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1.
BMC Infect Dis ; 14: 222, 2014 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-24758157

RESUMO

BACKGROUND: A good correlation between HCV core antigen (HCVAg) and different HCV-RNA assays has been described, but little data are available in HCV/HIV co-infection. We aimed to evaluate HCVAg in comparison with HCV-RNA and to determine their kinetics during antiviral treatment in selected HCV/HIV co-infected patients. METHODS: 355 samples from 286 HCV/HIV co-infected subjects for whom HCV-RNA (Abbott RealTime) was requested were analysed also for HCVAg (Abbott ARCHITECT) in order to evaluate the correlation between the two parameters both in patients treated or untreated for chronic hepatitis C and according to different HCV genotypes. The differences between percentages were evaluated by chi square or Fisher's exact test, while mean and median values were compared by Student's t test or the Mann-Whitney test, respectively. All differences were considered significant for a p value <0.05. RESULTS: HCVAg was detectable on 288/315 sera (91.4%) positive for HCV-RNA and in 5 out of40 (12.5%) sera with undetectable HCV-RNA for a total concordance of 90.1%. The correlation was fair both in untreated (r = 0.742) and in treated (r = 0.881) patients and stronger for genotypes 1 and 4 than for genotype 3. Both HCV-RNA and HCVAg levels were significantly higher (p = 0.028 and p = 0.0098, respectively) in patients infected by genotype 1 than by genotype 3. The mean ratio of Log values between HCV-RNA (IU/mL) and HCVAg (fmol/liter) was 2.27 ± 1.09 in untreated and 2.20 ± 0.82 in treated patients (p = n.s.),consistent with a sensitivity of HCVAg corresponding to about 1,000 IU/mL of HCV-RNA, and ranged from 2.21 to 2.32 among HCV genotypes with no significant differences; five samples (1.4%; 2 genotype 1a or 1c, 3 genotype 3a) showed highly divergent values. The analysis of 18 monitoring profiles from patients treated with PEG-IFN and Ribavirin showed similar trends, except in one case in which relapse could be predicted by HCVAg and not by HCV-RNA. CONCLUSION: These results suggest that HCVAg represents an adequate tool for determining an ongoing HCV infection also in HIV co-infected patients, with lower costs and faster turnaround time than HCV-RNA.


Assuntos
Infecções por HIV/imunologia , Hepatite C Crônica/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Coinfecção , Feminino , Genótipo , Infecções por HIV/sangue , Infecções por HIV/complicações , Hepacivirus/genética , Hepacivirus/imunologia , Antígenos da Hepatite C/sangue , Hepatite C Crônica/sangue , Hepatite C Crônica/complicações , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Ribavirina/uso terapêutico
2.
BMC Infect Dis ; 11: 310, 2011 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-22054111

RESUMO

BACKGROUND: Occult hepatitis B virus (HBV) infection (OBI) is characterized by HBV DNA persistence even though the pattern of serological markers indicates an otherwise resolved HBV infection. Although OBI is usually clinically silent, immunocompromised patients may experience reactivation of the liver disease. CASE PRESENTATION: We report the case of an individual with human immunodeficiency virus (HIV) infection and anti-HBV core antibody positivity, who experienced severe HBV reactivation after discontinuation of lamivudine-including antiretroviral therapy (ART). HBV sequencing analysis showed a hepatitis B surface antigen escape mutant whose presence in an earlier sample excluded reinfection. Molecular sequencing showed some differences between two isolates collected at a 9-year interval, indicating HBV evolution. Resumption of ART containing an emtricitabine/tenofovir combination allowed control of plasma HBV DNA, which fell to undetectable levels. CONCLUSION: This case stresses the ability of HBV to evolve continuously, even during occult infection, and the effectiveness of ART in controlling OBI reactivation in HIV-infected individuals.


