progression, but two patients harbouring MddNR virus responded well to dual protease inhibitor associations. CONCLUSIONS: MddNR resistant HIV-1 can be found in European patients. MddNR is characterized by a specific set of drug resistance mutations, cross-resistance to most ddN analogues and a fast clinical progression. MddNR can be associated with protease inhibitor or NNRTI resistance.
Assuntos
Fármacos Anti-HIV/farmacologia , Didesoxinucleosídeos/farmacologia , Resistência a Múltiplos Medicamentos/genética , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Sequência de Aminoácidos , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/uso terapêutico , Didesoxinucleosídeos/administração & dosagem , Didesoxinucleosídeos/uso terapêutico , Quimioterapia Combinada , Europa (Continente) , Infecções por HIV/tratamento farmacológico , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Dados de Sequência Molecular , Fenótipo , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/uso terapêutico , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
The level of HIV-1 RNA in plasma has become one of the most important markers in the follow-up of HIV-infected patients. Three techniques are commercially available: both the Amplicor HIV Monitor and the NASBA HIV-1 RNA QT are target amplification methods, whereas the Quantiplex HIV RNA assay is a branched DNA signal amplification technique. Detection in both target amplification techniques is based on a single primer pair and a single probe in the gag region, whereas multiple probes capture the pol region of the viral RNA in the branched DNA assay. We investigated the discrepant observation of an undetectable viral load in an immunodeficient pregnant HIV-1-infected patient of African origin with no prior antiretroviral treatment. Although clinical progression was present in this patient with tuberculosis and a low CD4 cell count, viral load determinations with both the Amplicor Monitor and NASBA assays revealed no detectable RNA levels. The presence of HIV-1 RNA in the plasma of the patient was demonstrated by an in-house RNA-PCR. Subsequent HIV-1 RNA quantification with the branched DNA method revealed a high viremia (460,000 copies/ml). DNA sequence analysis of the gag gene identified a subtype G HIV-1 strain (HIV-1BL). To our knowledge this is the first report of a patient harboring an HIV-1 genotype of the main group with a high viral load as quantified by the branched DNA assay, but undetectable with the two commercial HIV RNA amplification techniques because of genetic divergence. In the case of discrepant low viral loads determined by one amplification technique in patients with advanced clinical stage one should use an alternative quantification technique for confirmation.
Assuntos
Síndrome da Imunodeficiência Adquirida/diagnóstico , HIV-1/isolamento & purificação , Complicações Infecciosas na Gravidez/diagnóstico , RNA Viral/isolamento & purificação , Carga Viral/instrumentação , Síndrome da Imunodeficiência Adquirida/virologia , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , HIV-1/genética , Humanos , Linfócitos/química , Reação em Cadeia da Polimerase , Gravidez , Complicações Infecciosas na Gravidez/virologia , Análise de Sequência de DNARESUMO
We evaluated the predictive value of baseline HIV-1 genotypic resistance mutations for failure of a nucleoside reverse transcriptase inhibitor (NRTI) containing therapy. The change in therapy of 88 HIV-1-infected patients was analyzed retrospectively, relating the genotypic resistance profile at baseline to the evolution of viral load and CD4+ T cell counts. Genotypic resistance at baseline and at 6 months was evaluated with the LiPA HIV-1 RT, which detects mutations at codons 41, 69, 70, 74, 184, and 215. At 1 to 3 months after change in therapy, patients without preexisting resistance mutations to the new drug (group S) had a significantly better evolution in viral load (reduction of 0.37 log(10)) compared with patients with known preexisting resistance mutation(s) (group R) (increase of 0.08 log(10)). This difference was particularly striking for patients with the baseline M184V mutation and whose treatment was modified by the addition of lamivudine. After 6 months the median difference in viral load evolution between the two groups increased to 0.61 log(10): the viral load of patients of group S was still 0.18 log(10) below baseline while patients of group R had an increase of 0.43 log(10) in viral load above baseline. Changes in CD4+ T cell counts were not significantly different. The evolution in viral load in HIV-1-infected patients with and without baseline resistance mutation(s) toward a newly added NRTI is significantly different at 1-3 months and at 6 months after changing or adding one NRTI.
