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2.
Artigo em Inglês | MEDLINE | ID: mdl-31940451

RESUMO

Mycobacteriology laboratories play a key role in tuberculosis (TB) control by providing phenotypic and molecular diagnostics, by performing molecular typing to aid contact tracing, and by supporting research and similar laboratories in Australia's neighbouring countries where TB is prevalent. The National Tuberculosis Advisory Committee (NTAC) published a set of laboratory guidelines in 2006 aiming to document the infrastructure, equipment, staffing and work practices required for safe high-quality work in Australian mycobacteriology laboratories. These revised guidelines have the same aims and have been through a similar extensive consultative peer-review process involving the Mycobacterium Reference Laboratory (MRL) network, the Mycobacterium Special Interest Group (SIG) of the Australian Society for Microbiology (ASM), and other relevant national bodies. This revised document contains several significant changes reflecting the publication of new biosafety guidelines and tuberculosis standards by various national and international organisations, technology developments - such as the MPT64-based immunochromatographic tests (ICTs) and the Xpert MTB/RIF assay, and updated work practices in mycobacteriology laboratories. The biosafety recommendations affirm the latest Australian/New Zealand Standard 2243.3: 2010 and promote a biorisk assessment approach that, in addition to the risk categorisation of the organism, also considers the characteristics of the procedure being performed. Using this biorisk assessment approach, limited manipulations, such as Ziehl-Neelsen (ZN) microscopy, MPT64 ICTs, and culture inactivation/DNA extraction for molecular testing, may be performed on a positive TB culture in a PC2 laboratory with additional features and work practices. Other significant changes include recommendations on the integration of MPT64 ICTs and novel molecular tests into TB laboratory workflows to provide rapid accurate results that improve the care of TB patients. This revised document supersedes the original 2006 publication. NTAC will periodically review these guidelines and provide updates as new laboratory technologies become available.


Assuntos
Guias de Prática Clínica como Assunto , Tuberculose/microbiologia , Austrália , Humanos , Laboratórios/normas , Mycobacterium tuberculosis , Tuberculose/diagnóstico
3.
Ann Lab Med ; 40(2): 169-173, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31650734

RESUMO

The GENEDIA MTB/NTM Detection Kit (GENEDIA MTB/NTM; Green Cross Medical Science Corp., Chungbuk, Korea) is a multiplex real-time PCR assay used for differential identification of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). While the importance of differential identification of MTB/NTM is recognized, there is limited data on the performance of GENEDIA MTB/NTM assay to date. A total of 687 consecutive sputum specimens were cultured and analyzed with the GENEDIA MTB/NTM and GENEDIA MTB assays. Nineteen specimens (2.8%) were MTBC-positive, and 69 (10.0%) were NTM-positive based on mycobacterial culture. All specimens showed concordant results for MTBC using both assays, with a kappa value of 1.00, overall sensitivity of 63.2% (12/19), and specificity of 100% (668/668). The overall NTM sensitivity and specificity were 23.2% (16/69) and 99.7% (616/618) for GENEDIA MTB/NTM. The association between NTM-positivity using GENEDIA MTB/NTM and the diagnosis of NTM pulmonary disease was not statistically significant. In conclusion, the two real-time PCR assays showed similar diagnostic performance for MTBC detection. However, the sensitivity for NTM detection was lower than that for MTBC detection.


Assuntos
Infecções por Micobactéria não Tuberculosa/diagnóstico , Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas/genética , Escarro/microbiologia , Tuberculose/diagnóstico , DNA Bacteriano/análise , Humanos , Reação em Cadeia da Polimerase Multiplex , Infecções por Micobactéria não Tuberculosa/microbiologia , Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Tuberculose/microbiologia
4.
BMC Infect Dis ; 19(1): 1048, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31829183

