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BACKGROUND: Detailed knowledge of the evolutionary potential of polymorphic sites in a viral protein is important for understanding the development of drug resistance in the presence of an inhibitor. We therefore set out to analyse the molecular evolution of the HIV-1 subtype B integrase at the inter-patient level in Germany during a 20-year period prior to the first introduction of integrase strand inhibitors (INSTIs). METHODS: We determined 337 HIV-1 integrase subtype B sequences (amino acids 1-278) from stored plasma samples of antiretroviral treatment-naïve individuals newly diagnosed with HIV-1 between 1986 and 2006. Shannon entropy was calculated to determine the variability at each amino acid position. Time trends in the frequency of amino acid variants were identified by linear regression. Direct coupling analysis was applied to detect covarying sites. RESULTS: Twenty-two time trends in the frequency of amino acid variants demonstrated either single amino acid exchanges or variation in the degree of polymorphy. Covariation was observed for 17 amino acid variants with a temporal trend. Some minor INSTI resistance mutations (T124A, V151I, K156 N, T206S, S230 N) and some INSTI-selected mutations (M50I, L101I, T122I, T124 N, T125A, M154I, G193E, V201I) were identified at overall frequencies >5%. Among these, the frequencies of L101I, T122I, and V201I increased over time, whereas the frequency of M154I decreased. Moreover, L101I, T122I, T124A, T125A, M154I, and V201I covaried with non-resistance-associated variants. CONCLUSIONS: Time-trending, covarying polymorphisms indicate that long-term evolutionary changes of the HIV-1 integrase involve defined clusters of possibly structurally or functionally associated sites independent of selective pressure through INSTIs at the inter-patient level. Linkage between polymorphic resistance- and non-resistance-associated sites can impact the selection of INSTI resistance mutations in complex ways. Identification of these sites can help in improving genotypic resistance assays, resistance prediction algorithms, and the development of new integrase inhibitors.
Assuntos
Farmacorresistência Viral/genética , Evolução Molecular , Infecções por HIV/virologia , Integrase de HIV/genética , HIV-1/enzimologia , HIV-1/genética , Polimorfismo Genético , Adulto , Substituição de Aminoácidos , Fármacos Anti-HIV/uso terapêutico , Feminino , Genótipo , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/uso terapêutico , HIV-1/classificação , HIV-1/efeitos dos fármacos , Humanos , Masculino , Mutação , Análise de Sequência de DNA , Fatores de TempoRESUMO
An increase in the proportion of ambiguous base calls in HIV-1 pol population sequences during the course of infection has been demonstrated in different study populations, and sequence ambiguity thresholds to classify infections as recent or nonrecent have been suggested. The aim of our study was to evaluate sequence ambiguities as a candidate biomarker for use in an HIV-1 incidence assay using samples from antiretroviral treatment-naive seroconverters with known durations of infection (German HIV-1 Seroconverter Study). We used 2,203 HIV-1 pol population sequences derived from 1,334 seroconverters to assess the sequence ambiguity method (SAM). We then compared the serological incidence BED capture enzyme immunoassay (BED-CEIA) with the SAM for a subset of 723 samples from 495 seroconverters and evaluated a multianalyte algorithm that includes BED-CEIA results, SAM results, viral loads, and CD4 cell counts for 453 samples from 325 seroconverters. We observed a significant increase in the proportion of sequence ambiguities with the duration of infection. A sequence ambiguity threshold of 0.5% best identified recent infections with 76.7% accuracy. The mean duration of recency was determined to be 208 (95% confidence interval, 196 to 221) days. In the subset analysis, BED-CEIA achieved a significantly higher accuracy than the SAM (84.6 versus 75.5%, P < 0.001) and results were concordant for 64.2% (464/723) of the samples. Also, the multianalyte algorithm did not show better accuracy than the BED-CEIA (83.4 versus 84.3%, P = 0.786). In conclusion, the SAM and the multianalyte algorithm including SAM were inferior to the BED-CEIA, and the proportion of sequence ambiguities is therefore not a preferable biomarker for HIV-1 incidence testing.