Assuntos
Terapia Antirretroviral de Alta Atividade/métodos , Antivirais/administração & dosagem , Infecções por HIV/tratamento farmacológico , Vírus da Hepatite B/classificação , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Lamivudina/administração & dosagem , DNA Viral/química , DNA Viral/genética , Evolução Molecular , Feminino , Infecções por HIV/complicações , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/complicações , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Análise de Sequência de DNA
3.
J Clin Virol ; 51(4): 264-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21621454

RESUMO

BACKGROUND: Testing for hepatitis C virus core antigen (HCV Ag) may represent a complementary tool to anti-HCV and HCV-RNA in the diagnosis and monitoring of HCV infection. OBJECTIVE: To evaluate the performance characteristics of the automated Abbott ARCHITECT HCV Ag assay. STUDY DESIGN: Five sites analyzed over 3000 routine serum samples from populations at different risk, comparing HCV Ag results with anti-HCV screening and supplemental assay results and with HCV-RNA. RESULTS: The HCV Ag assay showed a specificity of 100%, a good precision (CV<10%) and excellent dilution linearity (r>0.999). The sensitivity (3 fmol/L) corresponds to 700-1100 IU/mL of HCV-RNA. A non-linear correlation with HCV-RNA was found: r=0.713 vs. Siemens bDNA (523 specimens), r=0.736 vs. Roche Cobas TaqMan (356 specimens) and r=0.870 vs. Abbott Real-Time PCR (273 specimens). HCV Ag quantitation was equally effective on different HCV genoypes (239 for genotype 1/1a/1b/1c, 108 for genotype 2/2a/2c, 86 for genotype 3/3a, 50 for genotype 4/4a/4c/4d). Testing of subjects at high risk for HCV and with potential or actual impairment of the immune system identified 2 cases negative for anti-HCV and positive for HCV Ag on 361 hemodialyzed (0.6%) and 7 cases on 97 (7.2%) among transplant recipients. HCV Ag positivity anticipated anti-HCV seroconversion in all three cases of acute hepatitis C. CONCLUSIONS: HCV Ag may be used as reflex testing on anti-HCV positive individuals to confirm or exclude an active infection, and on subjects with acute hepatitis or belonging to high risk groups.


Assuntos
Automação/métodos , Técnicas de Laboratório Clínico/métodos , Hepatite C/diagnóstico , Proteínas do Core Viral/sangue , Viremia/diagnóstico , Virologia/métodos , Monitoramento de Medicamentos/métodos , Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Anticorpos Anti-Hepatite C/sangue , Humanos , Imunoensaio/métodos , RNA Viral/sangue , RNA Viral/genética , Sensibilidade e Especificidade
4.
New Microbiol ; 33(4): 293-302, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21213587

RESUMO

Despite the widespread use of molecular biology techniques, standardized methods for the measurement of HIV-1 proviral DNA are currently lacking and several discordant results are still present in different studies. To assess the clinical meaning of the proviral DNA load, a study group comprising seven different laboratories was set up to standardize a HIV-1 proviral DNA quantification method able to assess the DNA proviral load of the most relevant circulating HIV-1 subtypes. Reference samples (24 cellular samples infected with HIV-1 clade B, and 40 samples of peripheral blood mononuclear cells containing different concentrations of plasmids expressing different HIV-1 clades) were distributed and tested blindly. All laboratories employed hTERT gene as housekeeping gene and primers within the gag gene to quantify different HIV-1 clades. Inter-laboratory results did not differ statistically but showed only minor variations concerning HIV-1 DNA amounts and different HIV clades, with a good agreement among the laboratories participating in the study. Since test standardization represents a key step for future application in clinical practice, further studies of the patients' samples are in progress to establish the real meaning and utility of the proviral DNA load for clinical management of HIV-1 infected patients.