RESUMO

BACKGROUND: Success of India's TB control program relies on rapid case detection, monitoring, care and treatment of drug resistance. Patients on multidrug resistance (MDR) treatment are monitored by follow up cultures. Discordant results (culture and smear positive while capilia negative) are usually declared negative Mycobacterium tuberculosis complex (MTBC). This study was designed to understand the possible causes of discordant results. METHODS: The capilia kit was evaluated to test its utility among 4737 follow up MDR patients enrolled during a period of 1 year. A total of 889 were liquid culture positive, 3375 were negative and 473 were contaminated. Of the 889 cultures positive, 829 were found positive by ZN smear, capilia test and MTBDR plus assay. The cultures which gave a positive result on Mycobacterium Growth Indicator Tube 960 (MGIT 960) and ZN smear but were negative on capilia test with no growth on Brain Heart Infusion agar (BHI) were included in this study. The conflicting results of capilia were compared with other molecular techniques; MTBDR plus assay and DNA sequence analysis of MPT64 gene. RESULTS: Out of 889 culture positive, 60 (6.7%) were found positive on liquid culture and ZN smear but were negative on capilia. Of these 60 cultures, 10 (16.7%) were found positive by both MTBDR plus assay and PCR. The sequencing analysis revealed that all of the capilia negative isolates had mutations within the MPT64 gene. CONCLUSION: Re-evaluation of culture positive but capilia negative isolates should be done before declaring them as Mycobacterium other than tuberculosis (MOTT) because such cases can act as chronic carriers of TB in the population which can lead to the rise of this lethal disease.


Assuntos
Antígenos de Bactérias/genética , Imunoensaio/normas , Mycobacterium tuberculosis/isolamento & purificação , Kit de Reagentes para Diagnóstico/normas , Tuberculose/diagnóstico , Adulto , Estudos Transversais , Confiabilidade dos Dados , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Feminino , Seguimentos , Humanos , Índia , Masculino , Mutação , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Tuberculose/microbiologia , Adulto Jovem
5.
BMC Infect Dis ; 19(1): 1058, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842773

RESUMO

BACKGROUND: Xpert® MTB/RIF (Xpert) has high sensitivity for diagnosing tuberculosis (TB) compared to sputum-smear microscopy (smear) and can reduce time-to-diagnosis, time-to-treatment and potentially unfavorable patient-level treatment outcome. METHODS: People living with HIV (PLHIV) initiating antiretroviral therapy at 22 HIV clinics were enrolled and underwent systematic screening for TB (August 2012-November 2014). GeneXpert instruments were deployed following a stepped-wedge design at 13 centers from October 2012-June 2013. Treatment outcomes classified as an unfavorable outcome (died, treatment failure or loss-to-follow-up) or favorable outcome (cured and treatment completed). To determine outcome, smear was performed at month 5 or 6. Empiric treatment was defined as initiating treatment without/before receiving TB-positive results. Adjusting for intra-facility correlation, we compared patient-level treatment outcomes between patients screened using smear (smear arm)- and Xpert-based algorithms (Xpert arm). RESULTS: Among 6041 patients enrolled (smear arm, 1816; Xpert arm, 4225), 256 (199 per 2985 and 57 per 1582 person-years of follow-up in Xpert and smear arms, respectively; adjusted incidence rate ratio, 9.07; 95% confidence interval [CI]: 4.70-17.48; p < 0.001) received TB diagnosis and were treated. TB treatment outcomes were available for 203 patients (79.3%; Xpert, 157; smear, 46). Unfavorable outcomes were reported for 21.7% (10/46) in the smear and 13.4% (21/157) in Xpert arm (adjusted hazard ratio, 1.40; 95% CI: 0.75-2.26; p = 0.268). Compared to smear, in Xpert arm median days from sputum collection to TB treatment was 6 days (interquartile range [IQR] 2-17 versus 22 days [IQR] 3-51), p = 0.005; patients with available sputum test result had microbiologically confirmed TB in 59.0% (102/173) versus 41.9% (18/43), adjusted Odds Ratio [aOR], 2.00, 95% CI: 1.01-3.96, p = 0.048). In smear arm empiric treatment was 68.4% (39/57) versus 48.7% (97/199), aOR, 2.28, 95% CI: 1.24-4.20, p = 0.011), compared to Xpert arm. CONCLUSIONS: TB treatment outcomes were similar between the smear and Xpert arms. However, compared to the smear arm, more patients in the Xpert arm received a TB diagnosis, had a microbiologically confirmed TB, and had a shorter time-to-treatment, and had a lower empiric treatment. Further research is recommended to identify potential gaps in the Botswana health system and similar settings. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02538952. Retrospectively registered on 2 September 2015.