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Marcadores Genéticos , Variação Genética , Infecções por HIV/diagnóstico , Infecções por HIV/virologia , HIV-1/genética , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genética , Adulto , Contagem de Linfócito CD4 , Estudos de Coortes , Feminino , Genótipo , HIV-1/classificação , HIV-1/isolamento & purificação , Humanos , Masculino , Estudos Prospectivos , Carga ViralRESUMO
The evolution of a sexually transmitted multiresistant HIV-1 in a linked transmission chain was followed for 33 months to assess its potential to persist in the absence of treatment. The multiresistant HIV reverted slowly to wild type in reverse transcriptase (positions 44, 67, 74, 118) rendering the virus only susceptible to lamivudine/emtricitabine. Persistence of the replication-competent resistant HIV increases its potential to spread further and strengthens the importance of resistance testing in newly infected patients.
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Farmacorresistência Viral/genética , Genes pol/genética , Infecções por HIV/genética , HIV-1/genética , Adulto , Antivirais/uso terapêutico , Contagem de Linfócito CD4 , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Emtricitabina , Infecções por HIV/tratamento farmacológico , Humanos , Lamivudina/uso terapêutico , Masculino , Mutação/genética , Inibidores da Transcriptase Reversa/uso terapêutico , Carga ViralRESUMO
BACKGROUND: Although HIV continues to spread globally, novel intervention strategies such as treatment as prevention (TasP) may bring the epidemic to a halt. However, their effective implementation requires a profound understanding of the underlying transmission dynamics. METHODS: We analyzed parameters of the German HIV epidemic based on phylogenetic clustering of viral sequences from recently infected seroconverters with known infection dates. Viral baseline and follow-up pol sequences (n = 1943) from 1159 drug-naïve individuals were selected from a nationwide long-term observational study initiated in 1997. Putative transmission clusters were computed based on a maximum likelihood phylogeny. Using individual follow-up sequences, we optimized our clustering threshold to maximize the likelihood of co-clustering individuals connected by direct transmission. RESULTS: The sizes of putative transmission clusters scaled inversely with their abundance and their distribution exhibited a heavy tail. Clusters based on the optimal clustering threshold were significantly more likely to contain members of the same or bordering German federal states. Interinfection times between co-clustered individuals were significantly shorter (26 weeks; interquartile range: 13-83) than in a null model. CONCLUSIONS: Viral intraindividual evolution may be used to select criteria that maximize co-clustering of transmission pairs in the absence of strong adaptive selection pressure. Interinfection times of co-clustered individuals may then be an indicator of the typical time to onward transmission. Our analysis suggests that onward transmission may have occurred early after infection, when individuals are typically unaware of their serological status. The latter argues that TasP should be combined with HIV testing campaigns to reduce the possibility of transmission before TasP initiation.
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Infecções por HIV/transmissão , Infecções por HIV/virologia , Soroconversão , Adulto , Análise por Conglomerados , Farmacorresistência Viral/genética , Feminino , Alemanha/epidemiologia , Infecções por HIV/prevenção & controle , HIV-1/classificação , HIV-1/enzimologia , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Masculino , Filogenia , Prevalência , Estudos Prospectivos , Fatores de Tempo , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genéticaRESUMO
INTRODUCTION: The aim of our study was to analyze the occurrence and evolution of HIV-1 integrase polymorphisms during the HIV-1 epidemic in Germany prior to the introduction of the first integrase inhibitor raltegravir in 2007. MATERIALS AND METHODS: Plasma samples from drug-naïve HIV-1 infected individuals newly diagnosed between 1986 and 2006 were used to determine PCR-based population sequences of the HIV-1 integrase (amino acids 1-278). The HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. We calculated the frequency of amino acids at each position of the HIV-1 integrase in 337 subtype B strains for the time periods 1986-1989, 1991-1994, 1995-1998, 1999-2002, and 2003-2006. Positions were defined as polymorphic if amino acid variation was >1% in any period. Logistic regression was used to identify trends in amino acid variation over time. Resistance-associated mutations were identified according to the IAS 2013 list and the HIVdb, ANRS and GRADE algorithms. RESULTS: Overall, 56.8% (158/278) amino acid positions were polymorphic and 15.8% (25/158) of these positions exhibited a significant trend in amino acid variation over time. Proportionately, most polymorphic positions (63.3%, 31/49) were detected in the N-terminal zinc finger domain of the HIV-1 integrase. Motifs and residues essential for HIV-1 integrase activity were little polymorphic, but within the minimal non-specific DNA binding region I220-D270 up to 18.1% amino acid variation was noticed, including four positions with significant amino acid variation over time (S230, D232, D256, A265). No major resistance mutations were identified, and minor resistance mutations were rarely observed without trend over time. E157Q considered by HIVdb, ANRS, and GRADE algorithms was the most frequent resistance-associated polymorphism with an overall prevalence of 2.4%. CONCLUSIONS: Detailed knowledge of the evolutionary variation of HIV-1 integrase polymorphisms is important to understand the development of resistance in the presence of the drug. Our results will contribute to define the relevance of integrase polymorphisms in HIV-strains resistant to integrase inhibitors and to improve resistance interpretation algorithms.
