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1.
J Antimicrob Chemother ; 72(6): 1769-1773, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28333232

RESUMO

Background: Surveillance of HIV-1 resistance in treated patients with a detectable viral load (VL) is important to monitor, in order to assess the risk of spread of resistant viruses and to determine the proportion of patients who need new antiretroviral drugs with minimal cross-resistance. Methods: The HIV-1 protease and reverse transcriptase (RT) and integrase genes were sequenced in plasma samples from 782 consecutive patients on failing antiretroviral regimens, seen in 37 specialized centres in 2014. The genotyping results were interpreted using the ANRS v24 algorithm. Prevalence rates were compared with those obtained during a similar survey conducted in 2009. Results: The protease and RT sequences were obtained in 566 patients, and the integrase sequence in 382 patients. Sequencing was successful in 60%, 78%, 78% and 87% of patients with VLs of 51-200, 201-500, 501-1000 and >1000 copies/mL, respectively. Resistance to at least one antiretroviral drug was detected in 56.3% of samples. Respectively, 3.9%, 8.7%, 1.5% and 3.4% of patients harboured viruses that were resistant to any NRTI, NNRTI, PI and integrase inhibitor (INI). Resistance rates were lower in 2014 than in 2009. Resistance was detected in 48.5% of samples from patients with a VL between 51 and 200 copies/mL. Conclusion: In France in 2014, 90.0% of patients in AIDS care centres were receiving antiretroviral drugs and 12.0% of them had VLs >50 copies/mL. Therefore, this study suggests that 6.7% of treated patients in France might transmit resistant strains. Resistance testing may be warranted in all treated patients with VL > 50 copies/mL.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Farmacorresistência Viral Múltipla , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Carga Viral , Adulto , Terapia Antirretroviral de Alta Atividade , Feminino , França , Genes Virais , Genótipo , Infecções por HIV/sangue , Integrase de HIV/sangue , Integrase de HIV/genética , Protease de HIV/sangue , Protease de HIV/genética , Transcriptase Reversa do HIV/sangue , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , Inibidores da Transcriptase Reversa/uso terapêutico , Análise de Sequência de DNA , Falha de Tratamento
2.
Infect Dis (Lond) ; 48(6): 467-71, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26654354

RESUMO

Viral load testing for human immunodeficiency virus 1 (HIV-1) in resource-poor settings continues to be a challenge. Although antiretroviral therapy (ART) is being made available in developing countries, monitoring of viral load is not being done on a regular basis. The purpose of this study was to assess the utility of Cavidi version 3.0, which measures the plasma reverse transcriptase (RT) activity and compare its performance with molecular HIV viral load assays. In all, 125 HIV-1 and 13 HIV-2 positive samples were analyzed. The overall sensitivity of the assay was 86.8% and 94.1% for viral load >1000 copies/ml measured by Qiagen Artus HIV-1 RG RT PCR and Abbott RealTime HIV-1 PCR assays, respectively. Compared with the routine molecular viral load assays, Cavidi version 3.0 is inexpensive, user-friendly, the expenditure on infrastructure is minimal, and it can be used for monitoring of both HIV types.


Assuntos
Infecções por HIV/sangue , Infecções por HIV/virologia , Transcriptase Reversa do HIV/sangue , HIV-1/enzimologia , HIV-2/enzimologia , Ativação Enzimática , Infecções por HIV/diagnóstico , HIV-1/isolamento & purificação , HIV-2/isolamento & purificação , Humanos , RNA Viral/análise , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Testes Sorológicos/economia , Testes Sorológicos/métodos , Carga Viral/economia , Carga Viral/métodos
3.
Biomed Res Int ; 2015: 240407, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26779533