Assuntos
Infecções por HIV/complicações , Microscopia/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Escarro/microbiologia , Tuberculose/complicações , Tuberculose/tratamento farmacológico , Adulto , Botsuana , Confiabilidade dos Dados , Feminino , Seguimentos , Humanos , Perda de Seguimento , Masculino , Programas de Rastreamento , Estudos Prospectivos , Sensibilidade e Especificidade , Tempo para o Tratamento , Resultado do Tratamento , Tuberculose/diagnóstico , Tuberculose/microbiologia
6.
BMC Infect Dis ; 19(1): 1063, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852545

RESUMO

BACKGROUND: Contacting patients with tuberculosis have a substantial risk of developing the disease. Household contact screening has recently been recommended as a strategy to enhance case detection in high-burden countries. But there is no enough information in Gondar town regarding household contact screening practice among TB patients. METHODS: An institution-based cross-sectional study was conducted from March 1 to 30, 2019 on 404 tuberculosis patients attending at health facilities in Gondar Town. Epi-Info version 7 for data entry and SPSS version 20 for data analysis were used. Descriptive statistics were carried out to illustrate the means, standard deviations, and frequencies. Bivariable and multivariable logistic regression analyses were used to identify significantly associated variables with the dependent variable. RESULTS: From 412 study populations, 404 were completed the study with 98.06% response rate. The overall household contact TB screening adherence was 47.5% (95% CI: 43.1, 52.5). In the multivariable analysis, having certificate and above educational level (AOR = 2.83, 95% CI:1.40,5.67), having sufficient knowledge about TB (AOR = 8.26, 95% CI:4.34,15.71), being satisfied with health care service (AOR = 3.26, 95% CI:1.58,6.76), health education given by health care workers (AOR = 2.60, 95% CI:1.54,4.40),and having HIV/AIDS co-infection (AOR = 3.54, 95% CI:1.70,7.39), were factors associated with household contact TB screening adherence. CONCLUSION: Compared to other previous studies, the current finding was high but it was low as compared with WHO and Ethiopian Ministry of Health recommendations (all persons having TB contact should be screened). Educational status, knowledge on TB, satisfaction with delivered health care service, health education given by HCWs about TB and HIV/AIDS co-infection were factors associated with household contact TB screening practice. Thus, strengthening household TB contact screening and educational programs regarding the risk of getting TB infection from household contacts is crucial.


Assuntos
Síndrome de Imunodeficiência Adquirida/epidemiologia , Infecções por HIV/epidemiologia , Cooperação do Paciente , Tuberculose/epidemiologia , Síndrome de Imunodeficiência Adquirida/complicações , Síndrome de Imunodeficiência Adquirida/virologia , Adulto , Coinfecção , Estudos Transversais , Etiópia/epidemiologia , Características da Família , Feminino , Infecções por HIV/complicações , Infecções por HIV/virologia , Educação em Saúde , Instalações de Saúde , Pessoal de Saúde/educação , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Tuberculose/complicações , Tuberculose/microbiologia , Adulto Jovem
7.
Mikrobiyol Bul ; 53(4): 355-363, 2019 Oct.
Artigo em Turco | MEDLINE | ID: mdl-31709933

RESUMO

One of the most important steps for the control of tuberculosis is rapid and accurate detection of Mycobacterium tuberculosis in clinical samples. The early and accurate diagnosis of tuberculosis allows the initiation of the effective treatment regimen as early as possible. However the early diagnosis of tuberculosis can be achieved by the integration of molecular methods into the diagnostic algorithm of tuberculosis together with the gold standard culture methods. For this reason, molecular methods have become valuable diagnostic tools in routine diagnostic laboratories in recent years. The aim of this study was to determine the diagnostic efficacy of Anyplex MTB/NTM test (Seegene, South Korea) used for the molecular diagnosis of tuberculosis in routine molecular diagnostic laboratories. In addition to this aim, a preliminary evaluation of in-house polymerase chain reaction (PCR) primers that was designed to produce a kit as an alternative against imported commercial kits was performed. Ten thousand six hundred fifthy two clinical specimens that were collected from suspected tuberculosis cases in three years were included in the study. All samples were tested by microscopic examination after staining, culture and real-time PCR (Rt-PCR) methods. The smears were examined by microscope after staining with Kinyoun method for the existence of acid resistant bacilli. For culture, following the N-acetyl-L-sistein-sodium hydroxide homogenization and decontamination procedure, the samples were inoculated into the MGIT (Mycobacteria Growth Indicator Tube) tubes (Becton Dickinson, USA). Rt-PCR method was performed by using Anyplex MTB/NTM test. In the first stage of the study, the performance of the Anyplex MTB/NTM test was compared with the gold standard culture method. M.tuberculosis was isolated in 178 specimens out of 10.652 (1.7%). After the comparison with the gold standard culture method, the sensitivity and specificity of Anyplex MTB/NTM test was found to be 84% and 99% respectively in pulmonary samples, and 74% and 99% respectively in extrapulmonary samples. In the second stage of the study, PCR method with laboratory designed primers was applied to 100 culture positive samples. The PCR results of 98 samples were found to be in agreement with the culture, while M.tuberculosis DNA was not detected in two samples. As a result of the study it was concluded that Anyplex MTB/NTM test is a rapid, practical and reliable method that can be used in routine tuberculosis diagnosis. The high agreement between PCR method using the laboratory-designed primers and the PCR method used in routine practice will lighten the way for the development of national tuberculosis molecular diagnostic kits with a relevant cost. In this way, it will be possible to perform rapid diagnosis in a more cost-effective manner in routine diagnosis laboratories.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tuberculose/diagnóstico , Tuberculose/microbiologia
8.
BMC Infect Dis ; 19(1): 934, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694567