Assuntos
DNA Viral/análise , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Reação em Cadeia da Polimerase/normas , Provírus/isolamento & purificação , Carga Viral/normas , Linhagem Celular , HIV-1/genética , Humanos , Itália , Provírus/genética , Sensibilidade e Especificidade , Telomerase/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
5.
J Antimicrob Chemother ; 54(6): 991-8, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15563518

RESUMO

OBJECTIVES: To analyse the distribution and molecular features of mef(A)-containing elements in a large collection of different Streptococcus pyogenes clinical isolates with efflux-mediated erythromycin resistance. To further characterize a tet(O)-mef(A) element. METHODS: Gene detection was carried out by PCR using primers designed from established sequences or from sequences in this study. From a tet(O)-mef(A) element (approximately 60 kb), an 11 972 bp region including the tet(O) and mef(A) genes was sequenced. RESULTS: In the tetracycline-susceptible isolates (n =28), the mef(A) gene was contained in a regular Tn1207.1 transposon (7.2 kb), which was inserted into one of two previously described elements, Tn1207.3 (approximately 52 kb) or a 58.8 kb chimeric element, both flanked by the comEC gene. In the tetracycline-resistant isolates (n =61), all of which carried the tet(O) gene, the mef(A) gene was part of a variable Tn1207.1-related transposon inserted into unique elements which contained the tet(O) gene approximately 2.3 to 5.5 kb upstream of the mef(A) gene and were not flanked by the comEC gene. In the Tn1207.1-like transposon of these tet(O)-mef(A) elements, only msr(D) (orf5) and a modified orf6, in addition to mef(A), were detected by PCR in all isolates tested; while orf1 and orf2 were always undetectable, orf3, orf7 and orf8 were found in variable percentages. In an orf3-positive element, sequencing identified four new open reading frames downstream of the tet(O) gene, followed by three short sequences with homology to sequences of the pneumococcal mega element. CONCLUSIONS: The mef(A) gene is carried on different chromosomal genetic elements depending on whether the isolates are susceptible or resistant to tetracycline.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Proteínas de Membrana/genética , Streptococcus pyogenes/efeitos dos fármacos , Resistência a Tetraciclina , Tetraciclina/farmacologia , Proteínas de Transporte/genética , Farmacorresistência Bacteriana/genética , Eritromicina/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Análise de Sequência de DNA , Streptococcus pyogenes/genética
6.
Antimicrob Agents Chemother ; 48(9): 3253-9, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15328081

RESUMO

The human immunodeficiency virus type 1 (HIV-1) fusion inhibitor enfuvirtide has recently been introduced into clinical practice and has exhibited efficient anti-HIV-1 activity in combination with other antiretroviral agents. In the present study, we addressed the effect of long-term treatment with enfuvirtide on the intrahost evolution of HIV-1. The genotype and phenotype patterns and the relative replication capacity (rRC) of enfuvirtide-resistant HIV-1 mutants were evaluated in samples from 11 subjects (7 virological nonresponders and 4 responders) who received the compound for more than 1 year in combination with different regimens. Selection of one or more mutations clustering in a sequence (amino acids 36 to 45) of the gp41 N-terminal heptad repeat was observed in samples from the seven virological nonresponders but not in those from responders. In two subjects who discontinued enfuvirtide, reversion of the resistant genotype was detected within 3 months. Recombinant clones bearing mutated gp41 sequences displayed reduced susceptibilities to enfuvirtide, with the 50% inhibitory concentrations (IC(50)s) ranging from 0.6 to 12.8 microg/ml, whereas the IC(50) for isolates with baseline sequences was 0.013 +/- 0.010 microg/ml. Interestingly, long-term monitoring of resistant variants provided evidence that ongoing adaptation to the drug is paralleled by phenotypic changes. A limited drop in the rRC in the absence of drug was observed for clones from four of the seven nonresponders bearing mutations associated with resistance. Overall, the data indicate that the different genotype patterns associated with a detectable degree of HIV-1 resistance to enfuvirtide generated during long-term treatments are characterized by a substantially low genetic barrier, possible ongoing adaptation with increased degrees of resistance, and limited influence on the viral rRC.