RESUMO

Monitoring antiretroviral therapy using measurements of viral load (VL) and the genotyping of resistance mutations is not routinely performed in low- to middle-income countries because of the high costs of the commercial assays that are used. The analysis of dried plasma spot (DPS) samples on filter paper may represent an alternative for resource-limited settings. Therefore, we evaluated the usefulness of analyzing DPS samples to determine VL and identify drug resistance mutations (DRM) in a group of HIV-1 patients. The VL was measured from 22 paired plasma and DPS samples. In these samples, the average VL was 4.7 log10 copies/mL in liquid plasma and 4.1 log10 copies/mL in DPS, with a correlation coefficient of R = 0.83. A 1.1 kb fragment of HIV pol could be amplified in 14/22 (63.6%) of the DPS samples and the same value was amplified in plasma samples. A collection of ten paired DPS and liquid plasma samples was evaluated for the presence of DRM; an excellent correlation was found in the identification of DRM between the paired samples. All HIV-1 pol sequences that were obtained corresponded to HIV subtype B. The analysis of DPS samples offers an attractive alternative for monitoring ARV therapy in resource-limited settings.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Farmacorresistência Viral/genética , Infecções por HIV/sangue , HIV-1/isolamento & purificação , Adulto , Feminino , Genótipo , Infecções por HIV/genética , Infecções por HIV/virologia , Protease de HIV/sangue , Protease de HIV/genética , Transcriptase Reversa do HIV/sangue , Transcriptase Reversa do HIV/genética , HIV-1/genética , HIV-1/patogenicidade , Humanos , Masculino , México , Mutação , Filogenia , RNA Viral/sangue , RNA Viral/genética , Carga Viral , Produtos do Gene pol do Vírus da Imunodeficiência Humana/sangue , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genética
4.
Talanta ; 85(1): 770-8, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21645772

RESUMO

This manuscript describes an electrochemical approach to the detection of the reverse transcriptase of the human immunodeficiency virus type-1 (HIV-1 RT) in serum exploiting an organometallic peptide conjugate that is chemically linked to a nanostructured gold surface. The assay format is based on the formation of a thin film of a ferrocene-labeled lipoic acid (Fc-LA) onto a gold nanoparticles-functionalized screen-printed carbon electrode (GNPs-SPCE). Time-of-Flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy were employed to confirm the binding of the Fc-LA to the electrode surface via formation of a gold-thiol bond. The RT biosensor was developed by covalent attachment of the peptide VEAIIRILQQLLFIH to the carboxylic acid group of Fc-LA. Square wave voltammetry offered a two-dimensional measurement of RT based on the anodic shift and reduction of current density of the Fc redox signal upon binding of RT to its specific peptide. This allowed a linear quantification of the target RT in the range of 1-500 pg mL(-1) equivalent to 0.9-427 fM, with a detection limit of 0.8 pg mL(-1) (0.7 fM) with a short response time.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Transcriptase Reversa do HIV/sangue , Nanoestruturas/química , Compostos Ferrosos , Humanos , Limite de Detecção , Metalocenos , Peptídeos , Ligação Proteica , Ácido Tióctico
5.
AIDS Res Hum Retroviruses ; 27(8): 921-4, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21114462

RESUMO

Human immunodeficiency virus type 1 (HIV-1) is characterized by high genetic variability due to its high replication rate and the lack of proofreading activity of the reverse transcriptase enzyme. On the basis of phylogenetic analysis performed on numerous isolates from all over the world, HIV-1 is subdivided into types, subtypes, subsubtypes, circulating recombinant forms, and unique recombinant forms. No data are currently available about the circulation of HIV-1 types in Montenegro. Here, we describe the genetic variability of HIV-1 strains identified in plasma samples of patients from Montenegro. Phylogenetic analysis on 32 HIV-1 sequences was carried out. The prevalent circulating HIV-1 subtype is B. The strains were interspersed within the tree. Two main clades (I and II) may suggest independent introductions of HIV-1 subtype B into Montenegro, although other epidemiological evidence will be needed to assume a small number of introductions. No obvious evidence of clustering by residence, age, or sex was found (data not shown). Nelfinavir resistance was found, though lopinavir is the only PI administered. Continuous monitoring of HIV-1-infected individuals is crucial to a better understand of the epidemiology of the B subtype in Montenegro.