RESUMO

BACKGROUND: Morbidity and mortality from Mycobacterium tuberculosis (Mtb) infection remain significant in cancer patients. We evaluated clinical characteristics, management, and outcomes in patients with active Mtb infection at our institution who had cancer or suspicion of cancer. METHODS: We retrospectively examined medical records of all patients with laboratory-confirmed active Mtb infection diagnosed between 2006 and 2014. RESULTS: A total of 52 patients with laboratory-confirmed active Mtb infection were identified during the study period, resulting in an average rate of 6 new cases per year. Thirty-two (62%) patients had underlying cancer, while 20 (38%) patients did not have cancer but were referred to the institution because of suspicion of underlying malignancy. Among patients with cancer, 18 (56%) had solid tumors; 8 (25%) had active hematologic malignancies; and 6 (19%) had undergone hematopoietic-cell transplantation (HCT). Patients with and without cancer were overall similar with the exception of median age (61 years in cancer patients compared to 53 years in noncancer patients). Pulmonary disease was identified in 32 (62%) patients, extrapulmonary disease in 10 (19%) patients, and disseminated disease in 10 (19%) patients. Chemotherapy was delayed in 53% of patients who were to receive such treatment. Eleven patients (all of whom had cancer) died; 3 of these deaths were attributable to Mtb infection. CONCLUSIONS: Although not common, tuberculosis remains an important infection in patients with cancer. Approximately one-third of patients were referred to our institution for suspicion of cancer but were ultimately diagnosed with active Mtb infection rather than malignancy.


Assuntos
Institutos de Câncer , Neoplasias Pulmonares/diagnóstico , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/diagnóstico , Tuberculose/epidemiologia , Centros Médicos Acadêmicos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Feminino , Neoplasias Hematológicas , Transplante de Células-Tronco Hematopoéticas , Humanos , Incidência , Leucemia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Texas/epidemiologia , Resultado do Tratamento , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Adulto Jovem
9.
Nat Struct Mol Biol ; 26(12): 1106-1113, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740853

RESUMO

T-box riboregulators are a class of cis-regulatory RNAs that govern the bacterial response to amino acid starvation by binding, decoding and reading the aminoacylation status of specific transfer RNAs. Here we provide a high-resolution crystal structure of a full-length T-box from Mycobacterium tuberculosis that explains tRNA decoding and aminoacylation sensing by this riboregulator. Overall, the T-box consists of decoding and aminoacylation sensing modules bridged by a rigid pseudoknot structure formed by the mid-region domains. Stem-I and the Stem-II S-turn assemble a claw-like decoding module, while the antiterminator, Stem-III, and the adjacent linker form a tightly interwoven aminoacylation sensing module. The uncharged tRNA is selectively recognized by an unexpected set of favorable contacts from the linker region in the aminoacylation sensing module. A complex structure with a charged tRNA mimic shows that the extra moiety dislodges the linker, which is indicative of the possible chain of events that lead to alternative base-pairing and altered expression output.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , RNA Bacteriano/metabolismo , RNA de Transferência/metabolismo , Proteínas com Domínio T/metabolismo , Aminoacilação , Proteínas de Bactérias/química , Pareamento de Bases , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/química , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA de Transferência/química , Riboswitch , Proteínas com Domínio T/química , Tuberculose/microbiologia
10.
Vet Microbiol ; 239: 108482, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31759775

