RESUMO
Inhibition of viral replication is the most important goal in patients with Hepatitis B virus chronic infection (CHB). Currently, five oral nucleo(t)side analogs (NAs), including Lamivudine, Adefovir, Telbivudine, Entecavir, and Tenofovir, have been approved for treatment. The widespread use of NAs has also been linked with a progressive growth of unlikely anomaly attributable to mitochondrial dysfunctions, not previously recognized. Here, we explore the hypothesis that NAs may cause persistent epigenetic changes during prolonged NAs therapy in CHB patients. We obtained peripheral blood mononuclear cells (PBMC) from whole blood samples of consecutive patients with chronic HBV infection, 18 receiving NAs and 20 untreated patients. All patients were Caucasian and Italians. Epigenetic analysis was performed by Bisulphite sequencing PCR to search the existence of methylated cytosine residues in the Light (L)-strands of mitochondrial DNA control region (D-loop). Gene expression analysis of DNA methyltransferases 1 was performed by a quantitative relative Real-Time Polymerase Chain Reaction (PCR). DNMT1 expression was significantly (P < 000001) higher in NA treated patients (4.09, IQR 3.52-5.15) when compared with HBV naives (0.61, IQR 0.34-0.82). Besides, DNMT1 expression was significantly correlated with NA therapy duration (Spearman Rho = 0.67; P < 0.05). Furthermore, NA therapy duration was the only significant predictor of DNMT1 expression at multivariate analysis (Beta = 0.95, P < 0.0000001). Bisulphite PCR sequencing showed that methylation of cytosine residues occurred in a higher percentage in patients treated with NAs in comparison with untreated patients and healthy controls. Our data showed a DNMT1 overexpression significantly correlated to NA therapy duration and an higher regional mtDNA hypermethylation. This might suggest an epigenetic alteration that could be involved in one of the possible mechanisms of mitochondrial gene regulation during NAs therapy.
Assuntos
Antivirais/efeitos adversos , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , DNA Mitocondrial/química , Hepatite B Crônica/tratamento farmacológico , Nucleosídeos/uso terapêutico , Inibidores da Transcriptase Reversa/efeitos adversos , Adenina/efeitos adversos , Adenina/análogos & derivados , Idoso , Estudos Transversais , Citosina/química , Feminino , Guanina/efeitos adversos , Guanina/análogos & derivados , Hepatite B Crônica/sangue , Hepatite B Crônica/virologia , Humanos , Lamivudina/efeitos adversos , Leucócitos Mononucleares , Masculino , Pessoa de Meia-Idade , Nucleosídeos/química , Organofosfonatos/efeitos adversos , Reação em Cadeia da Polimerase em Tempo Real , Telbivudina , Timidina/efeitos adversos , Timidina/análogos & derivadosRESUMO
Our study purpose was to evaluate mitochondrial (mt)DNA and RNA in peripheral blood mononuclear cells (PBMCs) and body shape changes (BSC) in HBV-infected patients. mtDNA and mtRNA were measured in PBMCs. The presence of BSC was evaluated through a questionnaire and clinical evaluation. A total of 157 subjects were enrolled, of these 107 were HBV-infected patients, 54 receiving nucleoside analogues (NAs, Group A), 53 naive to antivirals (Group B) and 50 age-sex matched controls (Group C). All HBV-treated patients had negative HBV-DNA. Twenty (37,0%) received lamivudine + adefovir, 20 (37.0%) tenofovir, 2 (3.7%) lamivudine and 12 (22.2%) entecavir. Therapy median duration was 38 months (IQR 20-60) in NA-treated patients. Group A showed significantly higher mtDNA/nuclear (n) DNA ratio (p = 0.000008) compared to Group C and Group B (p = 0.002). Group B showed significantly higher mtDNA/nDNA ratio compared to Group C (p = 0.017). Group A and B had significantly lower mtRNA/nRNA ratio compared to Group C (p = 0.00003 and p = 0.00006, respectively). Tenofovir and entecavir showed less impact compared to lamivudine + adefovir. mtDNA/nDNA ratio positively (Rho = 0.34, p < 0.05) and mtRNA/nRNA ratio negatively (Rho = -0.34, p < 0.05) correlated with therapy duration. BSC were significantly more frequent in Group A [10/54 (18.5%)] compared to Group B [3/53 (5.6%, p = 0.04)] and Group C [0/50, (p = 0.0009)]. In conclusion, long-term NA therapy was associated both to mitochondrial toxicity and BSC, showing significant differences in mtDNA and mtRNA levels. Tenofovir and entecavir showed lower impact on alterations, compared to 1st generation NA.