Assuntos
Infecções por HIV/sangue , Inibidores da Protease de HIV/administração & dosagem , Protease de HIV/genética , HIV-1/genética , Lopinavir/administração & dosagem , Adulto , Terapia Antirretroviral de Alta Atividade/efeitos adversos , Sequência de Bases , Farmacorresistência Viral , Feminino , Variação Genética , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , Protease de HIV/sangue , Inibidores da Protease de HIV/uso terapêutico , Transcriptase Reversa do HIV/sangue , Transcriptase Reversa do HIV/genética , HIV-1/classificação , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/isolamento & purificação , Humanos , Lopinavir/uso terapêutico , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Montenegro , Nelfinavir/farmacologia , Filogenia , Vigilância da População
6.
Diagn Microbiol Infect Dis ; 68(3): 208-13, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20846816

RESUMO

The quantitation of HIV viral load using an assay that measures the activity of reverse transcriptase (RT) may provide an alternative strategy for the monitoring of HIV viral load within resource-limited areas in China. Plasma viral load analyses of 215 samples from 87 patients infected with HIV were detected using the RT activity assay (ExaVir Load versions 2 and 3; Cavidi, Uppsala, Sweden) and RT polymerase chain reaction (PCR) (COBAS TaqMan 48, Amplink version 3.2; Roche Molecular Systems, Branchburg, NJ). The RT activity assay versions 3 (RT3) and 2 (RT2) could detect 95.3% and 86.9% of samples with measurable RNA by RT-PCR, respectively. A stronger correlation was observed between viral loads detected by RT3 and RT-PCR than between RT2 and RT-PCR (r = 0.95, P < 0.001, and r = 0.92, P < 0.001, respectively). The correlation between serial samples collected from 6 patients at 1, 3, 6, 12, 18, and 24 months after beginning triple combination antiretroviral therapy, using the 2 different methodologies, was also strong (r = 0.99, P < 0.001, for RT3 and RT-PCR, r = 0.98, P < 0.001, for RT2 and RT-PCR). The viral loads detected by RT activity assay were inversely correlated with CD4(+) T-cell counts. Reproducibility of the RT activity assay was assessed by testing 3 samples in triplicate by 3 different operators. Viral load testing using assays that measure HIV RT activity is an affordable, feasible, simple, and reliable alternative for HIV RNA viral load determination in laboratories in China and other developing countries.


Assuntos
Infecções por HIV/virologia , Transcriptase Reversa do HIV/sangue , HIV/enzimologia , HIV/isolamento & purificação , Carga Viral , Fármacos Anti-HIV/uso terapêutico , Contagem de Linfócito CD4 , China , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , RNA Viral/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatística como Assunto
7.
Artigo em Inglês | MEDLINE | ID: mdl-19822735

RESUMO

The reverse transcriptase (RT) enzyme of HIV type 1 (HIV-1) is largely targeted by the host immune selection pressure and would differ in the anatomical compartments, thereby having a drastic impact on viral quasi-species evolution. The HIV-1 RT region sequenced from plasma and genital secretions of 8 antiretroviral treatment (ART)-naive females was analyzed for the pattern of amino acid mutations and the ratio of synonymous and nonsynonymous substitutions to determine whether it is under different selection pressure in both the compartments. Phylogenetic and mutational analysis of the HIV-1 RT in plasma and genital secretions of HIV-1-infected ART-naive females showed limited variation likely reflecting the absence of differential selection pressure and therefore genetic variation in these compartments.


Assuntos
Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , HIV-1/genética , RNA Viral/genética , Adulto , Algoritmos , Contagem de Linfócito CD4 , Códon , Bases de Dados de Ácidos Nucleicos , Feminino , Infecções por HIV/enzimologia , Infecções por HIV/transmissão , Transcriptase Reversa do HIV/análise , Transcriptase Reversa do HIV/sangue , Transcriptase Reversa do HIV/química , HIV-1/classificação , HIV-1/enzimologia , Humanos , Índia , Mutação , Filogenia , Reação em Cadeia da Polimerase , RNA Viral/análise , RNA Viral/sangue , Alinhamento de Sequência , Esfregaço Vaginal , Adulto Jovem
8.
J Clin Microbiol ; 47(10): 3266-70, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19656978