RESUMO

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis, a chronic infectious disease that can affect cattle, other domesticated species, wild animals and humans. This disease produces important economic losses worldwide. Two M. bovis strains (04-303 and 534) have been isolated in Argentina. Whereas the 04-303 strain was isolated from a wild boar, the 534 strain was obtained from cattle. In a previous study, six weeks after infection, the 04-303 strain induced 100% mortality in mice. By contrast, mice infected with the 534 strain survived, with limited tissue damage, after four months. In this study we compared all predictive proteins encoded in both M. bovis genomes. The comparative analysis revealed 141 polymorphic proteins between both strains. From these proteins, nine virulence proteins showed polymorphisms in 04-303, whereas five did it in the 534 strain. Remarkably, both strains contained a high level of polymorphism in proteins related to phthiocerol dimycocerosate (PDIM) synthesis or transport. Further experimental evidence indicated that only mutations in the 534 strain have an impact on PDIM synthesis. The observed reduction in PDIM content in the 534 strain, together with its low capacity to induce phagosome arrest, may be associated with the reported deficiency of this strain to replicate and survive inside bovine macrophages. The findings of this study could contribute to a better understanding of pathogenicity and virulence aspects of M. bovis, which is essential for further studies aiming at developing new vaccines and diagnostic techniques for bovines.


Assuntos
Mycobacterium bovis/genética , Mycobacterium bovis/patogenicidade , Tuberculose/microbiologia , Virulência/genética , Animais , Bovinos , Camundongos , Mutação , Mycobacterium bovis/classificação , Análise de Sobrevida , Sus scrofa/microbiologia , Tuberculose/mortalidade , Tuberculose Bovina/microbiologia
11.
Int J Nanomedicine ; 14: 6707-6719, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31692512

RESUMO

Background: Tuberculosis is the leading cause of death by an infectious microorganism worldwide. Conventional treatment lasts at least six months and has adverse effects; therefore, it is important to find therapeutic alternatives that reduce the bacterial load and may reduce the treatment duration. The immune response against tuberculosis can be modulated by several mechanisms, including extracellular vesicles (EVs), which are nano-sized membrane-bound structures that constitute an efficient communication mechanism among immune cells. Methods: The EVs released by the J774A.1 mouse macrophage cell line, both spontaneously (S-EV) and after infection with Mycobacterium tuberculosis H37Rv (Mtb-EV), were purified by ultra-centrifugation and size-exclusion chromatography. The size distribution and chemical composition of these EVs were evaluated, and their effect on the bacterial load and the production of cytokines was determined in both in vitro and in vivo models of M. tuberculosis infection. Results: Mtb-EV are larger than S-EV, they contain M. tuberculosis-specific antigens (not detected in EVs released from M. fortuitum-infected J774A.1 cells) and are rich in phosphatidylserine, present in their outer membrane layer. S-EV, but not Mtb-EV, reduced the bacterial load and the production of MCP-1 and TNF-α in M. tuberculosis-infected macrophages, and these effects were reversed when phosphatidylserine was blocked with annexin V. Both S-EV and Mtb-EV significantly reduced the lung bacterial load in mice infected with M. tuberculosis after 60 days of treatment, but they had no effect on survival or on the lung pneumonic area of these mice. Conclusion: J774A.1 macrophages infected with M. tuberculosis H37Rv released EVs that differed in size and phosphatidylserine content from spontaneously released EVs, and these EVs also had different biological effects: S-EV reduced the mycobacterial load and the cytokine production in vitro (through a phosphatidylserine-dependent mechanism), while both EVs reduced the lung bacterial load in vivo. These results are the basis for further experiments to evaluate whether EVs improve the efficiency of the conventional treatment for tuberculosis.