Assuntos
Proteína gp41 do Envelope de HIV/farmacologia , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Contagem de Linfócito CD4 , Clonagem Molecular , Farmacorresistência Viral , Enfuvirtida , Genótipo , Proteína gp41 do Envelope de HIV/efeitos dos fármacos , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/uso terapêutico , Inibidores da Fusão de HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Humanos , Assistência de Longa Duração , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos/uso terapêutico , Fenótipo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
7.
Virology ; 307(2): 328-40, 2003 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-12667802

RESUMO

The human immunodeficiency virus type 1 (HIV-1) gp120 V3 loop plays a predominant role in chemokine receptor usage; however, other linear and nonlinear gp120 domains are involved in this step of the HIV-1 replication cycle. At present, the functional relationship between V3 and these domains with regard to coreceptor usage is unclear. To gain insights into the nature of this relationship in naturally selected viral variants, we developed a recombinant strategy based on two different gp120 backbones derived from CXCR4 (X4)- and CCR5 (R5)-tropic viral strains, respectively. Using this recombinant model system, we evaluated the phenotype patterns conferred to chimeric viruses by exogenous V3 loops from reference molecular clones and samples from infected subjects. In 13 of 17 recombinants (76%), a comparable phenotype was observed independently of the gp120 backbone, whereas in a minority of the recombinant viruses (4/17, 24%) viral infectivity depended on the gp120 context. No case of differential tropism using identical V3 sequence in the two gp120 contexts was observed. Site-directed mutagenesis experiments were performed to evaluate the phenotypic impact of specific V3 motifs. The data indicate that while the interaction of HIV-1 with chemokine receptors is driven by V3 loop and influenced by its evolutionary potential, the gp120 context plays a role in influencing the replication competence of the variants, suggesting that compensatory mutations occurring at sites other than V3 are necessary in some cases.


Assuntos
Proteína gp120 do Envelope de HIV/química , HIV-1/fisiologia , Receptores de HIV/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Proteína gp120 do Envelope de HIV/fisiologia , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Receptores de Quimiocinas/fisiologia , Recombinação Genética , Relação Estrutura-Atividade , Replicação Viral
8.
AIDS ; 17(5): 663-71, 2003 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-12646788

RESUMO

OBJECTIVE: To investigate the role of processivity and drug-dependence of HIV-1 protease as fitness determinants in variants resistant to protease inhibitors (PI). DESIGN AND METHODS: HIV-1 protease sequences from 32 infected subjects (27 patients who failed PI-treatments and five PI-naive controls) were evaluated using a recombinant method. The HIV-1 phenotype to seven PI was analysed together with the replication capacity of recombinants and the processivity and drug-dependence of the HIV-1 proteases. Protease mutants (positions 10, 46, 54, 82, 84, 90, and combinations thereof) were generated in vitro and studied under identical experimental conditions. RESULTS: In the absence of PI, 24 of 27 (89%) resistant proteases from treated subjects showed decreased processivity compared with the wild type. Processivity was lower in sequences bearing fewer mutations, than in more mutated ones. Twelve sequences (44%) conferred slower replication kinetics to the recombinant viruses. Seven sequences (26%) showed higher processivity levels in the presence of PI than in their absence, suggesting that drug-dependence influences PI-resistant variants. Among the mutants generated in vitro, mutations 82A and 90M determined broad cross-resistance to PI in association with 10I. A drop of processivity was observed for the 82A+90M variants; 10I allowed partial recovery for 82A and 84V, and marked recovery for 90M mutants. CONCLUSIONS: A decrease in HIV-1 protease processivity parallels early selection of primary mutations, whereas its recovery is driven by compensatory mutations. Furthermore, a PI may select drug-dependent, besides resistant, HIV-1 protease variants. Changes in processivity and drug-dependence of HIV-1 proteases have implications in the replication capacity of PI-resistant viruses.


Assuntos
Farmacorresistência Viral , Inibidores da Protease de HIV/farmacologia , Protease de HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Clonagem Molecular , Protease de HIV/genética , HIV-1/genética , HIV-1/patogenicidade , Humanos , Mutagênese Sítio-Dirigida , Fenótipo , Mutação Puntual , Replicação Viral
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