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BACKGROUND: Transmission of drug-resistant HIV-1 (TDR) can impair the virologic response to antiretroviral combination therapy. Aim of the study was to assess the impact of TDR on treatment success of resistance test-guided first-line therapy in the German HIV-1 Seroconverter Cohort for patients infected with HIV between 1996 and 2010. An update of the prevalence of TDR and trend over time was performed. METHODS: Data of 1,667 HIV-infected individuals who seroconverted between 1996 and 2010 were analysed. The WHO drug resistance mutations list was used to identify resistance-associated HIV mutations in drug-naïve patients for epidemiological analysis. For treatment success analysis the Stanford algorithm was used to classify a subset of 323 drug-naïve genotyped patients who received a first-line cART into three resistance groups: patients without TDR, patients with TDR and fully active cART and patients with TDR and non-fully active cART. The frequency of virologic failure 5 to 12 months after treatment initiation was determined. RESULTS: Prevalence of TDR was stable at a high mean level of 11.9% (198/1,667) in the HIV-1 Seroconverter Cohort without significant trend over time. Nucleotide reverse transcriptase inhibitor resistance was predominant (6.0%) and decreased significantly over time (ORâ=â0.92, CIâ=â0.87-0.98, pâ=â0.01). Non-nucleoside reverse transcriptase inhibitor (2.4%; ORâ=â1.00, CIâ=â0.92-1.09, pâ=â0.96) and protease inhibitor resistance (2.0%; ORâ=â0.94, CIâ=â0.861.03, pâ=â0.17) remained stable. Virologic failure was observed in 6.5% of patients with TDR receiving fully active cART, 5,6% of patients with TDR receiving non-fully active cART and 3.2% of patients without TDR. The difference between the three groups was not significant (pâ=â0.41). CONCLUSION: Overall prevalence of TDR remained stable at a rather high level. No significant differences in the frequency of virologic failure were identified during first-line cART between patients with TDR and fully-active cART, patients with TDR and non-fully active cART and patients without TDR.