Assuntos
Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Tuberculose/terapia , Animais , Carga Bacteriana , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Vesículas Extracelulares/química , Vesículas Extracelulares/transplante , Masculino , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologia
12.
Infect Immun ; 88(1)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31636137

RESUMO

Mycobacterium tuberculosis Rv3775 (LipE) was annotated as a putative lipase. However, its lipase activity has never been characterized, and its precise role in tuberculosis (TB) pathogenesis has not been thoroughly studied to date. We overexpressed and purified the recombinant LipE (rLipE) protein and demonstrated that LipE has a lipase/esterase activity. rLipE prefers medium-chain ester substrates, with the maximal activity on hexanoate. Its activity is the highest at 40°C and pH 9. We determined that rLipE hydrolyzes trioctanoate. Using site-directed mutagenesis, we confirmed that the predicted putative activity triad residues Ser97, Gly342, and His363 are essential for the lipase activity of rLipE. The expression of the lipE gene was induced under stressed conditions mimicking M. tuberculosis' intracellular niche. The gene-disrupting mutation of lipE led to significantly reduced bacterial growth inside THP-1 cells and human peripheral blood mononuclear cell-derived macrophages and attenuated M. tuberculosis infection in mice (with ∼8-fold bacterial load reduction in mouse lungs). Our data suggest that LipE functions as a lipase and is important for M. tuberculosis intracellular growth and in vivo infection.


Assuntos
Esterases/metabolismo , Lipase/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose/microbiologia , Fatores de Virulência/metabolismo , Animais , Análise Mutacional de DNA , Modelos Animais de Doenças , Estabilidade Enzimática , Esterases/deficiência , Esterases/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Lipase/deficiência , Lipase/genética , Camundongos , Modelos Teóricos , Mutagênese Sítio-Dirigida , Células THP-1 , Temperatura Ambiente , Fatores de Virulência/genética
13.
World J Microbiol Biotechnol ; 35(11): 168, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31654206

RESUMO

DNA methylation has been introduced as a promising biomarker for different diseases. Alterations in macrophage DNA methylation status have been documented during Mycobacterium tuberculosis (Mtb) infection. We conducted this study using a human methylation PCR array kit, which comprised a panel of 22 genes in TLR2 signaling pathway, in order to gain insights into epigenetic interactions between drug-susceptible and -resistant Mtb strains and THP-1-derived macrophages (one of the main host immunity cells during TB infection). We also evaluated the expression of Rv1988 gene in the studied isolates. It was found that the methylation level of all of the studied inflammatory genes, except Irak-2 and Tbk-1, increased in THP-1 macrophages, which were infected by extensively drug-resistant (XDR) Mtb strains, compared with the mock cells (P < 0.05). In susceptible strains, we only found hypomethylation in Irak-2 gene, in addition to a slight increase in the methylation levels of Ubev, Ube2n, and Traf6 genes. The present findings provide new insights into the potential role of resistant and susceptible Mtb strains in promoting aberrant epigenetic modifications in macrophages. Further investigations on the host epigenomes, infected with different Mtb isolates, are needed to elucidate their functions in immunological responses and to introduce new effective tools against Mtb infection.


Assuntos
Metilação de DNA , Epigênese Genética , Macrófagos/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose/genética , Tuberculose/metabolismo , Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Tuberculose Extensivamente Resistente a Medicamentos , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Macrófagos/microbiologia , Metiltransferases/genética , Mycobacterium tuberculosis/patogenicidade , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Células THP-1 , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 2 Toll-Like/genética , Tuberculose/microbiologia , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
14.
EMBO J ; 38(22): e101876, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31583725

RESUMO

Clonal microbial populations are inherently heterogeneous, and this diversification is often considered as an adaptation strategy. In clinical infections, phenotypic diversity is found to be associated with drug tolerance, which in turn could evolve into genetic resistance. Mycobacterium tuberculosis, which ranks among the top ten causes of mortality with high incidence of drug-resistant infections, exhibits considerable phenotypic diversity. In this study, we quantitatively analyze the cellular dynamics of DNA damage responses in mycobacteria using microfluidics and live-cell fluorescence imaging. We show that individual cells growing under optimal conditions experience sporadic DNA-damaging events manifested by RecA expression pulses. Single-cell responses to these events occur as transient pulses of fluorescence expression, which are dependent on the gene-network structure but are triggered by extrinsic signals. We demonstrate that preexisting subpopulations, with discrete levels of DNA damage response, are associated with differential susceptibility to fluoroquinolones. Our findings reveal that the extent of DNA integrity prior to drug exposure impacts the drug activity against mycobacteria, with conceivable therapeutic implications.