RESUMO

In resource-limited settings, the virological monitoring of antiretroviral therapy is limited by high cost and the lack of infrastructure. The Cavidi ExaVir Load assay employs a simple and inexpensive enzyme-linked immunosorbent assay format to measure human immunodeficiency virus (HIV) reverse transcriptase activity, which correlates with plasma RNA load. The version 3 assay has been described as having improved precision and sensitivity. There are limited data on its performance relative to those of current real-time assays. Our objective was to compare HIV type 1 (HIV-1) RNA load measurement in plasma by ExaVir Load version 3 (designated ExaVir), Abbott M2000sp/M2000rt RealTime HIV-1 assay (designated RealTime), and Roche COBAS Ampliprep/COBAS TaqMan HIV-1 version 1 assay (designated TaqMan). Plasma from 119 patients (34 with subtype B infection, 85 with non-subtype B infection [A-H, CRF01, CRF02, CRF06, CRF12, CRF14, and complex]; 48 subjects were treatment experienced, 71 were naive) and serial dilutions of the second international standard (IS) were tested. Assay relationship and agreement were determined by linear regression, correlation analysis, and the Bland-Altman method. The ExaVir assay quantified 77/83 (92.8%) samples with viral loads of >2.3 log10 copies/ml by the molecular assays. Results were linearly associated and strongly correlated with RealTime and TaqMan measurements (R of 0.94 and 0.92, respectively) for both subtype B (R of 0.97 and 0.95, respectively) and non-subtype B (R of 0.93 and 0.91, respectively) samples. Mean differences were 0.28 and 0.18 log10 copies/ml in favor of the two molecular assays; 7/119 (5.9%) and 5/119 (4.2%) samples were outside the 95% level of agreement. ExaVir underquantified the IS by a mean of 0.2 (range, 0.0 to 0.5) log10 copies/ml. The ExaVir assay showed excellent concordance with real-time molecular assays, offering a suitable option for virological monitoring in settings with limited infrastructure.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Infecções por HIV/virologia , Transcriptase Reversa do HIV/sangue , HIV-1/enzimologia , Carga Viral/métodos , Humanos , Plasma/química , Plasma/virologia , Kit de Reagentes para Diagnóstico
9.
J Acquir Immune Defic Syndr ; 52(3): 387-90, 2009 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-19617845

RESUMO

BACKGROUND: Viral load (VL) is a critical marker for monitoring HIV disease progression and response to antiretroviral therapy. In resource-constrained settings, there is a need for a simple and inexpensive assay to monitor infected adults and children. METHODS: We compared versions 2 and 3 of the ExaVir Load assay, Cavidi AB (HIV RT) with the Roche, COBAS Amplicor HIV-1 Monitor assay (HIV RNA) for quantifying HIV VL. RESULTS: The HIV RT version 2 assay showed good sensitivity with detection in 94% of samples with HIV RNA >1000 copies per milliliter. Adult samples were tested using HIV RT version 2 (n = 35) and version 3 (n = 23) assays with plasma volumes of 1 mL (recommended), 0.5 mL and 0.25 mL in comparison with HIV RNA. The HIV RT and HIV RNA assay results were comparable when tested using different volumes. Comparison of results from pediatric samples (n = 27), tested using 1 mL and a smaller volume by HIV RT version 2 were not significantly different. CONCLUSIONS: The HIV RT assay was comparable to the HIV RNA assay with sensitivity approaching that of HIV RNA. Smaller volumes than the recommended 1 mL can be used, improving utility of this assay for pediatric monitoring.


Assuntos
Infecções por HIV/virologia , Transcriptase Reversa do HIV/sangue , Carga Viral/métodos , Adulto , Criança , Infecções por HIV/sangue , Humanos , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
10.
HIV Med ; 8(6): 388-95, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17661847