Assuntos
Adiposidade/efeitos dos fármacos , Antivirais/efeitos adversos , Hepatite B Crônica/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Adenina/efeitos adversos , Adenina/análogos & derivados , Estudos Transversais , DNA Mitocondrial/isolamento & purificação , DNA Viral/isolamento & purificação , Farmacorresistência Viral , Quimioterapia Combinada/efeitos adversos , Quimioterapia Combinada/métodos , Feminino , Guanina/efeitos adversos , Guanina/análogos & derivados , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/sangue , Hepatite B Crônica/virologia , Humanos , Lamivudina/efeitos adversos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Organofosfonatos/efeitos adversos , RNA Mitocondrial/isolamento & purificação , Tenofovir/efeitos adversosRESUMO
This study compared the effectiveness of the QuantiFERON-TB Gold (QFT) assay with the Mantoux tuberculin skin test to detect Mycobacterium tuberculosis infection in 29 children during a school outbreak of tuberculosis. Of the 21 children with M. tuberculosis infection, 11 had a radiograph suggestive of the infection. The QFT assay was positive in all 21 of the children, and the Mantoux test was negative at first testing in 2 children (1 of whom was the sentinel case). The findings demonstrate that the QFT test is extremely useful in accurately identifying infected and uninfected children, permitting rapid intervention.
Assuntos
Surtos de Doenças , Imunoensaio , Interferon gama/análise , Mycobacterium tuberculosis/isolamento & purificação , Teste Tuberculínico , Tuberculose Pulmonar/diagnóstico , Antígenos de Bactérias/análise , Criança , Feminino , Humanos , Pulmão/diagnóstico por imagem , Masculino , Mycobacterium tuberculosis/imunologia , Radiografia , Sensibilidade e Especificidade , Linfócitos T/química , Linfócitos T/imunologia , Tuberculose Pulmonar/epidemiologiaRESUMO
In this study, we characterized the humoral responses in cattle of Sardinia. The animals were divided into three groups: 1) 28 cattle infected with Mycobacterium bovis; 2) 48 cattle from herds in which foci of infection was notified; 3) 50 cattle from herds that were TB-free. Levels of IgG antibody were measured against the following antigens of M. tuberculosis: Heparin-Binding-Haemagglutin (HBHA), Ag85B, PPE44, and PE_PGRS33 to investigate their potential to diagnose TB in animals. Our results indicated that HBHA is a potential candidate for the development of a serological assay for rapid diagnosis of cattle infected with M. bovis.
Assuntos
Lectinas/sangue , Mycobacterium bovis/metabolismo , Tuberculose Bovina/diagnóstico , Animais , Formação de Anticorpos , Antígenos de Bactérias/sangue , Proteínas de Bactérias/sangue , Biomarcadores/sangue , Bovinos , Lectinas/imunologia , Técnicas de Diagnóstico Molecular , Mycobacterium bovis/imunologia , Sensibilidade e Especificidade , Tuberculose Bovina/microbiologiaRESUMO
During a six-month period a region of Northern Sardinia was monitored to check the presence of mycobacterial infections in wild boars. Forty-eight serum and 229 biopsy samples were collected from different animals and examined by both traditional diagnostic techniques (culture, bacterioscopic and molecular tests) and enzyme-linked immunosorbent assay (ELISA). The latter was used to determine the antibody response against both methylated and nonmethylated Heparin-Binding Haemagglutinin (HBHA) protein. Nine mycobacterial strains were isolated: three M. avium ssp. paratuberculosis (Map), three M. avium, one M. interjectum and two M. scrofulaceum strains. By PCR, only one animal was positive for M. bovis, whereas 10 animals were positive for Map. Out of the 48 sera tested, 19 showed a good humoral response to methylated HBHA and 17 to nonmethylated HBHA. Our data provide new information on the prevalence of mycobacterial infection among wild boars in Northern Sardinia and suggest that a more effective program should be developed to monitor mycobacterial infections in the wild animal population.