Assuntos
Proteínas de Bactérias/metabolismo , Ciprofloxacino/farmacologia , Dano ao DNA/genética , Mycobacterium tuberculosis/genética , Análise de Célula Única , Estresse Fisiológico , Tuberculose/patologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Dano ao DNA/efeitos dos fármacos , Humanos , Microfluídica , Mycobacterium tuberculosis/efeitos dos fármacos , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
16.
Internist (Berl) ; 60(11): 1155-1175, 2019 Nov.
Artigo em Alemão | MEDLINE | ID: mdl-31641790

RESUMO

Tuberculosis is a bacterial infectious disease that is usually transmitted by inhalation of droplets containing the bacteria. The World Health Organization (WHO) estimates that approximately 10 million patients were newly diagnosed with tuberculosis in 2017. Rapid diagnosis relies on a combination of imaging and microbiological, molecular, and, rarely, immunological tests. Genotypic methods enable early diagnosis and allow highly accurate prediction of drug resistance. Phenotypic (culture-based) methods are the diagnostic gold standard. Standard management of patients with pan drug-susceptible pulmonary tuberculosis includes a combination of rifampicin, isoniazid, ethambutol and pyrazinamide for 2 months followed by rifampicin and isoniazid for additional 4 months, which leads to cure rates of >80%. With individualized treatment schemes, similar cure rates can be achieved for patients with multidrug-resistant tuberculosis.


Assuntos
Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose/diagnóstico , Tuberculose/tratamento farmacológico , Etambutol/uso terapêutico , Humanos , Isoniazida/uso terapêutico , Pirazinamida/uso terapêutico , Rifampina/uso terapêutico , Tuberculose/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia
17.
Mymensingh Med J ; 28(4): 920-924, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31599261

RESUMO

The diagnosis of mycotic lung infection in pulmonary TB patients remains misdiagnosed because of its non-specific clinical manifestations which mimics the symptoms of TB. Physicians have to rely on the investigation but as radiology and pathology cannot probe the appropriate diagnosis, conventional microbiology or PCR testing continue as an essential mode for the diagnosis. In developing country like India PCR is not cost effective. Thus, Direct microscopy by KOH (10%), Gram's staining & Culture remains only option for identification. A three-year cross-sectional study was carried out in the Department of Microbiology, Maharishi Markandeshwar Institute of Medical Science & Research, Mullana, India from August 2015 to August 2018. On 300 LED positive sputum samples collected from previously treated cases of pulmonary TB. Early morning sputum was collected and subjected to KOH 10%, Gram's staining afterwards cultured on Sabouraud Dextrose Agar and species identification was done by LPCB preparation. In 300 LED smear positive samples, the dominant pathogens were C. albicans (43.3%), followed by C. non-albicans (26.7%), A. fumigatus (21.7%) etc. ATT administration for 5-8 months' duration of illness showed highest fungal infection (45%) and maximum growth of fungus was seen in the Autumn season (45%). The co-occurrence of fungi with tubercle bacteria adds fatal consequences thus routine screening is recommended for proper diagnosis and early treatment of mycotic infection in the patients of Pulmonary TB on ATT.


Assuntos
Coinfecção , Tuberculose/microbiologia , Estudos Transversais , Fungos , Humanos , Índia/epidemiologia , Sensibilidade e Especificidade , Tuberculose/epidemiologia
18.
Pak J Pharm Sci ; 32(4): 1529-1536, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31608871

RESUMO

Tuberculosis (TB) is among the 10 most common worldwide causes of mortality. In Pakistan, estimated 510,000 tuberculosis patients had been diagnosed with an occurrence of 276/100,000. As per most recent global TB report 2018, Pakistan is amongst the 30 countries high TB with drug-resistant Mycobacterium tuberculosis particularly MDR (multi-drug resistant strains). A retrospective study had been designed using DR-TB patients' records from January 2013 to the December 2017 year from a public sector hospital in Karachi. Overall 315 drug-resistant tuberculosis patient's data had been incorporated in the study. All data had been analyzed using SPSS version 16 software. Chi-square test had been used to analyze the data with CI (confidence interval) 95% and level of significance 5%. The study result showed that 64.1% MDR patients, 27.9% MTB rifampicin resistance, 4.8% mono-drug resistant , XDR(1.6%), 1% poly-drug resistant and only 0.6% are MDR suspects showing no association of DR-TB with gender (p-value 0.787), age group (p-value 0.757), treatment outcomes (p-value 0.549), year of registration( p-value 0.206), first line treatment history(p-value 0.643) with a 95% confidence interval. The drug resistance TB cases have been periodically rising every year. Early identification is required to reduce the percent mortality and inhibit the disease transmission.