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Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1 , Adulto , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Feminino , Genótipo , Infecções por HIV/epidemiologia , Infecções por HIV/transmissão , HIV-1/genética , Humanos , Masculino , Prevalência , Fatores de Risco , Resultado do Tratamento , Carga ViralRESUMO
BACKGROUND: The variety and limitations of current laboratory methods for estimating HIV-incidence has driven attempts to improve and standardize the performance of serological 'Tests for Recent HIV-Infections' (TRI). Primary and follow-up HIV-1 positive plasma samples from individuals with well-defined dates of infection collected as part of the German Seroconverter Cohort provided specimens highly suitable for use in comparing the performance of three TRIs: the AWARE™ BED™ EIA HIV-1 Incidence test (BED-CEIA), Genetic systems HIV-1/HIV-2 Plus O EIA antibody avidity-based assay (BioRad Avidity) and Sedia™ HIV-1 LAg Avidity EIA (LAg Avidity). METHODS: The evaluation panel included 180 specimens: 44 from antiretroviral (ARV)-naïve individuals with recently acquired HIV-infection (≤ 130 days; 25 B and 19 non-B subtypes) and 136 from long-term (>12 months) infected individuals [101 ARV-naïve subtype B, 16 non-B subtypes, 14 ARV-treated individuals, 5 slow progressors (SLP)]. RESULTS: For long-term infected, ARV-naïve individuals the false recent rates (FRR) of both the BioRad and LAg Avidity assays were 2% (2/101 for subtype B) and 6% (1/16 for subtype 'non-B'), while the FRR of the BED-CEIA was 7% (7/101 for subtype B) and 25% (4/16 for subtype 'non-B') (all p>0.05). Misclassification of ARV-treated individuals and SLP was rare by LAg (1/14, 0/5) and BioRad Avidity assays (2/14, 1/5) but more frequent by BED-CEIA (5/14, 3/5). Among recently-infected individuals (subtype B), 60% (15/25) were correctly classified by BED-CEIA, 88% (22/25) by BioRad Avidity and significantly fewer by LAg (48%, 12/25) compared to BioRad Avidity (p = 0.005) with a higher true-recency rate among non-B infections for all assays. CONCLUSIONS: This study using well-characterized specimens demonstrated lower FRRs for both avidity methods than with the BED-CEIA. For recently infected individuals the BioRad Avidity assay was shown to give the most accurate results.
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Afinidade de Anticorpos/imunologia , Antígenos HIV/imunologia , Soropositividade para HIV/diagnóstico , Soropositividade para HIV/imunologia , HIV-1/imunologia , Técnicas Imunoenzimáticas/métodos , Técnicas Imunoenzimáticas/normas , Estudos de Coortes , Alemanha , Anticorpos Anti-HIV/imunologia , Humanos , Kit de Reagentes para Diagnóstico , Padrões de ReferênciaRESUMO
BACKGROUND: Nevirapine single-dose (NVP-SD) reduces mother-to-child transmission of HIV type-1 (HIV-1), but frequently induces resistance mutations in the HIV-1 genome. Little is known about drug-resistant HIV-1 variants in the breast milk of women who have taken NVP-SD. METHODS: Blood and breast milk samples of 39 HIV-1-infected Ugandan women were taken 6-12 weeks after NVP-SD intake. Samples were analysed by population sequencing and allele-specific real-time PCR (AS-PCR) with detection limits for NVP-resistant HIV-1 variants (K103N and Y181C) of < 1% of the total viral population. RESULTS: AS-PCR results for both plasma and breast milk were obtained for 19 women who constituted the final study group (HIV-1 subtype frequencies were A1 n = 11, D n = 5, G n = 2 and C n = 1). A total of 7 (37%) and 10 (53%) women carried NVP-resistant virus in breast milk and plasma, respectively. Overall, 71% (5/7) women with NVP-resistant HIV-1 in breast milk displayed >1 drug-resistant variant. Resistance in breast milk was higher at week 6 (6/13 samples [46%]) compared with week 12 (1/6 samples [17%]). In total, 10 drug-resistant populations harbouring the K103N and/or Y181C mutation were detected in the 19 breast milk samples; 7 (70%) were caused by resistant minorities (< 5% of the total HIV-1 population). In the four women with drug-resistant virus in both plasma and breast milk, the mutation patterns differed between the two compartments. CONCLUSIONS: Minor populations of drug-resistant HIV-1 were frequently found in breast milk of Ugandan women after exposure to NVP-SD. Further studies need to explore the role of minor drug-resistant variants in the postnatal transmission of (resistant) HIV-1.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , HIV-1/genética , Nevirapina/administração & dosagem , Profilaxia Pós-Exposição , Adulto , Fármacos Anti-HIV/uso terapêutico , Estudos de Coortes , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Feminino , Variação Genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/transmissão , Infecções por HIV/virologia , Humanos , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Leite Humano/química , Leite Humano/efeitos dos fármacos , Leite Humano/virologia , Mutação/efeitos dos fármacos , Nevirapina/uso terapêutico , Gravidez , Complicações Infecciosas na Gravidez/tratamento farmacológico , Complicações Infecciosas na Gravidez/prevenção & controle , Complicações Infecciosas na Gravidez/virologia , UgandaRESUMO