Assuntos
Infecções por Mycobacterium/veterinária , Mycobacterium/isolamento & purificação , Sus scrofa , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/microbiologia , Animais , Feminino , Itália/epidemiologia , Masculino , Mycobacterium/genética , Infecções por Mycobacterium/epidemiologia , Infecções por Mycobacterium/microbiologia , Estudos SoroepidemiológicosRESUMO
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease (JD), a chronic gastroenteritis of ruminants and other animals, including primates. Many evidences suggested association of MAP to Crohn's disease, a chronic granulomatous gastrointestinal disease of humans with strong similarities with JD. The present study attempts to evaluate global gene regulation in MAP, which has not been addressed previously, despite the availability of MAP genome sequence. For this purpose, we investigated: (i) the presence of sigma factors and their relationship to sigma factors of other mycobacteria (M. avium subsp.avium, M. tuberculosis, M. bovis, M. leprae and M. smegmatis), and (ii) their expression during different growth conditions and in vitro infection of intestinal epithelial Caco2 cells. MAP genome contains 19 putative sigma factor, but only 12 belong to gene families common to other mycobacteria. Gene expression was evaluated with Real-Time PCR during growth in 7H9 medium and mycobactin J, in 7H9 medium plus mycobactin J and lisozyme, and during infection of Caco2 cells: very different expression patterns were observed and, on the whole, only 7 sigma factors were found to be expressed. sigJ was upregulated during the infection of Caco2 cells. Even if only few sigma factors were expressed in the three conditions tested, the overall high numbers of MAP sigma factors suggests a noteworthy flexibility of this pathogen. Thus, this first report on expression of MAP sigma factors opens the way to an extensive characterization of global gene regulation, as a key to understand strategies of survival and mechanisms of infections used by this organism.
Assuntos
Genoma Bacteriano/genética , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium/genética , Fator sigma/genética , Fator sigma/metabolismo , Transcrição Gênica , Células CACO-2 , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Intestinos/citologia , Intestinos/efeitos dos fármacos , Intestinos/microbiologia , Muramidase/metabolismo , Mycobacterium avium/efeitos dos fármacos , Mycobacterium avium/crescimento & desenvolvimento , Mycobacterium avium subsp. paratuberculosis/efeitos dos fármacos , Mycobacterium avium subsp. paratuberculosis/crescimento & desenvolvimento , Oxazóis/farmacologia , Filogenia , Transcrição Gênica/efeitos dos fármacosRESUMO
Linezolid, an oxazolidinone that acts by inhibiting protein synthesis, was evaluated in strains of tuberculosis and non-tubercular mycobacteria resistant to one or more drugs isolated in northern Sardinia. The in vitro activity of Linezolid (Pfizer) was assessed on different isolates of Mycobacterium spp. from clinical samples by the Proportional Method. Linezolid demonstrated an excellent activity against the 24 strains of M. tuberculosis and against M. gordonae, M. marinum, M. aurum, M. phlei, and M. avium, with MIC values ranging from 0.5 to 2 microg/ml. Linezolid can be used in combination with the standard antitubercular medications, or as an effective therapeutic alternative in infections caused by M. tuberculosis or by other species of non-tubercular mycobacteria.
Assuntos
Acetamidas/farmacologia , Anti-Infecciosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium/efeitos dos fármacos , Oxazolidinonas/farmacologia , Farmacorresistência Bacteriana , Humanos , Itália , Linezolida , Testes de Sensibilidade Microbiana/métodos , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
We have developed a Real-Time PCR assay to detect M. tuberculosis using the iCycler iQ detection system by TaqMan assay directly on the clinical specimen. A total of 513 clinical samples were taken from patients with suspected tuberculosis and other patients that had an active mycobacterial infection, as well as patients with diagnosed tuberculosis who were receiving antitubercular therapy. The sensitivity and specificity of this assay, 10% and 100%, respectively, were compared to those of conventional microbiological methods.