Assuntos
Tuberculose/tratamento farmacológico , Tuberculose/epidemiologia , Adolescente , Adulto , Idoso , Antituberculosos/uso terapêutico , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Paquistão/epidemiologia , Prevalência , Saúde Pública , Estudos Retrospectivos , Rifampina/uso terapêutico , Tuberculose/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adulto Jovem
19.
Pak J Pharm Sci ; 32(4): 1709-1714, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31608894

RESUMO

Resistance pattern both in newly and previously treated-TB patients and risk factors associated in spread of tuberculosis are investigated in the current study. A total 244 Mycobacterium tuberculosis isolates were used for drug-susceptibility test against four drugs. Environmental risk factors were assessed by using self-designed history proforma. Among 244 TB-isolates, 64% were categorized as MDR-TB in drug-susceptibility test. Male proportion was 51% while 32% belonged to 15-34 years age group and 49% were from city Lahore whereas majority of people (31%) was working on daily wages. Divergent drug-resistance pattern was obtained; RIF (68%), SM (52%), EMB (51%). INH showed only (27%) resistance against first-line anti-TB drug. Drug-resistance prevalence for two drug combination was highest (50%) for (INH+SM) and (INH+EMB) followed by (RIF+SM) (49%) whereas for three drugs combination (INH+RIF+EMB) and (INH+RIF+SM) the prevalence was almost same 50% and 49% respectively while 66% patients were categorized as previously treated and 34% as new TB cases. In drug susceptibility test, 71% were identified as MDR-TB among New TB cases, while 63% were identified as MDR-TB from previously treated cases. Surprisingly DST results displayed that percentage prevalence of MDR-TB both in newly and previously treated cases was almost same.


Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/microbiologia , Adolescente , Adulto , Idoso , Antituberculosos/uso terapêutico , Meio Ambiente , Feminino , Humanos , Isoniazida/farmacologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Paquistão/epidemiologia , Prevalência , Rifampina/farmacologia , Fatores de Risco , Tuberculose/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adulto Jovem
20.
Rev Inst Med Trop Sao Paulo ; 61: e51, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31531629

RESUMO

A drug resistance survey involving Mycobacterium tuberculosis isolated from patients of a tertiary Hospital in the Rio de Janeiro city (RJ), Brazil, between the years 1996 and 1998 revealed a high frequency of isoniazid (HR) resistance. These isolates were revisited and genotyped. Patients came from different RJ neighborhoods and municipalities, and 70% were outpatients. Applying the 3' and 5' IS 6110 -RFLP and the Spoligotype genotyping methods, the clonal structure of this population was investigated obtaining a snapshot of past epidemiological events. The 3' clusters were subsequently 5' IS 6110 -RFLP typed. Spoligotyping was analyzed in the SITVIT2 database. Epidemiological relationships were investigated. The major lineage was T (54.4%), and SIT 53/T1 and SIT 535/T1 were the most frequent. The T1 sublineage comprises 12.8% of resistant strains and SIT 535 were assigned for 31.8% of them. Orphan patterns corresponded to 12% and 73.3% and belonged to the T lineage. One pattern was unlisted in the SITVIT2. The 5' IS 6110 -RFLP did not confirm 3/12 of the 3' IS 6110 -RFLP clusters. A combination of all methods decreased the number of clusters to three. Nosocomial transmission was associated with one cluster involving a hospital cupbearer. This event was suspected in a multidrug resistant-TB inpatient caregiver who harbored a mixed infection. The 3' IS 6110 clusters were associated with HR (p=0.046). These genotypic retrospective data may reflect a fraction of more extensive recent transmission in different communities that may be corroborated by the concentration of HR patients, and may serve as a database for further evolutionary and characterization evaluation of circulating strains and together with epidemiological data favors a more effective transmission control.


Assuntos
Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana/genética , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Adolescente , Adulto , Técnicas de Tipagem Bacteriana , Brasil , DNA Bacteriano/genética , Feminino , Genótipo , Técnicas de Genotipagem , Humanos , Masculino , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Polimorfismo de Fragmento de Restrição , Estudos Retrospectivos
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