BACKGROUND: The aim of this study is to analyse the prevalence of transmitted drug resistance, TDR, and the impact of TDR on treatment success in the German HIV-1 Seroconverter Cohort. METHODS: Genotypic resistance analysis was performed in treatment-naïve study patients whose sample was available 1,312/1,564 (83.9% October 2008). A genotypic resistance result was obtained for 1,276/1,312 (97.3%). The resistance associated mutations were identified according to the surveillance drug resistance mutations list recommended for drug-naïve patients. Treatment success was determined as viral suppression below 500 copies/ml. RESULTS: Prevalence of TDR was stable at a high level between 1996 and 2007 in the German HIV-1 Seroconverter Cohort (Nâ=â158/1,276; 12.4%; CI(wilson) 10.7-14.3; p(for trend)â=â0.25). NRTI resistance was predominant (7.5%) but decreased significantly over time (CI(Wilson): 6.2-9.1, p(for trend)â=â0.02). NNRTI resistance tended to increase over time (NNRTI: 3.5%; CI(Wilson): 2.6-4.6; p(for trend)=â0.07), whereas PI resistance remained stable (PI: 3.0%; CI(Wilson): 2.1-4.0; p(for trend)â=â0.24). Resistance to all drug classes was frequently caused by singleton resistance mutations (NRTI 55.6%, PI 68.4%, NNRTI 99.1%). The majority of NRTI-resistant strains (79.8%) carried resistance-associated mutations selected by the thymidine analogues zidovudine and stavudine. Preferably 2NRTI/1PIr combinations were prescribed as first line regimen in patients with resistant HIV as well as in patients with susceptible strains (susceptible 45.3%; 173/382 vs. resistant 65.5%; 40/61). The majority of patients in both groups were treated successfully within the first year after ART-initiation (susceptible: 89.9%; 62/69; resistant: 7/9; 77.8%). CONCLUSION: Overall prevalence of TDR remained stable at a high level but trends of resistance against drug classes differed over time. The significant decrease of NRTI-resistance in patients newly infected with HIV might be related to the introduction of novel antiretroviral drugs and a wider use of genotypic resistance analysis prior to treatment initiation.
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Farmacorresistência Viral , Infecções por HIV/tratamento farmacológico , Adulto , Feminino , Genótipo , Alemanha , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Masculino , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
OBJECTIVES: HIV protease - as well as gag cleavage site (CS) - mutations occur in HIV with PI resistance but little is known about the prevalence of CS mutations in drug-naïve patients. PATIENTS AND METHODS: HIV samples (collected before 1997: n=94, after 1997: n=1617) from drug-naïve patients were analysed in the C-terminal gag and pol gene. Additionally, sequences from HIV Stanford database were included according to the collection date of the blood sample (before 1997: n=200, after 1997: n=375). RESULTS: Only CS mutations 431V and 452S were correlated with primary PI resistance in drug-naïve HIV. Previously described therapy-associated CS mutations (431V/449F/449H/451T/452S/453A) were found in less than 0.5% of therapy-naïve HIV without primary drug resistance and were totally absent in HIV isolates collected before 1997. The detection of 431V in the absence of PR mutations was significantly correlated with the presence of 429K. The treatment-associated CS mutations (436R/437V/453L) were generally found in more than 1% of drug-naïve HIV with differences between HIV subtypes. Natural polymorphisms were frequently found and also differed between HIV subtype B and non-B subtypes. CONCLUSIONS: The majority of therapy-associated CS mutations were rarely detected in drug-naïve HIV, but can be found in the absence of protease mutations. Moreover, the prevalence of these CS mutations seemed to have increased in recent years. The presence of treatment-associated CS mutations in drug-naïve patients might lower the genetic barrier of first-line therapies with protease inhibitors.