Assuntos
Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/uso terapêutico , Contagem de Linfócito CD4 , Didanosina/farmacologia , Didanosina/uso terapêutico , Resistência Microbiana a Medicamentos , Feminino , Genótipo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Lamivudina/farmacologia , Lamivudina/uso terapêutico , Masculino , Mutação/efeitos dos fármacos , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , RNA Viral/análise , Estudos Retrospectivos , Inibidores da Transcriptase Reversa/farmacologia , Carga Viral , Zalcitabina/farmacologia , Zalcitabina/uso terapêutico , Zidovudina/farmacologia , Zidovudina/uso terapêuticoRESUMO
The introduction of potent combinations of antiviral drugs is a major breakthrough in the treatment of HIV. We investigated the long-term virologic outcome and the development of resistance after initiating highly active antiretroviral therapy (HAART) in drug-naive patients in daily clinical practice. Twenty-five treatment-naive HIV-1 patients were started on HAART. Fifteen patients responded with a drop in viral load below the limit of detection during 35.5 (interquartile range: 7) months of therapy. In 6 of 10 patients with virologic failure, virus with resistance-related mutations against the received drugs emerged. Compared with responders (R), nonresponding (NR) patients were in a later disease stage at therapy start (p = 0.0089) with lower CD4 cell counts at baseline (p = 0.040), and a lower proportion of nonresponders showed protease inhibitor (PI) levels above C(min) (p = 0.049). More NR patients showed secondary PI mutations at baseline (p = 0.079), and the CCR2-64I coreceptor polymorphism was absent among NR patients, compared with 38.5% of R patients displaying CCR2-64I (p = 0.053), although the differences were not significant. In conclusion, starting HAART in antiretroviral drug-naive HIV-infected patients followed in daily clinical practice prevented viral breakthrough for up to 44 months in 60% of the patients. Virologic failure was associated with the development of resistance-related mutations, a later stage of disease at start of therapy and lower PI drug levels.
Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Terapia Antirretroviral de Alta Atividade , HIV-1 , Síndrome da Imunodeficiência Adquirida/genética , Síndrome da Imunodeficiência Adquirida/imunologia , Contagem de Linfócito CD4 , Feminino , Genótipo , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , Humanos , Masculino , Mutação , Polimorfismo Genético , Receptores CCR2 , Receptores CCR5/genética , Receptores de Quimiocinas/genética , Fatores de TempoRESUMO
HIV-1 samples from six patients undergoing diverse anti-HIV therapies possessed the E138A mutation in their reverse transcriptase (RT) genome. Patients were receiving the following therapies: TIBO monotherapy (one patient); zidovudine plus didanosine combination therapy (one); zidovudine monotherapy (one); sequential therapy with zidovudine, then stavudine and finally zalcitabine plus didanosine (one); and two were drug naive. E138K, not E138A, is a known TSAO-specific resistance mutation, emerging under selective pressure in vitro. Our phenotypic data on the patient isolates, confirmed by data on an E138A mutant acquired through in vitro mutagenesis, indicated that an alanine substitution for glutamate at codon 138 of the HIV-1 RT renders the virus TSAO resistant, confirming the importance of this amino acid residue in the activity of TSAO derivatives. In addition, we have demonstrated through phenotypic analysis of the E138A and A98S mutants (after in vitro mutagenesis) that the mutation A98S, found in one of these patients, could be partially responsible for the phenotypic reversal of TSAO resistance. This reversal could be explained by the restoration of a hydrogen bond between 98S and the main-chain residue L349, which compensates for the loss of the E138-G99 main-chain hydrogen bond. As TSAO derivatives have not been used in the clinical setting, the presence of the E138A mutation at a frequency of 6.7% in our study of 90 TSAO-inexperienced HIV-seropositive individuals implies that 138A of the RT must be a natural variant and that the mutant virus is replication competent. Our observations suggest that the E138A mutation may likely arise in patients under the selective pressure of TSAO or related compounds that show a decreased antiviral potency toward the E138A variant.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Compostos de Espiro/farmacologia , Timidina/análogos & derivados , Sequência de Bases , Primers do DNA/genética , Resistência Microbiana a Medicamentos/genética , Genes Virais , Variação Genética , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , HIV-1/genética , Humanos , Modelos Moleculares , Fenótipo , Mutação Puntual , Conformação Proteica , Inibidores da Transcriptase Reversa/farmacologia , Timidina/farmacologiaRESUMO
Current recommendations for the treatment of HIV-infected patients advise highly active antiretroviral therapy (HAART) consisting of combinations of 3 or more drugs to provide long-term clinical benefit. This is because only a complete suppression of virus replication will be able to prevent virus drug resistance, the main cause of drug failure. Virus drug resistance may remain a cause of concern in patients who have already received suboptimal mono- or bitherapy, or for patients who do not experience complete shut-down of virus replication under HAART. For these patients, replacement of one combination therapy regimen by another at drug failure, taking into account the existing resistance profile, will be needed. The development of new drugs will remain necessary for those patients who have failed to respond to all currently available drugs, as will be the institution of more effective and less toxic HAART regimens.
Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Fármacos Anti-HIV/uso terapêutico , Resistência a Medicamentos/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Algoritmos , Quimioterapia Combinada , Genótipo , Humanos , FenótipoRESUMO
Since the development of the highly sensitive polymerase chain reaction, PCR has been increasingly used for the diagnosis of viral infections, including the detection of human immunodeficiency virus (HIV), the causative agent of AIDS. In our laboratory a diagnostic PCR is carried out on proviral HIV-1 DNA using a standardised algorithm based on three HIV-1 primer sets. The three primer sets, amplifying a fragment in the LTR-gag gene, in the pol gene and in the env gene, are situated within conserved regions of the HIV-1 genome. These primers allow us to detect not only HIV strains from Belgian patients but also from African patients, who are, for historical reasons, a substantial part of the HIV-positive patients in Belgium. We are able to detect 1-5 copies of proviral HIV-1 DNA with each of the three nested primer sets. A sensitivity and specificity of 92 and 100%, respectively, were achieved when testing 24 Belgian and African HIV-1 seropositive samples. In our lab, the same PCRs are also used for the detection of viral RNA in cases of a doubtful undetectable viral load when using a commercial HIV-1 viral load assay. This is because present-day commercial assays are not entirely reliable with divergent strains. Both our 'in-house' diagnostic DNA and RNA-PCR can also be used semiquantitatively with limiting dilutions.
Assuntos
Síndrome da Imunodeficiência Adquirida/diagnóstico , DNA Viral/isolamento & purificação , HIV-1/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Síndrome da Imunodeficiência Adquirida/epidemiologia , Síndrome da Imunodeficiência Adquirida/virologia , África/epidemiologia , Bélgica/epidemiologia , Doadores de Sangue , Primers do DNA , Estudos de Avaliação como Assunto , Genes Virais/genética , HIV-1/genética , HumanosRESUMO
The performance to detect drug resistance mutations in the reverse transcriptase gene of HIV-1 was compared for direct solid phase sequencing, selective polymerase chain reaction (PCR) using the amplification refractory mutation system (ARMS) and the new line probe assay (LIPA) HIV-1 RT. The three tests were undertaken on 50 plasma samples from 25 treatment-experienced patients under combination therapy with dideoxynucleoside analogues. LiPA HIV-1 RT gave interpretable results in 80 to 96% of the samples depending on the codon of interest. In 2% of the samples a failure to amplify resulted in uninterpretable results for sequencing. ARMS gave no result in seven samples (14%). Overall, there was a 73 to 100% concordance between the three methods. In this study, LiPA HIV-1 RT proved to be an accurate and reliable alternative to DNA sequencing for the detection of drug resistance mutations in patient samples.
Assuntos
Fármacos Anti-HIV/farmacologia , Sondas de DNA , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação , Zidovudina/farmacologia , Códon , Didanosina/uso terapêutico , Resistência Microbiana a Medicamentos , Quimioterapia Combinada , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Lamivudina/uso terapêutico , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Zalcitabina/uso terapêutico , Zidovudina/uso terapêuticoRESUMO
The high replication rate of HIV, together with the low fidelity of its reverse transcriptase, provides the virus with an unprecedented genomic flexibility. This allows a fast adaptation to selective pressure, including antiviral drugs, resulting in the development of drug-resistant strains. The present improvements in the treatment of AIDS patients are at least partly owing to antiviral therapy. To assess the implications of HIV drug resistance on patient management, drug resistance assays for clinical HIV isolates are widely being used. Ideally, monitoring drug resistance should help clinicians in their treatment decisions. If patients would really benefit clinically from this strategy, then the gain from clinical improvement and from omitting drugs to which the virus is already resistant would outweigh the cost of drug resistance testing. In the next few years, researchers should consolidate the clinical benefit of antiviral drug resistance testing, and for this they need fast, reliable and cheap assays. All present assays are in vitro assays, which can only partly mimic the in vivo situation with confounding factors, such as cellular resistance (1). Efforts are presently made to establish in vivo assays (2; see also Chapter 10 ).
RESUMO
OBJECTIVES: The amino acid position 70 in HIV-1 reverse transcriptase (RT) plays an important role in nucleoside RT inhibitor (NRTI) resistance. K70R is part of the thymidine analog mutations, but also other amino acid changes have been associated with NRTI resistance, such as K70E and K70G. In this study, we investigated the in vivo selection of the HIV-1 RT mutations K70S and K70T and their in vitro effect on drug resistance and replication capacity. METHODS: Recombinant viruses with RT mutations were generated to measure the in vitro drug susceptibility and replication capacity. Bayesian network analysis and three-dimensional modeling were performed to understand the selection and impact of the RT70 mutations. RESULTS: K70S and K70T were found at a low frequency in RTI-experienced HIV-1 patients (0.10% and 0·20%). Baeyesian network learning identified no direct association with the in vivo exposure to any specific RTI. However, direct associations of K70S with mutations within the Q151M-complex and of K70T with K65R were observed. In vitro phenotypic testing revealed only minor effects of K70R/S/T as single mutations, associated with Q151M and within the context of the Q151M-complex. DISCUSSION: These results suggest that the selection of K70S/T and their phenotypic impact are influenced by the presence of other mutations in RT. However, the low impact on in vitro phenotype here observed, alongside with the low in vivo prevalence, the exclusive direct association with known major RTI mutations and the unknown correlation with in vivo response, do not yet necessitate the inclusion of K70S/T in drug resistance interpretation systems.