RESUMO

OBJECTIVE: The aim of the study was to investigate the influence of highly active antiretroviral therapy (HAART) on iron status and, conversely, the influence of iron status on the response to HAART. METHODS: Ferritin levels were retrospectively determined in stored plasma from 138 HAART-naïve, moderately immunosuppressed HIV-infected Thai patients participating in a structured treatment interruption trial. Ferritin levels were determined at three predefined time-points: (1) HAART initiation; (2) HAART discontinuation; and (3) HAART resumption. RESULTS: At baseline, 31% and 16% of the HIV-infected patients included in the study had high (>200 ng/mL) and low (<30 ng/mL) ferritin levels, respectively. Ninety-five per cent of patients with low ferritin levels were female. Ferritin decreased significantly during the interruption phase of HAART (-8.8 ng/mL; P=0.0005) but remained elevated in 62% of the patients with high baseline levels. A low baseline ferritin level was associated with a shorter time (P=0.041) to reach the CD4 cell target for HAART interruption (350 cells/microL), compared with a normal or high baseline ferritin level. Moreover, in a multivariate model, the relative risk (RR) of arriving at this CD4 cell target was significantly higher [RR 1.81; 95% confidence interval (CI) 1.05-3.14] in patients with low baseline ferritin. It is unlikely that inflammation affected ferritin in our patients, as mean levels of C-reactive protein were not elevated in patients with either high or low ferritin levels. CONCLUSIONS: Both high and low ferritin levels were highly prevalent in moderately immunosuppressed HIV-positive Thai patients. Structured treatment interruption of HAART resulted in a significant decrease in overall ferritin levels. Furthermore, subjects with low baseline ferritin levels had a faster and greater CD4 response to HAART, suggesting a potential beneficial effect of iron deficiency on immunological recovery after initiation of HAART.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Ferritinas/metabolismo , Infecções por HIV/tratamento farmacológico , Transcriptase Reversa do HIV/sangue , Inibidores da Transcriptase Reversa/sangue , Adulto , Terapia Antirretroviral de Alta Atividade/métodos , Contagem de Linfócito CD4 , Feminino , Infecções por HIV/sangue , Infecções por HIV/imunologia , Transcriptase Reversa do HIV/imunologia , Humanos , Masculino , Análise Multivariada , Estudos Retrospectivos , Inibidores da Transcriptase Reversa/imunologia , Resultado do Tratamento , Carga Viral
11.
Genome Res ; 17(8): 1195-201, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17600086

RESUMO

The detection of mutant spectra within a population of microorganisms is critical for the management of drug-resistant infections. We performed ultra-deep pyrosequencing to detect minor sequence variants in HIV-1 protease and reverse transcriptase (RT) genes from clinical plasma samples. We estimated empirical error rates from four HIV-1 plasmid clones and used them to develop a statistical approach to distinguish authentic minor variants from sequencing errors in eight clinical samples. Ultra-deep pyrosequencing detected an average of 58 variants per sample compared with an average of eight variants per sample detected by conventional direct-PCR dideoxynucleotide sequencing. In the clinical sample with the largest number of minor sequence variants, all 60 variants present in > or =3% of genomes and 20 of 35 variants present in <3% of genomes were confirmed by limiting dilution sequencing. With appropriate analysis, ultra-deep pyrosequencing is a promising method for characterizing genetic diversity and detecting minor yet clinically relevant variants in biological samples with complex genetic populations.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Análise Mutacional de DNA/métodos , Infecções por HIV/tratamento farmacológico , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/genética , DNA Viral/metabolismo , Farmacorresistência Viral/genética , Variação Genética , Protease de HIV/sangue , Transcriptase Reversa do HIV/sangue , HIV-1/enzimologia , Humanos , Mutação
13.
J Med Virol ; 78(2): 161-8, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16372295

RESUMO

The present study monitored the changes in human immunodeficiency virus (HIV) viral load using a reverse transcriptase (RT) assay and an HIV-1 RNA based assay, and relates these data to the dynamics of CD4 cell counts. The samples examined originate from a prospective study of HIV-1 subtype C infected, untreated Ethiopians followed twice yearly over a period of up to 5 years. The ExaVir Load test, version 1, was used for isolation and quantitation of HIV-1 RT in plasma. The RT activities recovered were compared to the HIV-1 RNA copy numbers, which had been determined previously by the NucliSens HIV-1 QT Test. There was a significant correlation between the data obtained in the two tests (r = 0.65, P < 0.0001). During follow-up, the median RT and RNA levels increased more or less in parallel up to approximately four times the values at admittance. CD4 cell counts, which had also been determined previously, decreased slowly but continuously from approximately 310 to 190 CD4 cells/ml. In the majority of individual patients, there was an inverse correlation between CD4 T-cell counts and RT activity, and with the RNA copy number, and the data obtained by either test could be used to predict CD4 T-cell counts. The ExaVir Load test thus provides data equivalent to the estimation of the number of HIV-1 RNA copies for the prediction of CD4 T-cell counts. It is based on a simple technique, can be run in any routine diagnostic laboratory, and is a competitive alternative for use in resource limited settings.


Assuntos
Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Carga Viral , Contagem de Linfócito CD4 , Etiópia , Infecções por HIV/metabolismo , Transcriptase Reversa do HIV/sangue , HIV-1/genética , HIV-1/metabolismo , Humanos , Estudos Prospectivos , RNA Viral/sangue
14.
Curr HIV Res ; 3(4): 371-6, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16250883

RESUMO

408 non-selected samples were obtained from healthy, adult individuals donating blood at the Ethiopian Red Cross Society-National Blood Transfusion Service. All samples were screened for HIV using the Vironostika Ag/Ab test, the Amplicor DNA PCR and examined for the presence of HIV reverse transcriptase (RT) using the ExaVir Load test (version 2). A panel of supplementary tests was used to evaluate the HIV status of the discordant samples and to confirm positivity. One aim was to assess an RT based test for screening for HIV in comparison with other more conventional tests. An HIV-prevalence of 3.4 % (14/408) was found. The Vironostika Ag/Ab test produced 391 negative, and according to the supplementary testing, 14 true- and three false- positive test results. The corresponding figures for the Amplicor DNA PCR test was 384 negative, 14 true- and two extra probably false -positive samples. In addition, the DNA PCR generated eight indeterminate results. The colorimetric version of the ExaVir load test exhibited 100 % specificity and detected RT in 13 of the true positive samples, but failed to detect one sample containing 200 HIV RNA copies /mL. This sample was detectable in the fluorimetric version of the test. The detection of RT activity in addition to the currently used markers would seem to have a potential for use in blood screening.


Assuntos
Infecções por HIV/diagnóstico , Transcriptase Reversa do HIV/sangue , HIV/enzimologia , Doadores de Sangue , DNA Viral/sangue , Reações Falso-Negativas , Reações Falso-Positivas , HIV/isolamento & purificação , Anticorpos Anti-HIV/sangue , Antígenos HIV/sangue , Humanos , Programas de Rastreamento/métodos , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Carga Viral
15.
J Clin Microbiol ; 43(8): 3793-6, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16081912

RESUMO

Measurement of human immunodeficiency virus type 1 (HIV-1) plasma RNA levels using Roche AMPLICOR version 1.5 (HIV RNA) is an integral part of monitoring HIV-infected patients in industrialized countries. These assays are currently unaffordable in resource-limited settings. We investigated a reverse transcriptase (RT) assay as a less expensive alternative for measuring viral burden that quantifies RT enzyme activity in clinical plasma samples. A comparison of RT and HIV RNA assays was performed on 29 paired plasma samples from patients living in the United States and 21 paired plasma samples from patients living in Cameroon. RT levels correlated significantly with plasma HIV RNA viral loads in plasma from U.S. patients (r = 0.898; P < 0.001) and Cameroonian patients, a majority of whom were infected with HIV-1 clade type CRF02_AG (r = 0.669; P < 0.01). Among 32 samples with HIV viral load of >2,000 copies/ml, 97% had detectable RT activity. One Cameroon sample had undetectable RNA viral load but detectable RT activity of 3 fg/ml. The RT assay is a simple and less expensive alternative to the HIV RNA assay. Field studies comparing these assays in resource-limited settings are warranted to assess the practicality and usefulness of this assay for monitoring HIV-infected patients on antiretroviral therapy.


Assuntos
Infecções por HIV/virologia , Transcriptase Reversa do HIV/sangue , RNA Viral/sangue , Estudos de Coortes , Humanos , Reação em Cadeia da Polimerase , Carga Viral
16.
J Med Virol ; 76(3): 291-6, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15902697

RESUMO

A more sensitive version of ExaVir Load, a test that utilizes reverse transcriptase (RT) activity from virions in plasma to determine HIV-1 viral load, is described. The virions were immobilized on a gel that was washed, followed by lysis of the virions, elution of purified RT, and finally RT activity determination. The changes made to the original test were: (1) improved washing of the immobilized virions by addition of a non-lytic detergent to the wash buffer, (2) improved virion lysis procedure, including changes in salt, detergent and pH, (3) the use of larger sample volumes in the RT assay, and (4) prolonged RT reaction time. The alterations gave a tenfold increased sensitivity compared to the original version. The correlation between RT load by the current test and RNA PCR was the same as previously (r=0.90). Using colorimetric product detection, the average detection limit in a panel of 262 patient plasma from Stockholm was 0.5 fg RT/ml, corresponding to approximately 170 RNA copies/ml. None of 54 HIV-1 RNA negative samples exhibited RT. The amount of RT load positive samples were 19% for samples containing 50-400 RNA, 71% for samples with 400-1,500, and 100% among samples with >8,000 copies/ml (according to Roche Amplicor). The sensitivity could be increased further using fluorimetric detection. In conclusion, the modifications of the test described result in an important increase in sensitivity. It can now be regarded as a competitive alternative method for HIV viral load determinations.


Assuntos
Infecções por HIV/virologia , Transcriptase Reversa do HIV/sangue , HIV-1/fisiologia , Carga Viral/métodos , Fluorometria , Transcriptase Reversa do HIV/isolamento & purificação , Humanos , RNA Viral/sangue , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , África do Sul , Espectrofotometria , Estatística como Assunto , Suécia
17.
Curr HIV Res ; 3(2): 183-90, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15853722

RESUMO

We evaluated a low cost manual reverse transcriptase assay (ExaVir Load V.1 and V.2; Cavidi Tech AB) against commercially available HIV RNA assays that quantify viral load to assess its suitability for use in resource-constrained settings. Frozen plasma samples previously tested for RNA by RT-PCR (Roche Diagnostics) and bDNA (Bayer Diagnostics) were retested for RT activity. Text sequence obtained from HIV genotype analysis was submitted to the Stanford HIV Resistance Database V.3.9 and were examined for resistant virus. Detectable RT was present in 98% of samples (V.1; n=127) and in 95% of samples (V.2; n=69) with RNA >10,000 and >1,000 copies/ml respectively. Positive association was found between the log10 RNA copies/ml and log10 RT copies/ml equivalents variables using Pearson's correlation (V.1: r=0.89, n=189; V.2: r=0.89, n=85). The RT activity over time closely followed the trend for RNA levels in samples from 10 HIV seropositive patients with progressive disease. A strong association between RT and RNA was also found with paired samples from 19 patients taken at initiation or change of antiretroviral therapy and again within 2 months. Current (n=40) or no (n=119) exposure to efavirenz therapy had no effect on RT assay performance despite efavirenz binding tightly to the RT enzyme. Samples that demonstrated resistance to the non-nucleoside RT inhibitors (n=112) had a decrease in RT of 0.20 log10 indicating a possible decrease in RT fitness. The RT assay showed good association with current molecular assays, and V.2 is sufficiently sensitive for monitoring HIV viral load in resource-constrained settings.


Assuntos
Infecções por HIV/diagnóstico , Transcriptase Reversa do HIV/sangue , HIV-1/isolamento & purificação , Carga Viral/métodos , Alcinos , Benzoxazinas , Ciclopropanos , Infecções por HIV/sangue , Infecções por HIV/virologia , Soropositividade para HIV/diagnóstico , Soropositividade para HIV/tratamento farmacológico , HIV-1/enzimologia , Humanos , Oxazinas/uso terapêutico , Inibidores da Transcriptase Reversa/uso terapêutico , Sensibilidade e Especificidade , Carga Viral/economia
18.
Clin Infect Dis ; 39(4): 552-8, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15356820

RESUMO

We evaluated phenotypic and genotypic markers of drug resistance in human immunodeficiency virus type 1 (HIV-1) at the time of virologic failure (VF) in subjects in the AIDS Clinical Trials Group Protocol 388 (ACTG 388) who received lamivudine-zidovudine (or lamivudine-stavudine) and either indinavir, efavirenz-indinavir, or nelfinavir-indinavir. At VF, phenotypically susceptible HIV-1 was found in 55% of subjects in the nelfinavir-indinavir arm, compared with 22% in the indinavir arm (P=.006). Phenotypic resistance to lamivudine was less common in the efavirenz-indinavir arm (33% of subjects; P=.002) and the nelfinavir-indinavir arm (43%; P=.003), compared with the indinavir arm (78%). Isolated phenotypic resistance to efavirenz at VF occurred in HIV-1 recovered from 33% of subjects in the efavirenz-indinavir arm; 24% of the subjects had HIV-1 with both efavirenz and lamivudine resistance. Results of genotypic tests were similar. The lower frequency of resistance in the nelfinavir-indinavir arm likely reflects decreased drug exposure that is due to intolerance, which is consistent with the lower potency and tolerability of this combination in ACTG 388. The lower frequency of lamivudine resistance in the efavirenz-indinavir arm is consistent with reports in other studies of potent regimens. Thus, although dual resistance to efavirenz and lamivudine occurred at VF in the efavirenz-indinavir arm, this risk was relatively low when evaluated in the context of the potency and tolerability of this regimen.


Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Terapia Antirretroviral de Alta Atividade/métodos , Farmacorresistência Viral/fisiologia , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Adulto , Feminino , Genótipo , Infecções por HIV/metabolismo , Transcriptase Reversa do HIV/sangue , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , Humanos , Indinavir/administração & dosagem , Indinavir/metabolismo , Lamivudina/administração & dosagem , Lamivudina/metabolismo , Masculino , Nelfinavir/administração & dosagem , Nelfinavir/metabolismo , Fenótipo , Estavudina/administração & dosagem , Estavudina/metabolismo , Falha de Tratamento , Zidovudina/administração & dosagem , Zidovudina/metabolismo
19.
AIDS ; 17(3): 331-6, 2003 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-12556686

RESUMO

BACKGROUND: Plasma viral load monitoring is an integral part of the standard of care for HIV-infected patients in industrialized countries. In developing countries, viral load assay is either unaffordable or hindered by on-site maintenance and/or technical problems. OBJECTIVES: To evaluate a new and simple quantitative assay for plasma HIV reverse transcriptase (RT) activity; and to compare RT activity-based and RNA-based quantification in plasma samples from patients infected by different subtypes of HIV-1 group-M, HIV-1 group-O and HIV-2. METHODS: The RT-based viral load assay involves separation of the virion-protected RT and quantification of its activity with an enzyme immunoassay. Plasma viraemia was quantified both by RT activity and by RNA copies in 322 samples from 236 HIV-1 group M-infected patients, including serial samples from 54 patients. Samples from 49 patients infected by HIV-1 group O or HIV-2 were also tested. RESULTS: RT activity and RNA copies were detected in 70% of plasma samples; respectively 25% and 1% of samples contained detectable RNA copies or RT activity alone. Measured RT activity corresponded to 48%, 96% and 100% of samples with 1.7-4.0 log(10), 4.1-4.8 log(10) and 4.9-6.7 log(10) RNA copies/ml, respectively. The values of the two assays correlated independently of the HIV subtype (P < 0.0001) and group/type (P < 0.03). Patient follow-up showed a similar pattern of viraemia with the two assays. CONCLUSION: Plasma RT activity assay is a simple, cheap and reliable alternative for HIV viral load determination. As such, it could be particularly valuable for diagnosis and treatment monitoring in developing countries.


Assuntos
Infecções por HIV/diagnóstico , Transcriptase Reversa do HIV/sangue , DNA Polimerase Dirigida por RNA/sangue , Carga Viral/métodos , Infecções por HIV/virologia , Humanos , RNA Viral/sangue , Sensibilidade e Especificidade
20.
J Virol Methods ; 106(1): 115-24, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12367736

RESUMO

A simple and highly sensitive reverse transcriptase (RT) assay was developed by combining a previously reported non-radioisotopic RT assay with the use of a template-primer-immobilized microplate, an enzyme capture protocol, product digestion and a chemiluminescent substrate. The assay was able to detect directly the RT activity in serum samples, plasma and cell culture medium without the need for concentration and extraction of the enzyme. The assay was able to detect RT activity equivalent to 100 virions/ml of HIV-1. These results suggest that this highly sensitive chemiluminescent RT assay can be used not only for virological investigation but also for routine screening of biopharmaceuticals.


Assuntos
Transcriptase Reversa do HIV/sangue , HIV-1/enzimologia , Células Cultivadas , Colorimetria , Desoxirribonuclease I/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Medições Luminescentes , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Linfócitos T/virologia
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